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Sample records for fibroblast proliferative activity

  1. Quiescent and proliferative fibroblasts exhibit differential p300 HAT activation through control of 5-methoxytryptophan production.

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    Huei-Hsuan Cheng

    Full Text Available Quiescent fibroblasts possess unique genetic program and exhibit high metabolic activity distinct from proliferative fibroblasts. In response to inflammatory stimulation, quiescent fibroblasts are more active in expressing cyclooxygenase-2 and other proinflammatory genes than proliferative fibroblasts. The underlying transcriptional mechanism is unclear. Here we show that phorbol 12-myristate 13-acetate (PMA and cytokines increased p300 histone acetyltransferase activity to a higher magnitude (> 2 fold in quiescent fibroblasts than in proliferative fibroblasts. Binding of p300 to cyclooxygenase-2 promoter was reduced in proliferative fibroblasts. By ultrahigh-performance liquid chromatography coupled with a quadrupole time of flight mass spectrometer and enzyme-immunoassay, we found that production of 5-methoxytryptophan was 2-3 folds higher in proliferative fibroblasts than that in quiescent fibroblasts. Addition of 5-methoxytryptophan and its metabolic precursor, 5-hydroxytryptophan, to quiescent fibroblasts suppressed PMA-induced p300 histone acetyltransferase activity and cyclooxygenase-2 expression to the level of proliferative fibroblasts. Silencing of tryptophan hydroxylase-1 or hydroxyindole O-methyltransferase in proliferative fibroblasts with siRNA resulted in elevation of PMA-induced p300 histone acetyltransferase activity to the level of that in quiescent fibroblasts, which was rescued by addition of 5-hydroxytryptophan or 5-methoxytryptophan. Our findings indicate that robust inflammatory gene expression in quiescent fibroblasts vs. proliferative fibroblasts is attributed to uncontrolled p300 histone acetyltransferase activation due to deficiency of 5-methoxytryptophan production. 5-methoxytryptophan thus is a potential valuable lead compound for new anti-inflammatory drug development.

  2. Relationship of serum somatomedin-like activity and fibroblast proliferative activity with age and growth in the rat.

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    Olsen, R F; Wangsness, P J; Patton, W H; Martin, R J

    1980-03-01

    An assay for fibroblast proliferative activity (FPA) using human lung fibroblasts, WI-38, was described. The assay was responsive to varying rat serum levels and was not influenced by direct growth hormone (GH) addition. The relationship of serum growth factors to age and body weight was examined in the rat. In study 1, serum was obtained from lean Zucker rats at 3, 5, 7, 9, 11 and 30 wk of age. Six samples were taken at each age and serum samples were analyzed for somatomedin-like activity (Sm) and fibroblast proliferative activity (FPA). Serum Sm was not different at any of the sampling ages. FPA was low at 3 wk, but was higher and constant from 5 wk to 30 wk. In study 2, 73 lean Zucker rats (7 wk of age) were maintained on laboratory chow and water ad libitum for 4 wk, and then serum was obtained by decapitation. The rats were ranked according to each of four different criteria: average daily gain (ADG) for the duration of the study, ADG for the fourth week, total body protein and total body fat. Serum Sm, FPA and insulin concentrations on the top 10 and bottom 10 rats of each ranking were compared. Neither FPA nor insulin was significantly different for any ranking. Serum Sm was significantly higher in the top 10 rats ranked by ADG for the duration of the study. Sm was not significantly different in rats ranked by body weight, total body protein or total body fat. The data suggest that serum somatomedin-like activity (Sm) may be important in the earlier stages of growth in rats.

  3. Effects of breed and zeranol implantation on serum insulin, somatomedin-like activity and fibroblast proliferative activity.

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    Wangsness, P J; Olsen, R F; Martin, R J

    1981-01-01

    Twenty-eight Suffolk-sired (Sx) and 28 Finnsheep-sired (Fx) lambs were implanted with either 0 or 12 mg zeranol. Zeranol significantly increased average daily gain over that of controls. Serum taken at biweekly intervals for 6 weeks was assayed for insulin, somatomedin-like activity (Sm) and fibroblast proliferative activity (FPA). Insulin appeared to increase with time, but there were no consistent time changes for FPA or Sm. Serum insulin concentration was higher (P less than .05) in implanted lambs than in controls (33.4 vs 25.6 microU/ml). Unlike insulin, serum Sm and FPA were not affected by zeranol implantation, and, thus, these serum factors appeared not to be involved in zeranol-stimulated growth. Sm was higher in the faster growing Sx lambs than in the slower growing Fx lambs. Thus, serum Sm activity may be involved in normal regulation of growth.

  4. Relationship of serum somatomedin-like activity and fibroblast proliferative activity with age and body weight gain in sheep.

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    Olsen, R F; Wangsness, P J; Patton, W H; Martin, R J

    1981-01-01

    The relationship between serum growth factors and body weight gain was examined in five Dorset lambs. The lambs were weighed and bled by jugular puncture at 2-week intervals between 2 and 18 weeks of age. Somatomedin-like activity (Sm) declined from initially high concentrations at 2 weeks to fairly constant concentrations between 6 and 18 weeks. Relative weight gain--i.e., gain expressed as a percentage of body weight--declined in a manner similar to that of Sm. Mean relative weight gain and mean Sm for the eight 2-week intervals were significantly related (r = .84). Absolute body weight gain--i.e., gain expressed in kilograms--remained fairly constant throughout the study and was not significantly correlated to Sm (r = .15). Serum fibroblast proliferative activity (FPA) was measured as a possible indicator of collective activities of serum growth factors. FPA initially followed a pattern similar to that of Sm, decreasing between 2 and 6 weeks and plateauing until 12 weeks. After 12 weeks, FPA increased to concentrations similar to those observed at 2 weeks. The increase in FPA after 12 weeks was apparently due to an increase in a non-Sm growth factor and had no obvious relationship to body weight changes. Results of the in vitro cell assay system might have been more meaningful if cell type(s) other than WI-38 fibroblasts (e.g., myogenic cells) had been used for estimating collective activities of serum mitogenic factors. The data suggest that serum Sm-like activity may be important in the regulation of growth in sheep.

  5. Proliferative Effects of Histamine on Primary Human Pterygium Fibroblasts

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    Zhenwei Qin

    2016-01-01

    Full Text Available Purpose. It has been confirmed that inflammatory cytokines are involved in the progression of pterygium. Histamine can enhance proliferation and migration of many cells. Therefore, we intend to investigate the proliferative and migratory effects of histamine on primary culture of human pterygium fibroblasts (HPFs. Methods. Pterygium and conjunctiva samples were obtained from surgery, and toluidine blue staining was used to identify mast cells. 3-[4, 5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT was performed to evaluate the proliferative rate of HPFs and human conjunctival fibroblasts (HCFs; ki67 expression was also measured by immunofluorescence analysis. Histamine receptor-1 (H1R antagonist (Diphenhydramine Hydrochloride and histamine receptor-2 (H2R antagonist (Nizatidine were added to figure out which receptor was involved. Wound healing model was used to evaluate the migratory ability of HPFs. Results. The numbers of total mast cells and degranulated mast cells were both higher in pterygium than in conjunctiva. Histamine had a proliferative effect on both HPFs and HCFs, the effective concentration (10 μmol/L on HPFs was lower than on HCFs (100 μmol/L, and the effect could be blocked by H1R antagonist. Histamine showed no migratory effect on HPFs. Conclusion. Histamine may play an important role in the proliferation of HPFs and act through H1R.

  6. Anti-proliferative effect of olmesartan on Tenon's capsule fibroblasts

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    Xuan Wang

    2016-05-01

    Full Text Available AIM: To evaluate the inhibitive effect of olmesartan to fibroblast proliferation and the anti-scarring effect in Tenon’s capsule, both in vitro and in vivo. METHODS: Human primary Tenon’s capsule fibroblasts were cultured in vitro, treated with up titrating concentrations of olmesartan. The rate of inhibition was tested with methyl thiazol tetrazolium (MTT method. Real-time PCR was performed to analyze changes in mRNA expressions of the fibrosis-related factors: matrix metalloproteinase-2 (MMP-2, tissue inhibitor of metalloproteinase (TIMP-1,2 and proliferating cell nuclear antigen (PCNA. Thirty rabbits were divided into 5 groups (3, 7, 14, 21, and 28d. A rabbit conjunctiva flap model was created in each eye. Olmesartan solution was injected subconjunctivally and then evaluated its anti-proliferation and anti-fibrosis effects through the histological morphology and immunohistochemistry of MMP-2 and PCNA in each group. Only the 7d group was treated with Masson’s trichrome to compare the neovascularization in the subconjunctiva area. RESULTS: In vitro, cultured Tenon's capsule human fibroblasts showed a dose dependent inhibition by olmesartan in MTT. Olmesartan reduced mRNA expressions of MMP-2 and PCNA but increased mRNA expressions of TIMP-1 and TIMP-2. In vivo, the rabbit eyes treated with olmesartan at 3rd, 7th, 14th and 21st days demonstrated a significant reduced expressions of MMP-2 and PCNA compared with control eye, no significant difference observed in 28th day group. The cellular proliferation and neovascularization was suppressed by olmesartan in Masson’s trichrome observation. CONCLUSION: By inhibiting fibroblasts in vitro and in vivo, olmesartan prevents the proliferation and activity of fibroblasts in scar tissue formation, which might benefit glaucoma filtering surgery.

  7. Anti-proliferative effect of olmesartan on Tenon's capsule fibroblasts.

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    Wang, Xuan; Fan, Ya-Zhi; Yao, Liang; Wang, Jian-Ming

    2016-01-01

    To evaluate the inhibitive effect of olmesartan to fibroblast proliferation and the anti-scarring effect in Tenon's capsule, both in vitro and in vivo. Human primary Tenon's capsule fibroblasts were cultured in vitro, treated with up titrating concentrations of olmesartan. The rate of inhibition was tested with methyl thiazol tetrazolium (MTT) method. Real-time PCR was performed to analyze changes in mRNA expressions of the fibrosis-related factors: matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase (TIMP-1,2) and proliferating cell nuclear antigen (PCNA). Thirty rabbits were divided into 5 groups (3, 7, 14, 21, and 28d). A rabbit conjunctiva flap model was created in each eye. Olmesartan solution was injected subconjunctivally and then evaluated its anti-proliferation and anti-fibrosis effects through the histological morphology and immunohistochemistry of MMP-2 and PCNA in each group. Only the 7d group was treated with Masson's trichrome to compare the neovascularization in the subconjunctiva area. In vitro, cultured Tenon's capsule human fibroblasts showed a dose dependent inhibition by olmesartan in MTT. Olmesartan reduced mRNA expressions of MMP-2 and PCNA but increased mRNA expressions of TIMP-1 and TIMP-2. In vivo, the rabbit eyes treated with olmesartan at 3(rd), 7(th), 14(th) and 21(st) days demonstrated a significant reduced expressions of MMP-2 and PCNA compared with control eye, no significant difference observed in 28(th) day group. The cellular proliferation and neovascularization was suppressed by olmesartan in Masson's trichrome observation. By inhibiting fibroblasts in vitro and in vivo, olmesartan prevents the proliferation and activity of fibroblasts in scar tissue formation, which might benefit glaucoma filtering surgery.

  8. The differential proliferative response of fetal and adult human skin fibroblasts to TGF-β is retained when cultured in the presence of fibronectin or collagen.

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    Armatas, Andreas A; Pratsinis, Harris; Mavrogonatou, Eleni; Angelopoulou, Maria T; Kouroumalis, Anastasios; Karamanos, Nikos K; Kletsas, Dimitris

    2014-08-01

    Transforming growth factor-β is a multifunctional and pleiotropic factor with decisive role in tissue repair. In this context, we have shown previously that TGF-β inhibits the proliferation of fetal human skin fibroblasts but stimulates that of adult ones. Given the dynamic reciprocity between fibroblasts, growth factors and extracellular matrix (ECM) in tissue homeostasis, the present study aims to investigate the role of fibronectin and collagen in the proliferative effects of TGF-β on fetal and adult cells. Human fetal and adult skin fibroblasts were grown either on plastic surfaces or on surfaces coated with fibronectin or collagen type-I, as well as, on top or within three-dimensional matrices of polymerized collagen. Their proliferative response to TGF-β was studied using tritiated thymidine incorporation, while the signaling pathways involved were investigated by Western analysis and using specific kinase inhibitors. Fetal skin fibroblast-proliferation was inhibited by TGF-β, while that of adult cells was stimulated by this factor, irrespective of the presence of fibronectin or collagen. Both inhibitory and stimulatory activities of TGF-β on the proliferation of fetal and adult fibroblasts, respectively, were abrogated when the Smad pathway was blocked. Moreover, inhibition of fetal fibroblasts was mediated by PKA activation, while stimulation of adult ones was effected through the autocrine activation of FGF receptor and the MEK-ERK pathway. Fetal and adult human skin fibroblasts retain their differential proliferative response to TGF-β when cultured in the presence of fibronectin and unpolymerized or polymerized collagen. The interplay between TGF-β and ECM supports the pleiotropic nature of this growth factor, in concordance with the different repair strategies between fetuses and adults. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Aging Impairs the Proliferative Capacity of Cardiospheres, Cardiac Progenitor Cells and Cardiac Fibroblasts: Implications for Cell Therapy

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    Jianqin Ye

    2013-09-01

    Full Text Available Introduction: Cardiospheres (CS are self-assembling clusters of cells that can be grown from cardiac tissue. They contain a heterogeneous cell population that includes cardiac progenitor cells (CPCs and cardiac fibroblasts. CS and CPCs have been shown to improve cardiac function after myocardial infarction (MI in experimental models and are now being studied in clinical trials. The effects of aging on the proliferative capacity of CS and CPCs, and the paracrine signaling between cell types, remain incompletely understood. Methods and Results: We compared the growth of CS from young and aging murine hearts at baseline and following MI. The number of CS from young and aging hearts was similar at baseline. However, after MI, young hearts had a dramatic increase in the number of CS that grew, but this proliferative response to MI was virtually abolished in the aging heart. Further, the proportion of cells within the CS that were CPCs (defined as Sca-1(stem cell antigen-1+/CD45− was significantly lower in aging hearts than young hearts. Thus the number of available CPCs after culture from aging hearts was substantially lower than from young hearts. Cardiac fibroblasts from aging hearts proliferated more slowly in culture than those from young hearts. We then investigated the interaction between aging cardiac fibroblasts and CPCs. We found no significant paracrine effects on proliferation between these cell types, suggesting the impaired proliferation is a cell-autonomous problem. Conclusions: Aging hearts generate fewer CPCs, and aging CPCs have significantly reduced proliferative potential following MI. Aging cardiac fibroblasts also have reduced proliferative capacity, but these appear to be cell-autonomous problems, not caused by paracrine signaling between cell types.

  10. Proliferative and inductive effects of Cyclosporine a on gingival fibroblast of child and adult

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    Bahareh Nazemi Salman

    2013-01-01

    Conclusions: The mechanism of a CsA-induced fibroblast overgrowth may converge on the steps involving fibroblast proliferation and cytokine network including IL-6, IL-8, IL-1β, TGF-β1, and PGE 2 , in both adults and pediatrics. As the prevalence and intensity of drug-induced gingival overgrowth is more serious in the pediatrics. As group than in adults, we suggest that more studies be conducted on the pediatric group.

  11. High inorganic phosphate causes DNMT1 phosphorylation and subsequent fibrotic fibroblast activation

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    Tan, Xiaoying [Department of Nephrology and Rheumatology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); Department of Cardiology and Pneumology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); Xu, Xingbo [Department of Cardiology and Pneumology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); Zeisberg, Elisabeth M. [Department of Cardiology and Pneumology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); German Center for Cardiovascular Research (DZHK), Göttingen (Germany); Zeisberg, Michael, E-mail: mzeisberg@med.uni-goettingen.de [Department of Nephrology and Rheumatology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); German Center for Cardiovascular Research (DZHK), Göttingen (Germany)

    2016-04-08

    Phosphate is an essential constituent of critical cellular functions including energy metabolism, nucleic acid synthesis and phosphorylation-dependent cell signaling. Increased plasma phosphate levels are an independent risk factor for lowered life-expectancy as well as for heart and kidney failure. Nevertheless, direct cellular effects of elevated phosphate concentrations within the microenvironment are poorly understood and have been largely neglected in favor of phosphor-regulatory hormones. Because interstitial fibrosis is the common determinant of chronic progressive kidney disease, and because fibroblasts are major mediators of fibrogenesis, we here explored the effect of high extracellular phosphate levels on renal fibroblasts. We demonstrate that high inorganic phosphate directly induces fibrotic fibroblast activation associated with increased proliferative activity, increased expression of α-smooth muscle actin and increased synthesis of type I collagen. We further demonstrate that such fibroblast activation is dependent on phosphate influx, aberrant phosphorylation of DNA methyltransferase DNMT1 and aberrant CpG island promoter methylation. In summary, our studies demonstrate that elevated phosphate concentrations induce pro-fibrotic fibroblast activation independent of phospho-regulatory hormones. - Highlights: • We exposed human kidney fibroblasts to media containing 1 mM or 3 mM phosphate. • Increased phosphate influx causes phosphorylation of DNA methyltransferase Dnmt1. • Phosphorylated Dnmt1 causes promoter methylation and transcriptional silencing of RASAL1. • Depletion of RASAL1 causes increased intrinsic Ras-GTP activity and fibroblast activation. • Inorganic phosphate causes fibroblast activation independent of phospho-regulatory hormones.

  12. Cancer-associated fibroblasts are an infrequent finding in the microenvironment of proliferative verrucous leukoplakia-associated squamous cell carcinoma.

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    Akrish, Sharon J; Rachmiel, Adi; Sabo, Edmond; Vered, Marilena; Ben-Izhak, Ofer

    2017-05-01

    Cancer-associated fibroblasts (CAFs) are generally associated with negative prognostic factors. This study compares the clinicopathologic impact of CAFs in oral squamous cell carcinoma in patients with a history of proliferative verrucous leukoplakia (p-scca) and patients with conventional squamous cell carcinoma of the buccal mucosa, gingiva, and palate (c-scca). A retrospective clinicopathologic and immunohistochemical analysis of 97 tumor specimens from 78 patients (13 patients with proliferative verrucous leukoplakia-associated squamous cell carcinoma (n = 32) and conventional squamous cell carcinoma from the buccal mucosa, gingiva, and palate (n = 65) was conducted. Immunostaining with anti-alpha-smooth muscle actin (α-SMA) antibody was used to evaluate the presence of CAFs. α-SMA expression was an infrequent finding in p-scca and seen in only 6% of p-scca compared to 40% of c-scca (P < 0.0004). In the c-scca subgroup, α-SMA significantly correlated with tumor size (T) (P = 0.009), tumor thickness (P < 0.0009), perineural invasion (P = 0.009), and microscopic grade (P = 0.018). The presence of CAFs was an infrequent finding in our p-scca cohort which may contribute to its seemingly slower growing and less invasive growth pattern. In the cohort of c-scca patients, higher levels of CAFs correlated with microscopic invasiveness, tumor size, and perineural invasion. Practically, these are important observations as targeting strategies are being developed to combat carcinoma types where CAFs significance has been validated. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Anti-proliferative activity of Fumaria vaillantii extracts on different cancer cell lines

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    Fatemeh Haji Abbas Tabrizi

    2016-01-01

    Full Text Available Plant-derived natural products are known to have cancer chemo-preventive and chemo-therapeutic properties. Plant extracts or their active constituents are used as folk medicine in traditional therapies by 80% of the world population. The aim of the present study was to determine the anti-proliferative potential of Fumaria vaillantii extracts on three different cancer cell lines including malignant melanoma SKMEL-3, human breast adenocarcinoma MCF-7 and human myelogenous leukemia K562 as well as human gingival fibroblast (HGF as normal cell line. Anti-proliferative activity was evaluated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT, flowcytometry and annexin methods. Total phenolics and flavonoids were determined by Folin-Ciocalteu and aluminum chloride methods. Chloroform fraction had the lowest IC 50 value at 72 h (0.1 μg/ml in MCF-7 cells. Flowcytometry and annexin-V analysis indicated that the chloroform fraction induced necrosis in MCF-7 cells. In addition, the colorimetric methods showed that the methanolic fraction possessed the highest amount of total phenolics (33.03 ± 0.75 mg/g of dry powder and flavonoids (10.5 ± 2.0 mg/g of dry powder.The collective data demonstrated that F. vaillantii chloroform fraction may contain effective compounds with chemo-therapeutic potential act through an apoptotic independent pathway.

  14. Polyphenols with Anti-Proliferative Activities from Penthorum Chinense Pursh

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    Doudou Huang

    2014-07-01

    Full Text Available Two new polyphenols, penthorumin C (1 and 2,6-dihydroxyacetophenone-4-O- [4ꞌ,6ꞌ-(S-hexahydroxydiphenoyl]-β-D-glucose (2, along with four known polyphenolic acids, pinocembrin-7-O-[4ꞌꞌ,6ꞌꞌ-hexahydroxydiphenoyl]-β-D-glucose(3, pinocembrin-7-O-[3ꞌꞌ-O- galloyl- 4ꞌꞌ,6ꞌꞌ-hexahydroxydiphenoyl]-β-D-glucose (4, thonningianin A (5, and thonningianin B (6 were isolated from Penthourm chinense. All compounds were evaluated for their anti-proliferative activity in HSC-T6 cells, and 2 and 5 showed significant activity, with IC50 values of 12.7 and 19.2 μM, respectively.

  15. The suppression of fibroblast growth factor 2/fibroblast growth factor 4-dependent tumour angiogenesis and growth by the anti-growth factor activity of dextran derivative (CMDB7).

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    Bagheri-Yarmand, R.; Kourbali, Y; Mabilat, C; Morère, J. F.; Martin, A; Lu, H; Soria, C; Jozefonvicz, J; Crépin, M

    1998-01-01

    Our previous studies showed that carboxymethyl benzylamide dextran (CMDB7) blocks basic fibroblast growth factor (FGF-2)-dependent cell proliferation of a human breast epithelial line (HBL100), suggesting its potential role as a potent antiangiogenic substance. The derived cell line (HH9), which was transformed with the hst/FGF4 gene, has been shown to be highly proliferative in vitro and to induce angiogenic tumours in nude mice. We show here that CMDB7 inhibits the mitogenic activities of t...

  16. Proliferative effect of ammodytin L from the venom of Vipera ammodytes on 208F rat fibroblasts in culture.

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    Rufini, S; Cesaroni, M P; Balestro, N; Luly, P

    1996-12-01

    Ammodytin L, purified from the venom of Vipera ammodytes, triggers a rapid and dramatic lytic process in myotubes in vitro, as well as in differentiated muscle cells in vivo, through a mechanism that is not well understood. Despite its great sequence similarity to phospholipase A2, it is devoid of any enzyme activity. Data on artificial membranes demonstrating a direct interaction between this toxin and the hydrophobic core of the lipid bilayer suggest that the toxin also acts on the lipid microenvironment in cell membranes. Recent experiments on living cells do not confirm this hypothesis, and a more intricate mechanism is proposed. In vitro, ammodytin L has necrotic effects only in well-differentiated myogenic cells, whereas other cell types such as platelets, red blood cells and lymphocytes show neither morphological nor functional alterations. In this work we demonstrate that rat 208F fibroblasts in culture after ammodytin L challenge increase [3H]thymidine incorporation, indicating that this toxin has a myogenic effect. Moreover, ammodytin L increases intracellular Ca2+ by acting on intracellular stores probably by activating a phosphatidylinositol-specific phospholipase C. Preincubation of the cells with ammodytin L did not prevent the massive Ca2+ release evoked by bradykinin, a phenomenon observed when fibroblasts were incubated with both thapsigargin and ionomycin. Heparin, an agent that inhibits the necrotic effect of the myotoxin in myotubes, also reduces the effect of ammodytin L on DNA synthesis. Heparin inhibits only the late sustained increase in intracellular Ca2+ induced by the toxin.

  17. In vitro anti-proliferative activity of clove extract on human gastric carcinoma

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    A. Karimi

    2017-10-01

    Full Text Available Background and objectives: Cancer cell resistance to common chemotherapy agents is on rise. Plants are considered valuable sources of herbal drugs for cancer therapy. The present study was conducted to investigate the in vitro antioxidant, anti-proliferative, and apoptosis-inducing properties of clove (Syzygium aromaticum L. extract in human gastric carcinoma (AGS. Methods: Crude ethanol extract of S. aromaticum dried buds was prepared and  in vitro anti-proliferative effects of the extract on AGS and normal Human dermal fibroblasts (HDF cell lines were studied by MTT assay. To examine apoptosis induction, AGS cells were incubated with IC50 concentrations of the extract, stained with propidium iodide (PI and annexin V-fluorescein isothiocyanate (FITC, and analyzed by flow cytometry. Antioxidant activity and total phenolics and flavonoids contents were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH assay, Folin-Ciocalteu method, and aluminum chloride colorimetric method, respectively. Results: The IC50 of DPPH and total phenolics and flavonoids contents of the extract were 10.05±1.93 μg/mL, 225.6±40 mg GAE/g, and 29.30±2.35 mgRUT/g, respectively. The IC50 of the extract against HDFs was 649 µg/mL, higher than AGS cells, which was 118.7 g/mL at 48 h after treatment. Flow cytometric analysis showed that the extract induced cell apoptosis. Conclusions: Crude ethanol S. aromaticum extract had high total phenolics content, and suppressed the proliferation of human gastric cancer cells, likely due to apoptosis induction. Further studies should be conducted to determine the mechanisms of its anticancer effects.

  18. RNA-Guided Activation of Pluripotency Genes in Human Fibroblasts

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    Xiong, Kai; Zhou, Yan; Blichfeld, Kristian Aabo

    2017-01-01

    fibroblasts. This SAM-mediated activation of LOS can be stably maintained for over 20 days in fibroblasts cultured in either fibroblasts or stem cell medium. However, when attempting to use the SAM-LOS activation as an approach for induced pluripotent stem cells-reprogramming, no embryonic stem-like colonies...

  19. Proliferative activity in oral pyogenic granuloma: A comparative immunohistochemical study

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    Rezvani Gita

    2010-07-01

    Full Text Available Context: Pyogenic granuloma (PG is one of the most common reactive vascular lesions in the oral mucosa, which has been divided into the lobular capillary hemangioma (LCH and the non lobular type (non-LCH as two distinct entities, on the basis of some investigations. Aims: This study aims to compare the proliferative and angiogenic activity of two histological types of PG to determine whether they have two distinct types of biological behavior. Settings and Design: In this retrospective cross-sectional study, immunostaining was performed on 10 cases of each type of PG. Materials and Methods: About 4μm sections were cut from formalin-fixed paraffin-embedded blocks and each specimen was stained with both anti-CD31 and anti-Ki-67 antibodies simultaneously. Labeling index (LI was determined for both types by counting Ki-67 and CD31 positive cells separately and simultaneously in 1000 stromal and luminal cells. Micro vessel count (MVC, the mean number of micro vessels in five areas at Χ200 magnification, was also determined for both groups. Statistical Analysis: The results were statistically compared using the Mann-Whitney U-test. Results: Ki-67 LI in LCH (5.4 ± 2.4 was higher than non-LCH (3.9 ± 3.9. The percentage of CD31 positive cells in LCH (28.5 ± 22 was lower than non-LCH (37.1 ± 20.8 and simultaneously immunostaining for both markers in LCH type (2.4 ± 2.1 was higher than non-LCH (1.2 ± 1. The MVC was approximately 77.35 ± 34.6 and 82.6 ± 42.7 in the lobular areas of LCH and central areas of non-LCH PG, respectively. These differences were not statistically significant. Conclusions: These results demonstrate a higher proliferation activity in endothelial cells of LCH PG than in non-LCH.

  20. Anti-proliferative activity of Monensin and its tertiary amide derivatives.

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    Huczyński, Adam; Klejborowska, Greta; Antoszczak, Michał; Maj, Ewa; Wietrzyk, Joanna

    2015-10-15

    New tertiary amide derivatives of polyether ionophore Monensin A (MON) were synthesised and their anti-proliferative activity against cancer cell lines was studied. Very high activity (IC50=0.09 μM) and selectivity (SI=232) of MON against human biphenotypic myelomonocytic leukemia cell line (MV4-11) was demonstrated. The MON derivatives obtained exhibit interesting anti-proliferative activity, high selectivity index and also are able to break the drug-resistance of cancer cell line.

  1. High inorganic phosphate causes DNMT1 phosphorylation and subsequent fibrotic fibroblast activation.

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    Tan, Xiaoying; Xu, Xingbo; Zeisberg, Elisabeth M; Zeisberg, Michael

    2016-04-08

    Phosphate is an essential constituent of critical cellular functions including energy metabolism, nucleic acid synthesis and phosphorylation-dependent cell signaling. Increased plasma phosphate levels are an independent risk factor for lowered life-expectancy as well as for heart and kidney failure. Nevertheless, direct cellular effects of elevated phosphate concentrations within the microenvironment are poorly understood and have been largely neglected in favor of phosphor-regulatory hormones. Because interstitial fibrosis is the common determinant of chronic progressive kidney disease, and because fibroblasts are major mediators of fibrogenesis, we here explored the effect of high extracellular phosphate levels on renal fibroblasts. We demonstrate that high inorganic phosphate directly induces fibrotic fibroblast activation associated with increased proliferative activity, increased expression of α-smooth muscle actin and increased synthesis of type I collagen. We further demonstrate that such fibroblast activation is dependent on phosphate influx, aberrant phosphorylation of DNA methyltransferase DNMT1 and aberrant CpG island promoter methylation. In summary, our studies demonstrate that elevated phosphate concentrations induce pro-fibrotic fibroblast activation independent of phospho-regulatory hormones.

  2. Hsp90 regulation of fibroblast activation in pulmonary fibrosis

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    Sontake, Vishwaraj; Wang, Yunguan; Kasam, Rajesh K.; Sinner, Debora; Reddy, Geereddy B.; Naren, Anjaparavanda P.; McCormack, Francis X.; Jegga, Anil G.; Madala, Satish K.

    2017-01-01

    Idiopathic pulmonary fibrosis (IPF) is a severe fibrotic lung disease associated with fibroblast activation that includes excessive proliferation, tissue invasiveness, myofibroblast transformation, and extracellular matrix (ECM) production. To identify inhibitors that can attenuate fibroblast activation, we queried IPF gene signatures against a library of small-molecule-induced gene-expression profiles and identified Hsp90 inhibitors as potential therapeutic agents that can suppress fibroblast activation in IPF. Although Hsp90 is a molecular chaperone that regulates multiple processes involved in fibroblast activation, it has not been previously proposed as a molecular target in IPF. Here, we found elevated Hsp90 staining in lung biopsies of patients with IPF. Notably, fibroblasts isolated from fibrotic lesions showed heightened Hsp90 ATPase activity compared with normal fibroblasts. 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), a small-molecule inhibitor of Hsp90 ATPase activity, attenuated fibroblast activation and also TGF-β–driven effects on fibroblast to myofibroblast transformation. The loss of the Hsp90AB, but not the Hsp90AA isoform, resulted in reduced fibroblast proliferation, myofibroblast transformation, and ECM production. Finally, in vivo therapy with 17-AAG attenuated progression of established and ongoing fibrosis in a mouse model of pulmonary fibrosis, suggesting that targeting Hsp90 represents an effective strategy for the treatment of fibrotic lung disease. PMID:28239659

  3. Respiratory activity and growth of human skin derma fibroblasts.

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    Papa, F; Scacco, S; Vergari, R; Bucaria, V; Dioguardi, D; Papa, S

    1998-09-01

    A study has been made on the speed of growth and respiratory activity of fibroblast cultures from control derma, cheloid (hypertrophic) scar and stabilized scar taken from human skin. The speed of growth and the efficiency of plaque formation of fibroblasts from cheloid scar were greater in comparison with those of fibroblasts from stabilized scar and were stimulated by the addition to the culture medium of the exudate from post-traumatic ulcer. Measurement of the contents of cytochromes showed a decrease in the content of cytochromes b562 and c + c1 in the fibroblast culture from both cheloid and stabilized scar as compared to the fibroblast culture from control derma. Cytochrome aa3 content did not show significant difference among the three types of fibroblast cultures. The respiratory activities supported by pyruvate plus malate, succinate or ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine did not show, however, significant difference among the three fibroblast cultures. These observations show that the speed of growth of skin fibroblasts does not depend on the overall respiratory capacity. The exudate stimulated the activity of cytochrome c oxidase in fibroblasts from control derma, and cheloid scar. This effect and the accompanying stimulation of fibroblast growth might be correlated with the balance of oxygen free radicals.

  4. Tumor-secreted LOXL2 activates fibroblasts through FAK signaling

    DEFF Research Database (Denmark)

    Barker, Holly E; Bird, Demelza; Lang, Georgina

    2013-01-01

    models. Here, we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. Genetic manipulation or antibody inhibition of LOXL2 in orthotopically grown mammary tumors reduced the expression of α-smooth muscle actin (α-SMA). Using a marker for reticular...... fibroblasts, it was determined that expression of α-SMA was localized to fibroblasts recruited from the host tissue. This marker also revealed that the matrix present in tumors with reduced levels of LOXL2 was more scattered compared with control tumors which exhibited matrices with dense, parallel alignments....... Importantly, in vitro assays revealed that tumor-derived LOXL2 and a recombinant LOXL2 protein induced fibroblast branching on collagen matrices, as well as increased fibroblast-mediated collagen contraction and invasion of fibroblasts through extracellular matrix. Moreover, LOXL2 induced the expression of α-SMA...

  5. Proliferative effect of green light emitting diode irradiation on chicken fibroblasts in hyperglycaemic circumstances: a preliminary in vitro study

    Science.gov (United States)

    Vinck, Elke; Cagnie, Barbara; Declercq, Heidi; Cornelissen, Ria; Cambier, Dirk

    2004-09-01

    A reduced mortality due to hyperglycaemia was noted since the development of insulin treatment for type I diabetes and various oral hypoglycaemic agents for type II diabetes. Nevertheless the chronic metabolic disorder, Diabetes Mellitus, remains an important cause of morbidity and mortality due to a series of common secondary metabolic complications. Patients with diabetes have an increased tendency to develop infections of the skin. Healing of skin lesions in diabetics evolves often relatively slow and the lesions tend to be more severe than in non-diabetics. Endeavouring to accelerate the healing process of skin lesions in diabetic patients, this preliminary in vitro study investigates the efficacy of green Light Emitting Diode (LED) irradiation on fibroblast proliferation of cells in hyperglycaemic circumstances. In an attempt to imitate the diabetic environment, embryonic chicken fibroblasts were cultured in hyperglycaemic medium (30.000mg Glucose per litre Hanks Medium). LED irradiation was performed three consecutive days with a wavelength of 540 nm and a power output of 10 mW, at 0,6 cm distance from the fibroblasts. Each treatment lasted 3 minutes, resulting in a surface energy density of 0,2 J/cm2. Statistical analysis revealed that LED irradiation at the applied parameters induced a higher rate of proliferation in hyperglycaemic circumstances after irradiation than in the same circumstances without irradiation. Regarding these results the effectiveness of green LED irradiation on cells in hyperglycaemic circumstances is proven. To ensure the effectiveness and to evaluate the value of LED irradiation in vivo, further research is required.

  6. [Proliferative Vitreoretinopathy].

    Science.gov (United States)

    Wiedemann, P; Bringmann, A

    2016-09-01

    Proliferative vitreoretinal retinopathy (PVR) is a very severe complication of vitreoretinal surgery. PVR is characterised by a complex cellular reaction. This corresponds to a vitreoretinal wound healing reaction and leads to tractional retinal detachment fixed by membranes. A rational goal of treatment is the removal of active cells and membranes, particularly the whole vitreous body; this can only be achieved surgically.

  7. Influence of pro-angiogenic cytokines on proliferative activity and survival of endothelial cells

    Directory of Open Access Journals (Sweden)

    Solyanik G. I.

    2010-04-01

    Full Text Available Aim. Tumor angiogenesis in contrast to physiological one is characterized by high level of malignant cell production of proangiogenic cytokines, which have different influence on functional activity of endothelial cells. The goal of the study – to carry out a comparative analysis of the influence of a vascular endothelial growth factor (VEGF and an epidermal growth factor (EGF on proliferative activity and survival of endothelial cells upon their confluent and exponential growth. Methods. The proliferative activity of endothelial cells was determined by MTT-test and their viability was detected by the trypane blue exclusion test. Results. It was shown that EGF (irrespectively of the level of serum factors in concentrations higher than 10 ng/ml activated the proliferative activity of confluent endotheliocytes in a concentration-dependent manner by 18–36 % (ð < 0.05 as compared to the control, while this cytokine didn’t affect the endothelial cells in the exponential growth phase. VEGF in wide concentration range didn’t display the mitogenic effect on endotheliocytes in both confluent and exponential growth phases. Furthermore, VEGF in concentrations higher than 100 ng/ml inhibited proliferative activity of confluent endothelial cells by 12 % (ð < 0.05. In case of deficiency of nutrients, EGF and VEGF promoted the survival of endothelial cells, considerably decreasing their death. Conclusions. EGF, in contrast to VEGF, stimulates proliferation and survival of the endothelial cells, whereas VEGF has significant influence only on the survival of the cells

  8. Epithelial cell proliferative activity of Barrett's esophagus : methodology and correlation with traditional cancer risk markers

    NARCIS (Netherlands)

    Peters, F T; Ganesh, S; Kuipers, E J; de Jager-Krikken, A; Karrenbeld, A; Harms, G; Sluiter, W J; Koudstaal, J; Klinkenberg-Knol, E C; Lamers, C B; Kleibeuker, J H

    1998-01-01

    Barrett's esophagus (BE) is a premalignant condition, due to chronic gastroesophageal reflux. Effective antireflux therapy may diminish cancer risk. To evaluate this option an intermediate marker is needed. We developed a methodology for measurement of epithelial cell proliferative activity of Barre

  9. Bacterial lipopolysaccharide promotes profibrotic activation of intestinal fibroblasts.

    LENUS (Irish Health Repository)

    Burke, J P

    2012-02-01

    BACKGROUND: Fibroblasts play a critical role in intestinal wound healing. Lipopolysaccharide (LPS) is a cell wall component of commensal gut bacteria. The effects of LPS on intestinal fibroblast activation were characterized. METHODS: Expression of the LPS receptor, toll-like receptor (TLR) 4, was assessed in cultured primary human intestinal fibroblasts using flow cytometry and confocal microscopy. Fibroblasts were treated with LPS and\\/or transforming growth factor (TGF) beta1. Nuclear factor kappaB (NFkappaB) pathway activation was assessed by inhibitory kappaBalpha (IkappaBalpha) degradation and NFkappaB promoter activity. Fibroblast contractility was measured using a fibroblast-populated collagen lattice. Smad-7, a negative regulator of TGF-beta1 signalling, and connective tissue growth factor (CTGF) expression were assessed using reverse transcriptase-polymerase chain reaction and western blot. The NFkappaB pathway was inhibited by IkappaBalpha transfection. RESULTS: TLR-4 was present on the surface of intestinal fibroblasts. LPS treatment of fibroblasts induced IkappaBalpha degradation, enhanced NFkappaB promoter activity and increased collagen contraction. Pretreatment with LPS (before TGF-beta1) significantly increased CTGF production relative to treatment with TGF-beta1 alone. LPS reduced whereas TGF-beta1 increased smad-7 expression. Transfection with an IkappaBalpha plasmid enhanced basal smad-7 expression. CONCLUSION: Intestinal fibroblasts express TLR-4 and respond to LPS by activating NFkappaB and inducing collagen contraction. LPS acts in concert with TGF-beta1 to induce CTGF. LPS reduces the expression of the TGF-beta1 inhibitor, smad-7.

  10. LIF Mediates Proinvasive Activation of Stromal Fibroblasts in Cancer

    Directory of Open Access Journals (Sweden)

    Jean Albrengues

    2014-06-01

    Full Text Available Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. Here, we identify leukemia inhibitory factor (LIF as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin (α-SMA expression. We demonstrate that a pulse of transforming growth factor β (TGF-β establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion in vitro and in vivo. Accordingly, carcinomas from multiple origins and melanomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Blockade of JAK activity by Ruxolitinib (JAK inhibitor counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. These findings establish LIF as a proinvasive fibroblast producer independent of α-SMA and may open novel therapeutic perspectives for patients with aggressive primary tumors.

  11. The effect of tranilast on fibroblast activation protein α (FAP-α expression in normal and keloid fibroblasts in vitro

    Directory of Open Access Journals (Sweden)

    Paweł P. Antończak

    2017-07-01

    Full Text Available Introduction . Tranilast (N-(3’,4’-demethoxycinnamoyl-anthranilic acid is an anti-allergic drug. Its mechanism of action is based on the inhibition of antigen-induced release of chemical mediators from mast cells and basophils. It also reveals antifibroproliferative activities. These properties of tranilast are used in the treatment of hypertrophic scars and keloids. Keloids are characterized by incorrect extracellular matrix components turnover. Fibroblasts derived from keloids reveal overproduction of collagen type I and decreased degradation of extracellular matrix in comparison with normal fibroblasts. Fibroblast activation protein α (FAP-α may play an important role in remodeling of extracellular matrix and the invasive properties of keloids. Objective . In the present study, the effect of tranilast on expression of FAP-α gene and its protein was evaluated in normal human dermal fibroblasts and fibroblasts derived from keloids cultured in vitro . Materials and methods. In the first stage of the study, the influence of tranilast on cell viability was estimated. The second stage of the study included the quantitative evaluation of FAP-α mRNA expression in normal and keloid fibroblasts treated with tranilast. The third stage of the study comprised fibroblast activation protein α expression analysis in the examined cells treated with tranilast. Results and conclusions . The expression of FAP-α gene and fibroblast activation protein α is higher in keloid fibroblasts. Tranilast at concentrations of 3 μM and 30 μM up-regulated mRNA FAP-α expression in normal fibroblasts but did not influence keloid fibroblasts. The drug, at concentrations of 30 μM and 300 μM up-regulated fibroblast activation protein α expression in normal fibroblasts and did not influence keloid fibroblasts. Tranilast antiproliferative effect is not associated with FAP-α expression in keloid fibroblasts.

  12. Proliferative response of fibroblasts expressing internalization-deficient epidermal growth factor (EGF) receptors is altered via differential EGF depletion effect.

    Science.gov (United States)

    Reddy, C C; Wells, A; Lauffenburger, D A

    1994-01-01

    We describe experiments comparing the proliferation responses to epidermal growth factor (EGF) by NR6 fibroblasts expressing genetically engineered epidermal growth factor receptors (EGFRs). These cells present either wild-type (WT) EGFR or a cytoplasmic domain-truncated (c'973) EGFR that exhibits a decreased ligand-induced internalization rate constant. In two distinct in vitro proliferation assays, with or without medium replenishment, we measured the specific cell proliferation rate constants and EGF depletion kinetics for both WT and c'973 cells. When EGF depletion is minimized by replenishment, the EGF concentration dependencies of the two cell types are similar, whereas when EGF depletion is not prevented, maximal proliferation of WT cells requires an initial EGF concentration that is approximately 10x that required by c'973 cells. However, when EGF depletion is accounted for, the dependencies of growth rate for the two cell types on the current EGF concentration in both assays are essentially identical. Our results demonstrate that diminished depletion of EGF from the extracellular medium is a major reason for increased mitogenic sensitivity to EGF by cells possessing internalization-deficient receptors.

  13. Tumor-secreted LOXL2 Activates Fibroblasts Through FAK Signaling

    OpenAIRE

    Barker, Holly E.; Bird, Demelza; Lang, Georgina; Janine T. Erler

    2013-01-01

    Cancer-associated fibroblasts enhance cancer progression when activated by tumor cells through mechanisms not yet fully understood. Blocking mammary tumor cell-derived lysyl oxidase-like 2 (LOXL2) significantly inhibited mammary tumor cell invasion and metastasis in transgenic and orthotopic mouse models. Here we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. Genetic manipulation or antibody inhibition of LOXL2 in orthotopically grown...

  14. Anti-proliferative activity of recombinant melittin expressed in ...

    African Journals Online (AJOL)

    kesiena

    2012-02-09

    Feb 9, 2012 ... activity, and then matured through deletion of N-terminal. 44 amino acid residues ... human renal, lung, liver, prostate, bladder and mammary cancer cells have been identified as targets of melittin. (Katoh, 1995; Son et al., ...

  15. Proliferative Activity in Libyan Breast Cancer with Comparison to European and Central African Patients

    Directory of Open Access Journals (Sweden)

    Jamela Boder

    2013-01-01

    Full Text Available Background. We evaluated the relation of proliferative indices with clinicopathological features and prognosis in breast cancer (BC of Libyan female patients. The data were compared with corresponding results in Finland and Nigeria. Patients and Methods. Histological samples of breast cancer from 130 patients were retrospectively studied. Mitotic activity index (MAI and standardized mitotic index (SMI were estimated. Results. There were statistically significant correlations between the proliferative indices and most clinicopathological features, with the strongest association observed for histological grade (P=0.01 for SMI and P=0.003 for MAI. The proliferative differences between Libyan, Nigerian, and Finnish population were prominent. The mean values of SMI and MAI in Libyan BC patients were 32.1 mitotic figures per square millimeter and 27.3 mitotic figures per 10 high-power fields, respectively. This is clearly lower than those in Nigeria but much higher than those in Finland. The differences between countries are seen in whole material and are also present in subgroups. The results indicated that mitotic activities can be reliable prognostic indicators in Libyan BCs, as they were among Finnish and Nigerian females. Univariate and multivariate analyses found at cut-offs of 19 and 44 mitosis/mm2 of SMI were the most significant prognostic factors. Conclusions. Proliferative indices with careful estimation of the MAI and SMI could be applied as quantitative criteria for Libyan BC to separate the patients into good, moderate, and bad prognosis groups.

  16. Suppressor cell activity in a proliferative disorder of T lymphocytes.

    Science.gov (United States)

    Kupa, A; Thomas, M E; Moore, H; Bradley, J; Zola, H; Hooper, M; Harding, P

    1981-06-01

    We report details of the immunological profile of a patient with the candidiasis endocrinopathy syndrome who has developed T-type chronic lymphocytic leukaemia. The patient is anergic to a panel of delayed hypersensitivity skin tests, and has poor in vitro mitogenic responses, but B cell function in vivo is not impaired. Subsequent functional studies have revealed that cells from the patient have a significant suppressive effect in coculture (P less than 0.05) on the responses of healthy donor lymphocytes (NR) to the mitogen phytohaemagglutinin (PHA). A degree of selectivity for the suppressive effect is suggested by the lack of similar effects on coculture responses to the mitogens concanavalin A (Con A) and pokeweed mitogen (PWM). Mitomycin C treatment of the patient's cells reduced their suppressive activity but significant suppression was still observed in the majority of PHA cocultures. The suppressor activity required the presence of the patient's cells in cocultures, as no suppression was observed when the patient's serum or cell culture supernatant were included instead of the patient's cells in NR cultures.

  17. [The study of proliferative epithelial activity in cholesteatoma of the middle ear during cytomorphophotometry].

    Science.gov (United States)

    Amador, J M; Esquivias, J J; Ciges, M

    1994-01-01

    Epithelial proliferation in colesteatoma and its influence on the subepithelial inflammatory reaction was studied using morphometry and nuclear photometry of specimens. Twenty specimens of colesteatoma and 15 specimens of skin from the external auditory canal were examined. Nuclear content was greater in the basal cells of colesteatoma epithelium than in the basal cells of external auditory canal epithelium, suggesting increased proliferative activity. This activity was found to be reted to the inflammatory infiltrate of the conjunctive tissue and varied in different specimens.

  18. Comparative antitumor and anti-proliferative activities ofHippophae rhamnoidesL. leaves extracts

    Institute of Scientific and Technical Information of China (English)

    Javid Ali; Bashir Ahmad

    2015-01-01

    Objective:To evaluate the antitumor and anti-proliferative activities of methanol, aqueous, acetone, ethyl acetate, ethanol, chloroform andn-hexane extracts ofHippophae rhamnoides leaves. Methods: Antitumor activities were evaluated by using the antitumor potato disc assay by using inoculums (Agrobacterium tumefaciens) with three different concentrations of test samples (10, 100 and 1 000 mg/L). Anti-proliferative activity was evaluated by the given method of methyl thiazolyl tetrazolium assay. The concentrations of the extract ranging from 0.039 to 10 mg/mL were tested against HeLa cells. Results: Highest tumors inhibition activity (60.9% and 55.8%) was shown by methanol and ethanol extracts, with EC50 values of 424.41 and 434.61 mg/L respectively. At 10 mg/mL, The highest cell inhibition 75.61% was observed in methanol extract and the lowest 36.59% were calculated inn-hexane extract. The difference in tumor and cell inhibition (%) may be due to the different concentration of active compounds responsible for antitumor and anti-proliferative activities. All extracts have considerable level of tumor and cell inhibitiory effect in a dose dependent manner. Conclusions:Our finding showed thatHippophae rhamnoidesleaves are a potent natural source of antitumor and antiproliferative agent.

  19. In vitro anti-proliferative and anti-angiogenic activities of thalidomide dithiocarbamate analogs.

    Science.gov (United States)

    El-Aarag, Bishoy Y A; Kasai, Tomonari; Zahran, Magdy A H; Zakhary, Nadia I; Shigehiro, Tsukasa; Sekhar, Sreeja C; Agwa, Hussein S; Mizutani, Akifumi; Murakami, Hiroshi; Kakuta, Hiroki; Seno, Masaharu

    2014-08-01

    Inhibition of angiogenesis is currently perceived as a promising strategy in the treatment of cancer. The anti-angiogenicity of thalidomide has inspired a second wave of research on this teratogenic drug. The present study aimed to investigate the anti-proliferative and anti-angiogenic activities of two thalidomide dithiocarbamate analogs by studying their anti-proliferative effects on human umbilical vein endothelial cells (HUVECs) and MDA-MB-231 human breast cancer cell lines. Their action on the expression levels of IL-6, IL-8, TNF-α, VEGF165, and MMP-2 was also assessed. Furthermore, their effect on angiogenesis was evaluated through wound healing, migration, tube formation, and nitric oxide (NO) assays. Results illustrated that the proliferation of HUVECs and MDA-MB-231 cells was not significantly affected by thalidomide at 6.25-100μM. Thalidomide failed to block angiogenesis at similar concentrations. By contrast, thalidomide dithiocarbamate analogs exhibited significant anti-proliferative action on HUVECs and MDA-MB-231 cells without causing cytotoxicity and also showed powerful anti-angiogenicity in wound healing, migration, tube formation, and NO assays. Thalidomide analogs 1 and 2 demonstrated more potent activity to suppress expression levels of IL-6, IL-8, TNF-α, VEGF165, and MMP-2 than thalidomide. Analog 1 consistently, showed the highest potency and efficacy in all the assays. Taken together, our results support further development and evaluation of novel thalidomide analogs as anti-tumor and anti-angiogenic agents.

  20. Anti-proliferative activities of terpenoids isolated from Alisma orientalis and their structure-activity relationships.

    Science.gov (United States)

    Xu, Wen; Li, Ting; Qiu, Jian-Fang; Wu, Shui-Sheng; Huang, Ming-Qing; Lin, Li-Gen; Zhang, Qing-Wen; Chen, Xiu-Ping; Lu, Jin-Jian

    2015-01-01

    This study aimed to isolate terpenoids from Alisma orientalis (Sam.) Juzep. and elucidate their antiproliferative activities, as well as structure-activity relationships. Fourteen protostane-type triterpenoids were isolated from the rhizome of A. orientalis. Among these triterpenoids, alisol A (1), alisol A 24-acetate (2), alisol B (3), alisol B 23-acetate (4), and alisol G (8) presented inhibitory effects on cancer cell lines tested. Compounds 3 and 4 showed the highest potential; IC50 values for HepG2, MDA-MB-231, and MCF-7 cells were 16.28, 14.47, and 6.66 μM for 3 and 18.01, 15.97, and 13.56 μM for 4, respectively. Based on these results, we concluded that the degree of C-16 oxidation and the double bond between C-13 and C-17 may be significant in anti-proliferative activities. Further study showed that 3 and 4 effectively induced apoptosis, as confirmed by flow cytometry. Increased intracellular calcium concentration and endoplasmic reticulum stress were detected after treatment with 4 in HepG2 cells. Although compounds 1 and 2 induced minimal apoptosis, they evidently delayed the G2/M phase in HepG2 cells. Further study showed that 1-4 also enhanced LC3II expression, indicating autophagy is occured.

  1. Dipeptides Increase Functional Activity of Human Skin Fibroblasts.

    Science.gov (United States)

    Malinin, V V; Durnova, A O; Polyakova, V O; Kvetnoi, I M

    2015-05-01

    We analyzed the effect of dipeptide Glu-Trp and isovaleroyl-Glu-Trp in concentrations of 0.2, 2 and 20 μg/ml and Actovegin preparation on functional activity of human skin fibroblasts. Dipeptides, especially Glu-Trp, produce a stimulating effect on human skin fibroblasts and their effect is equivalent to that of Actovegin. Dipeptides stimulate cell renewal processes by activating synthesis of Ki-67 and reducing expression of caspase-9 and enhance antioxidant function of the cells by stimulating the expression of Hsp-90 and inducible NO-synthase. These findings suggest that dipeptides are promising candidates for preparations stimulating reparative processes.

  2. Rho A and the Rho kinase pathway regulate fibroblast contraction: Enhanced contraction in constitutively active Rho A fibroblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nobe, Koji, E-mail: kojinobe@pharm.showa-u.ac.jp [Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo (Japan); Nobe, Hiromi [Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo (Japan); Department of Physical Therapy, Bunkyo-Gakuin University (Japan); Yoshida, Hiroko [Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo (Japan); Kolodney, Michael S. [Dermatology Division, Department of Medicine, UCLA, Los Angeles, CA (United States); Paul, Richard J. [Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, OH (United States); Honda, Kazuo [Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo (Japan)

    2010-08-20

    Research highlights: {yields} Mechanisms of fibroblast cell contraction in collagen matrix. {yields} Assessed an isometric force development using 3D-reconstituted-fibroblast fiber. {yields} Constitutively active Rho A induced the over-contraction of fibroblast cells. {yields} Rho A and Rho kinase pathway has a central role in fibroblast cell contraction. -- Abstract: Fibroblast cells play a central role in the proliferation phase of wound healing processes, contributing to force development. The intracellular signaling pathways regulating this non-muscle contraction are only partially understood. To study the relations between Rho A and contractile responses, constitutively active Rho A (CA-Rho A) fibroblast cells were reconstituted into fibers and the effects of calf serum (CS) on isometric force were studied. CS-induced force in CA-Rho A fibroblast fibers was twice as large as that in wild type (NIH 3T3) fibroblast fibers. During this response, the translocation of Rho A from the cytosol to the membrane was detected by Rho A activity assays and Western blot analysis. Pre-treatment with a Rho specific inhibitor (C3-exoenzyme) suppressed translocation as well as contraction. These results indicate that Rho A activation is essential for fibroblast contraction. The Rho kinase inhibitor ( (Y27632)) inhibited both NIH 3T3 and CA-Rho A fibroblast fiber contractions. Activation of Rho A is thus directly coupled with Rho kinase activity. We conclude that the translocation of Rho A from the cytosol to the membrane and the Rho kinase pathway can regulate wound healing processes mediated by fibroblast contraction.

  3. Association of Pro12Ala polymorphism in peroxisome proliferator activated receptor gamma with proliferative diabetic retinopathy

    Science.gov (United States)

    Tariq, Khadija; Malik, Saira Bano; Ali, Syeda Hafiza Benish; Maqsood, Sundas Ejaz; Azam, Aisha; Muslim, Irfan; Khan, Muhammad Shakil; Azam, Maleeha; Waheed, Nadia Khalida

    2013-01-01

    Purpose The association of non-synonymous substitution polymorphism rs1801282 (c.34C>G, p.Pro12Ala) in exon 4 of the peroxisome proliferator activated receptor gamma gene with diabetic retinopathy (DR) has been reported inconsistently. Therefore, the purpose of the present study was to understand the population-specific role of the Pro12Ala polymorphism in DR susceptibility in Pakistani subjects. Methods A total of 180 subjects with DR, 193 subjects with type 2 diabetes mellitus (T2DM) with no diabetic retinopathy, and 200 healthy normoglycemic non-retinopathic Pakistani individuals were genotyped for the rs1801282 (c.34C>G) polymorphism using polymerase chain reaction-restriction fragment length polymorphism. Results We found the individuals with T2DM carrying 12Ala were at a reduced risk of developing DR (odds ratio [OR]=0.53; 95% confidence interval [CI]=0.33–0.87). Upon stratified analysis regarding disease severity, we observed this protective effect was confined to proliferative DR (OR=0.4; 95% CI=0.2–0.8) with non-significant effects on the susceptibility of non-proliferative DR (OR=0.67; 95% CI=0.37–1.19). Conclusions We report a protective role of the 12Ala polymorphism against proliferative DR in individuals with T2DM in Pakistan. PMID:23559865

  4. Retinol induces morphological alterations and proliferative focus formation through free radicalmediated activation of multiple signaling pathways

    Institute of Scientific and Technical Information of China (English)

    Daniel Pens GELAIN; Matheus Augusto de Bittencourt PASQUALI; Fernanda Freitas CAREGNATO; Mauro Antonio Alves CASTRO; José Claudio Fonseca MOREIRA

    2012-01-01

    Aim:Toxicity of retinol (vitamin A)has been previously associated with apoptosis and/or cell malignant transformation.Thus,we investigated the pathways involved in the induction of proliferation,deformation and proliferative focus formation by retinol in cultured Sertoli cells of rats.Methods:Sertoli cells were isolated from immature rats and cultured.The cells were subjected to a 24-h treatment with different concentrations of retinol.Parameters of oxidative stress and cytotoxicity were analyzed.The effects of the p38 inhibitor SB203580(10 μmol/L),the JNK inhibitor SP600125 (10 μmol/L),the Akt inhibitor LY294002 (10 μmol/L),the ERK inhibitor U0126 (10 μmol/L)the pan-PKC inhibitor G(O)6983 (10 μmol/L)and the PKA inhibitor H89 (1 μmol/L)on morphological and proliferative/transformationassociated modifications were studied.Results:Retinol (7 and 14 μmol/L)significantly increases the reactive species production in Sertoli cells,inhibition of p38,JNK,ERK1/2,Akt,and PKA suppressed retinol-induced[3H]dT incorporation into the cells,while PKC inhibition had no effect.ERK1/2 and p38 inhibition also blocked retinol-induced proliferative focus formation in the cells,while Akt and JNK inhibition partially decreased focus formation.ERK1/2 and p38 inhibition hindered transformation-associated deformation in retinol-treated cells,while other treatments had no effect.Conclusion:Our results suggest that activation of multiple kinases is responsible for morphological and proliferative changes associated to malignancy development in Sertoli cells by retinol at the concentrations higher than physiological level.

  5. Autoantigen microarrays reveal autoantibodies associated with proliferative nephritis and active disease in pediatric systemic lupus erythematosus.

    Science.gov (United States)

    Haddon, D James; Diep, Vivian K; Price, Jordan V; Limb, Cindy; Utz, Paul J; Balboni, Imelda

    2015-06-17

    Pediatric systemic lupus erythematosus (pSLE) patients often initially present with more active and severe disease than adults, including a higher frequency of lupus nephritis. Specific autoantibodies, including anti-C1q, anti-DNA and anti-alpha-actinin, have been associated with kidney involvement in SLE, and DNA antibodies are capable of initiating early-stage lupus nephritis in severe combined immunodeficiency (SCID) mice. Over 100 different autoantibodies have been described in SLE patients, highlighting the need for comprehensive autoantibody profiling. Knowledge of the antibodies associated with pSLE and proliferative nephritis will increase the understanding of SLE pathogenesis, and may aid in monitoring patients for renal flare. We used autoantigen microarrays composed of 140 recombinant or purified antigens to compare the serum autoantibody profiles of new-onset pSLE patients (n = 45) to healthy controls (n = 17). We also compared pSLE patients with biopsy-confirmed class III or IV proliferative nephritis (n = 23) and without significant renal involvement (n = 18). We performed ELISA with selected autoantigens to validate the microarray findings. We created a multiple logistic regression model, based on the ELISA and clinical information, to predict whether a patient had proliferative nephritis, and used a validation cohort (n = 23) and longitudinal samples (88 patient visits) to test its accuracy. Fifty autoantibodies were at significantly higher levels in the sera of pSLE patients compared to healthy controls, including anti-B cell-activating factor (BAFF). High levels of anti-BAFF were associated with active disease. Thirteen serum autoantibodies were present at significantly higher levels in pSLE patients with proliferative nephritis than those without, and we confirmed five autoantigens (dsDNA, C1q, collagens IV and X and aggrecan) by ELISA. Our model, based on ELISA measurements and clinical variables, correctly identified patients with proliferative

  6. Changes in retinal venular oxygen saturation predict activity of proliferative diabetic retinopathy 3 months after panretinal photocoagulation

    DEFF Research Database (Denmark)

    Torp, Thomas Lee; Kawasaki, Ryo; Wong, Tien Yin

    2017-01-01

    BACKGROUND/AIMS: Proliferative diabetic retinopathy (PDR) is a severe blinding condition. We investigated whether retinal metabolism, measured by retinal oximetry, may predict PDR activity after panretinal laser photocoagulation (PRP). METHODS: We performed a prospective, interventional, clinical...

  7. Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

    Energy Technology Data Exchange (ETDEWEB)

    Berndt, Alexander, E-mail: alexander.berndt@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Büttner, Robert, E-mail: Robert-Buettner@gmx.net [Institute of Biochemistry and Biophysics, Friedrich Schiller University Jena, 07740 Jena (Germany); Gühne, Stefanie, E-mail: stefanie_guehne@gmx.net [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Gleinig, Anna, E-mail: annagleinig@yahoo.com [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Richter, Petra, E-mail: P.Richter@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Chen, Yuan, E-mail: Yuan.Chen@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Franz, Marcus, E-mail: Marcus.Franz@med.uni-jena.de [Clinic of Internal Medicine I, Jena University Hospital, 07740 Jena (Germany); Liebmann, Claus, E-mail: Claus.Liebmann@uni-jena.de [Institute of Biochemistry and Biophysics, Friedrich Schiller University Jena, 07740 Jena (Germany)

    2014-04-01

    Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM{sub TGF}, FCM{sub PDGF}) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM{sub B}). FCM{sub TGF} stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM{sub TGF}≫FCM{sub PDGF} induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM{sub TGF}>FCM{sub PDGF}) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin

  8. Anti-proliferative and mutagenic activities of aqueous and methanol extracts of leaves from Pereskia bleo (Kunth) DC (Cactaceae).

    Science.gov (United States)

    Er, Hui Meng; Cheng, En-Hsiang; Radhakrishnan, Ammu Kutty

    2007-09-25

    The anti-proliferative effects of the aqueous and methanol extracts of leaves of Pereskia bleo (Kunth) DC (Cactaceae) against a mouse mammary cancer cell line (4T1) and a normal mouse fibroblast cell line (NIH/3T3) were evaluated under an optimal (in culture medium containing 10% foetal bovine serum (FBS)) and a sub-optimal (in culture medium containing 0.5% FBS) conditions. Under the optimal condition, the aqueous extract showed a significant (pCactaceae) do not have appreciable anti-proliferative effect on the 4T1 and NIH/3T3 cells as the EC(50) values obtained are greater than 50 microg/mL when tested under optimal culture condition. Moreover, the aqueous extract may form mutagenic compound(s) upon the metabolisation by liver enzymes.

  9. Evaluation of in vitro anti-proliferative and immunomodulatory activities of compounds isolated from Curcuma longa

    Science.gov (United States)

    Yue, Grace G. L.; Chan, Ben C. L.; Hon, Po-Ming; Lee, Mavis Y. H.; Fung, Kwok-Pui; Leung, Ping-Chung; Lau, Clara B. S.

    2010-01-01

    The rhizome of Curcuma longa (CL) has been commonly used in Asia as a potential candidate for the treatment of different diseases, including inflammatory disorders and cancers. The present study evaluated the anti-proliferative activities of the isolated compounds (3 curcuminoids and 2 turmerones) from CL, using human cancer cell lines HepG2, MCF-7 and MDA-MB-231. The immunomodulatory activities of turmerones (α and aromatic) isolated from CL were also examined using human peripheral blood mononuclear cells (PBMC). Our results showed that the curcuminoids (curcumin, demethoxycurcumin and bisdemethoxycurcumin) and α-turmerone significantly inhibited proliferation of cancer cells in dose-dependent manner. The IC50 values of these compounds in cancer cells ranged from 11.0–41.8 μg/ml. Alpha-turmerone induced MDA-MB-231 cells to undergo apoptosis, which was confirmed by annexin-V & propidium iodide staining, and DNA fragmentation assay. The caspase cascade was activated as shown by a significant decrease of procaspases-3, -8 and -9 in α-turmerone treated cells. Both α-turmerone and aromatic-turmerone showed stimulatory effects on PBMC proliferation and cytokine production. The anti-proliferative effect of α-turmerone and immunomodulatory activities of ar-turmerone were shown for the first time. The findings revealed the potential use of CL crude extract (containing curcuminoids and volatile oil including turmerones) as chemopreventive agent. PMID:20438793

  10. The effects of altered fractionation schedules on the survival of human cell lines differing in their proliferative activity and repair capacity

    Energy Technology Data Exchange (ETDEWEB)

    Konefal, J.B.; Taylor, Y.C. (Washington Univ. School of Medicine, St. Louis, MO (USA))

    1989-11-01

    Plateau phase cultures of human cell lines were used as model systems to study the relative influences of proliferation and repair on the effectiveness of altered fractionation schedules. A human normal diploid fibroblast cell line (AG1522) which has a high capacity to repair potentially lethal radiation damage (PLD) and very little proliferative activity when grown to confluence was compared to human tumor cell lines which maintain significant cell-cycle activity in plateau phase. The human fibrosarcoma cell line, HT1080, used in the present study exhibited a much greater rate of turnover than the normal fibroblasts as determined from tritiated thymidine incorporation (5 day labeling index of 66% vs 20%) and no PLD repair, as determined by delayed plating experiments, in plateau phase. Twenty Gy were delivered to both cell lines over 5 days in 3 regimens: one 4 Gy fraction/day, two 2 Gy fractions/day with a 2 hr interval between doses, and two 2 Gy fractions/day with a 6 hr interval between doses. Although the normal fibroblasts demonstrated the greatest sparing between acute single doses and one 4 Gy fraction/day, there was little additional benefit (increased survival) from the increased dose fractionation. In contrast, the twice daily fractionation schedules resulted in significant differential sparing of the fibrosarcoma cells compared to the normal fibroblasts. With the 6 hr interval between doses, the survival advantages of the cell line with the slow turnover rate and high PLD repair capacity were completely lost. Split-dose experiments indicated slightly less sublethal damage repair in the fibrosarcoma cell line, but for both cell lines recovery was complete in 2 hr. DNA distributions were measured by flow cytometry and long term labeling index measurements performed in parallel with the multifraction radiation survival studies.

  11. Evidence of Anti-Proliferative Activities in Blue Mussel (Mytilus edulis By-Products

    Directory of Open Access Journals (Sweden)

    Marie-Elise Carbonneau

    2013-03-01

    Full Text Available Shellfish waste components contain significant levels of high quality protein and are therefore a potential source for biofunctional high-value peptides. The feasibility of applying a pilot scale enzymatic hydrolysis process to whole Mytilus edulis and, by fractionation, recover hydrolysates presenting a biological activity of interest, was evaluated. Fractions were tested on four immortalized cancerous cell lines: A549, BT549, HCT15 and PC3. The 50 kDa fraction, enriched in peptides, presented anti-proliferative activity with all cell lines and results suggest a bioactive molecule synergy within the fraction. At a protein concentration of 44 µg/mL, the 50 kDa fraction induced a mortality of 90% for PC3, 89% for A549, 85% for HCT15 and of 81% for BT549 cell lines. At the low protein concentration of only 11 µg/mL the 50 kDa fraction still entails a cell mortality of 76% for A549 and 87% for PC3 cell lines. The 50 kDa fraction contains 56% of proteins, 3% of lipids and 6% of minerals on a dry weight basis and the lowest levels detected of taurine and methionine and highest levels of threonine, proline and glycine amino acids. The enzymatic hydrolysis process suggests that Mytilus edulis by-products should be viewed as high-valued products with strong potential as anti-proliferative agent and promising active ingredients in functional foods.

  12. [Synthesis and anti-proliferative activity of fluoroquinolone (rhodanine unsaturated ketone) amide derivatives].

    Science.gov (United States)

    Gao, Liu-zhou; Xie, Yu-suo; Yan, Qiang; Wu, Shu-min; Ni, Li-li; Zhao, Hui; Huang, Wen-long; Hu, Guo-qiang

    2015-08-01

    To discover novel antitumor rhodanine unsaturated ketones, a series of fluoroquinolone (rhodanine α, β-unsaturated ketone) amine derivatives (5a-5r) were designed and synthesized with fluoroquinolone amide scaffold as a carrier. The structures of eighteen title compounds were characterized by elemental analysis, 1H NMR and MS. The in vitro anti-proliferative activity against Hep-3B, Capan-1 and HL60 cells was evaluated by MTT assay. The results showed that the title compounds not only had more significant anti-proliferative activity against three tested cancer cell lines than that of the parent ciprofloxacin 1, but also exhibited the highest activity against Capan-1 cells. The SAR revealed that some compounds carrying aromatic heterocyclic rings or phenyl attached to an electron-withdrawing carboxyl or sulfonamide substituent were comparable to or better than comparison doxorubicin against Capan-1 cells. As such, it suggests that fluoroquinolone (rhodanine α, β-unsaturated ketone) amines are promising leads for the development of novel antitumor fluoroquinolones or rhodanine analogues.

  13. Hyper-activation of Notch3 amplifies the proliferative potential of rhabdomyosarcoma cells.

    Directory of Open Access Journals (Sweden)

    Maria De Salvo

    Full Text Available Rhabdomyosarcoma (RMS is a pediatric myogenic-derived soft tissue sarcoma that includes two major histopathological subtypes: embryonal and alveolar. The majority of alveolar RMS expresses PAX3-FOXO1 fusion oncoprotein, associated with the worst prognosis. RMS cells show myogenic markers expression but are unable to terminally differentiate. The Notch signaling pathway is a master player during myogenesis, with Notch1 activation sustaining myoblast expansion and Notch3 activation inhibiting myoblast fusion and differentiation. Accordingly, Notch1 signaling is up-regulated and activated in embryonal RMS samples and supports the proliferation of tumor cells. However, it is unable to control their differentiation properties. We previously reported that Notch3 is activated in RMS cell lines, of both alveolar and embryonal subtype, and acts by inhibiting differentiation. Moreover, Notch3 depletion reduces PAX3-FOXO1 alveolar RMS tumor growth in vivo. However, whether Notch3 activation also sustains the proliferation of RMS cells remained unclear. To address this question, we forced the expression of the activated form of Notch3, Notch3IC, in the RH30 and RH41 PAX3-FOXO1-positive alveolar and in the RD embryonal RMS cell lines and studied the proliferation of these cells. We show that, in all three cell lines tested, Notch3IC over-expression stimulates in vitro cell proliferation and prevents the effects of pharmacological Notch inhibition. Furthermore, Notch3IC further increases RH30 cell growth in vivo. Interestingly, knockdown of Notch canonical ligands JAG1 or DLL1 in RMS cell lines decreases Notch3 activity and reduces cell proliferation. Finally, the expression of Notch3IC and its target gene HES1 correlates with that of the proliferative marker Ki67 in a small cohort of primary PAX-FOXO1 alveolar RMS samples. These results strongly suggest that high levels of Notch3 activation increase the proliferative potential of RMS cells.

  14. Anti-proliferative activities of sinigrin on carcinogen-induced hepatotoxicity in rats.

    Science.gov (United States)

    Jie, Meng; Cheung, Wan Man; Yu, Vivian; Zhou, Yanling; Tong, Pak Ho; Ho, John W S

    2014-01-01

    Liver cancer is one of the leading causes of cancer death worldwide. A very high incidence of new liver cancer cases is diagnosed every year, and metastasis has been found to correlate to poor prognoses in humans. Better treatments for liver cancer are thus clearly needed. Sinigrin is one of the major ingredients present in Brassica nigra, which has been used in combination with other herbs for treatment of various diseases. The anti-proliferative activities of sinigrin were studied in a model of carcinogen-induced hepatotoxicity in rats. Rats were orally administered with sinigrin on a daily basis for three months before sacrifice. Sinigrin was found to significantly inhibit the proliferation of liver tumor cells; the number of surface tumors in the rat liver was dramatically reduced. Sinigrin induced apoptosis of liver cancer cells through up-regulation of p53 and down-regulation of Bcl-2 family members and caspases. Our findings indicated that the liver functions were gradually restored after treatment with sinigrin and that the agent did not cause liver toxicity. Cell cycle analysis indicated that sinigrin caused cell cycle arrest in G0/G1 phase. The results suggest that sinigrin exerts important anti-proliferative activities in carcinogen-induced hepatocarcinogenesis in rats, and highlight the potential of sinigrin as an anti-cancer agent for liver cancer.

  15. Analysis of proliferative activity in oral gingival epithelium in immunosuppressive medication induced gingival overgrowth

    Directory of Open Access Journals (Sweden)

    Özdemir B Handan

    2006-05-01

    Full Text Available Abstract Background Drug-induced gingival overgrowth is a frequent adverse effect associated principally with administration of the immunosuppressive drug cyclosporin A and also certain antiepileptic and antihypertensive drugs. It is characterized by a marked increase in the thickness of the epithelial layer and accumulation of excessive amounts of connective tissue. The mechanism by which the drugs cause gingival overgrowth is not yet understood. The purpose of this study was to compare proliferative activity of normal human gingiva and in cyclosporine A-induced gingival overgrowth. Methods Gingival samples were collected from 12 generally healthy individuals and 22 Cyclosporin A-medicated renal transplant recipients. Expression of proliferating cell nuclear antigen was evaluated in formalin-fixed, paraffin-embedded gingival samples using an immunoperoxidase technique and a monoclonal antibody for this antigen. Results There were differences between the Cyclosporin A group and control group in regard to proliferating cell nuclear antigen and epithelial thickness. In addition, the degree of stromal inflammation was higher in the Cyclosporin A group when compared with the control group. Conclusion The results suggest that the increased epithelial thickness observed in Cyclosporin A-induced gingival overgrowth is associated with increased proliferative activity in keratinocytes.

  16. H3K4 demethylase activities repress proliferative and postmitotic aging

    Science.gov (United States)

    Alvares, Stacy M; Mayberry, Gaea A; Joyner, Ebony Y; Lakowski, Bernard; Ahmed, Shawn

    2014-01-01

    Homeostasis of postmitotic and proliferating cells is maintained by pathways that repress stress. We found that the Caenorhabditis elegans histone 3 lysine 4 (H3K4) demethylases RBR-2 and SPR-5 promoted postmitotic longevity of stress-resistant daf-2 adults, altered pools of methylated H3K4, and promoted silencing of some daf-2 target genes. In addition, RBR-2 and SPR-5 were required for germ cell immortality at a high temperature. Transgenerational proliferative aging was enhanced for spr-5; rbr-2 double mutants, suggesting that these histone demethylases may function sequentially to promote germ cell immortality by targeting distinct H3K4 methyl marks. RBR-2 did not play a comparable role in the maintenance of quiescent germ cells in dauer larvae, implying that it represses stress that occurs as a consequence of germ cell proliferation, rather than stress that accumulates in nondividing cells. We propose that H3K4 demethylase activities promote the maintenance of chromatin states during stressful growth conditions, thereby repressing postmitotic aging of somatic cells as well as proliferative aging of germ cells. PMID:24134677

  17. ROLE OF THE MORPHOMETRIC PARAMETERS OF INTRATUMORAL MICROVESSELS AND THE PROLIFERATIVE ACTIVITY OF TUMOR CELLS IN RENAL CELL CARCINOMA

    Directory of Open Access Journals (Sweden)

    N. A. Gorban

    2014-08-01

    Full Text Available Tumor cell proliferation and angiogenesis are essential factors for tumor growth, progression, and metastasis.Objective: to assess the relationship between the values of proliferative activity and the morphometric parameters of intratumoral microvessels in metastatic and localized carcinomas of the kidney.Materials and methods. Surgical specimens taken from 54 patients (32 men and 22 women aged 26 to 69 years (mean age 55 ± 1.5 years with the verified diagnosis of clear-cell renal cell carcinoma (RCC were studied.Conclusion. Proliferative activity and angioarchitectonics are an important biological characteristic of a tumor of unequal clinical value in RCC. Metastatic carcinoma has a higher proliferative activity and a low tumor vascularization than those of localized carcinoma.

  18. [Proliferative activity parameters and their correlation with genetic damage of blood lymphocytes during cultivation under the conditions of cytokinetic block].

    Science.gov (United States)

    Ingel', F I; Iurchenko, V V; Gus'kov, A S; Krivtsova, E K; Iurtseva, N A

    2006-01-01

    The subjects of the study were 15 volunteers aged 22 to 25 years, who underwent 25 air ionization sessions. The effects of genome instability were evaluated, and correlations between indicators of genome damage (lesions of micronuclei and nucleoplasmatic bridges) and parameters of proliferative and replicative activity (mitotic index, proliferative pool, the fraction of rapidly dividing cells, and replication index) of blood lymphocytes in the culture were studied. In order to establish the associations between the parameters, the parallel cultures were exposed to 0.07 mM of the standard mutagen MNNG during 5 hours. The study showed that the course of air ionization did not induce the micronuclei and nucleoplasmatic bridges in binuclear cells, but increased proliferative cell activity. This effect was accompanied by an increase in the fraction of rapidly dividing cells among all the dividing cells, and an increase in the dispersion of all proliferation parameters. MNNG induced a constant level of micronuclei in binuclear cells during the whole course, but not before the beginning of air ionization. The changes in the parameter "the fraction of dividing cells" (proliferative pool) were the most prominent manifestation of the suppression of proliferation by MNNG. MNNG loading inhibited the formation of binuclear cells most of all. The results demonstrate a non-random character of the correlation between the level of micronuclei in binuclear cells and proliferative activity parameters during cell cultivation under the conditions of cytokinetic block.

  19. New ursane triterpenoids from Salvia urmiensis Bunge: Absolute configuration and anti-proliferative activity.

    Science.gov (United States)

    Farimani, Mahdi Moridi; Bahadori, Mir Babak; Koulaei, Sheyda Ahmadi; Salehi, Peyman; Ebrahimi, Samad Nejad; Khavasi, Hamid Reza; Hamburger, Matthias

    2015-10-01

    Two new triterpenoids, urmiensolide B (1) and urmiensic acid (2), with rare carbon skeletons together with three known compounds were isolated from the aerial parts of Salvia urmiensis Bunge, an endemic species of Iran. The structures were established by a combination of 1D and 2D NMR, and HRESIMS, and in the case of 2 and 3, their structures were confirmed by single-crystal X-ray analysis. The absolute configuration of 2 was established by electronic circular dichroism (ECD) spectra. The new compounds were evaluated for their anti-proliferative activities against A549 and MCF-7 human cancer cell lines. Compounds 1 and 2 showed IC50 values of 2.8 and 1.6 μM against MCF-7 cells, respectively.

  20. [Immunohistochemical description of proliferative activity and apoptosis of lung squamous cell carcinoma (literature review)].

    Science.gov (United States)

    Филенко, Борис Н; Ройко, Наталия В; Степанчук, Алла П; Проскурня, Сергей А

    2016-01-01

    The analysis of the publications are describe immunohistochemical study of proliferative activity and apoptosis of lung squamous cell carcinoma. Established that the imbalance between proliferation and cell death is a key process in the development of tumors. However, the value of tumor markers in histogenesis and morfogenesis of tumors and forecast their occurrence is not studied enough. Despite the significant amount of scientific literature devoted to this issue, has not yet established a clear link expression of immunohistochemical markers of proliferation and apoptosis with the degree of differentiation of squamous cell lung cancer. Analysis of the literature shows that the morphology of this histogenetics type lung cancer at the cellular, subcellular structural and functional levels are controversial and require detailed investigation.

  1. Isolation of lignans from Schisandra chinensis with anti-proliferative activity in human colorectal carcinoma: Structure-activity relationships.

    Science.gov (United States)

    Gnabre, John; Unlu, Irem; Chang, Tso-Cheng; Lisseck, Paul; Bourne, Bryan; Scolnik, Ryan; Jacobsen, Neil E; Bates, Robert; Huang, Ru Chih

    2010-10-15

    Separate benzocyclooctadiene lignans were isolated from the berries of Schisandra chinensis in milligram quantities on analytical reverse phase (RP) HPLC by an automated repeat-injection method and shown to have anti-proliferative activity against human colorectal cancer cells. Structures of the compounds were determined by a combination of NMR and mass spectrometry. Stereospecific NMR assignments for gomisin-N and deoxyschisandrin, gave more complete and accurate data than previously reported, based on 600MHz 2D HSQC, DQF-COSY and HMBC data. Comparison of coupling constants and HMBC crosspeak intensities with calculated and X-ray crystal structures confirmed their stereochemistry and conformation. Analysis of structure-activity relationships revealed the importance of key structural determinants. The S-biphenyl configuration of gomisin N, the most active lignan, correlated with increased anti-proliferative activity, while the presence of a hydroxyl group at the C7 position reduced or abolished this activity. Increased activity was also observed when a methylenedioxy group was present between C12 and C13. The percent yield of the most active compounds relative to the starting plant materials was 0.0156% for deoxyschisandrin and 0.0173% for gomisin N. The results of these studies indicate that automated repeat-injection method of analytical HPLC may provide a superior alternative to the standard semi-preparative HPLC techniques for separation of complex mixtures.

  2. Chitin and chitosan from the Norway lobster by-products: Antimicrobial and anti-proliferative activities.

    Science.gov (United States)

    Sayari, Nadhem; Sila, Assaâd; Abdelmalek, Baha Eddine; Abdallah, Rihab Ben; Ellouz-Chaabouni, Semia; Bougatef, Ali; Balti, Rafik

    2016-06-01

    Chitin was recovered through enzymatic deproteinization of the Norway lobster (Nephrops norvegicus) processing by-products. The obtained chitin was characterized and converted into chitosan by N-deacetylation, the acid-soluble form of chitin. Chitosan samples were then characterized by Fourier transform infrared spectroscopy (FTIR) and 13 Cross polarization magic angle spinning nuclear magnetic resonance (CP/MAS)-NMR spectroscopy. The antimicrobial activity and anti-proliferative capacity of chitosan were evaluated. Antimicrobial activity assays indicated that prepared chitosan exhibited marked inhibitory activity against the bacterial and fungal strains tested. Further, cytotoxic effects of chitosan samples on human colon carcinoma cells HCT116 was evaluated using the MTT assay. Chitosan showed the antiproliferative capacity against the colon-cancer-cell HCT116 in a dose dependent manner with IC50 of 4.6mg/ml. Indeed, HCT116 cell proliferation was significantly inhibited (p<0.05) between 13.5 and 67.5% at 0.5-6mg/mL of chitosan after 24h of cell treatment. The chitosan showed high antitumor activity which seemed to be dependent on its characteristics such as acetylation degree.

  3. Roles of tissue plasminogen activator and its inhibitor in proliferative diabetic retinopathy

    Institute of Scientific and Technical Information of China (English)

    Shu-Ling; Wu; Dong-Mei; Zhan; Shu-Hong; Xi; Xiang-Lian; He

    2014-01-01

    AIM:To investigate the role of tissue plasminogen activator(t-PA) and plasminogen activator inhibitor(PAI)in proliferative diabetic retinopathy(PDR) and to discuss the correlations among t-PA, PAI and vascular endothelial growth factor(VEGF) expressions.METHODS:A total of 36 vitreous samples were collected from 36 patients with PDR(PDR group), and 17 vitreous samples from 17 patients with idiopathic macular hole were used as control. The concentrations of t-PA, PAI and VEGF in samples were determined by ELISA method. The correlations among t-PA, PAI and VEGF expressions were discussed.RESULTS:The concentrations of t-PA, PAI and VEGF in the PDR group were significantly higher than those in the control group(P <0.001). The t-PA and PAI expressions were highly correlated with the VEGF expression(P <0.001).CONCLUSION:In addition to VEGF, a variety of bioactive substances, such as t-PA and PAI, are involved in the pathogenesis involved in the angiogenesis of PDR.VEGF can activate t-PA expression, resulting in collagen tissue degradation and angiogenesis. VEGF may also activate the mechanism for endogenous anti-neovascularization.

  4. Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Werb, Z.; Aggeler, J.

    1978-04-01

    We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2 to 24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or ..cap alpha..-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16 to 48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10/sup 6/ cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, ..cap alpha../sub 1/-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.

  5. Two new diterpene derivatives from Euphorbia lunulata Bge and their anti-proliferative activities.

    Science.gov (United States)

    Liu, Chao; Liao, Zhi-xin; Liu, Shi-jun; Qu, Yan-bo; Wang, Heng-shan

    2014-07-01

    A new ent-abietane-type diterpene lactone (1) and a new jatrophane-type diterpenoid (2), together with twelve known compounds including three diterpenes (3-5), five triterpenes (6-10) and four sterides (11-14) were isolated from the ethanol extract of the whole plant of Euphorbia lunulata Bge. The structure of compounds 1 and 2 was elucidated on the basis of 1D and 2D NMR spectra and the HR-ESI-MS data. The structure of compound 2 was further analyzed by an X-ray crystallographic study. The in vitro anti-proliferative activities against MCF-7 and NCI-H460 cell lines for compounds 1-5 (diterpene) were evaluated. The results showed marked activity for compound 1 against the two cell lines with the IC50 values 19.5 (NCI-H460) and 18.6 (MCF-7) μM, while for cis-platinum (a positive cytotoxic control agent) 29.7 (NCI-H460) and 27.7 (MCF-7) μM. Compounds 2-5 exhibited moderate cytotoxic activities for the two cell lines with the IC50 values ranging from 32.1 to 58.2 μM.

  6. Effect of Enterococcus faecium EF 55 on morphometry and proliferative activity of intestinal mucosa in broilers infected with Salmonella Enteritidis

    Directory of Open Access Journals (Sweden)

    Ševčíková Zuzana

    2016-09-01

    Full Text Available Introduction: The present study aimed to investigate the effect of Enterococcus faecium EF55 on chickens, as well as its influence on proliferative activity of epithelial intestinal cells after infection with Salmonella enterica serovar Enteritidis phage type 4 (SE PT4. Moreover, the length and area of duodenal and jejunal villi of the birds were examined.

  7. Evaluation of viability and proliferative activity of human urothelial cells cultured onto xenogenic tissue-engineered extracellular matrices.

    LENUS (Irish Health Repository)

    Davis, Niall F

    2011-04-01

    To evaluate the viability and proliferative activity of human urothelial cells (HUCs) cultured on tissue-engineered extracellular matrix scaffolds and to assess the potential of extracellular matrixes to support the growth of HUCs in their expected in vivo urine environment.

  8. Feedback Activation of Basic Fibroblast Growth Factor Signaling via the Wnt/β-Catenin Pathway in Skin Fibroblasts.

    Science.gov (United States)

    Wang, Xu; Zhu, Yuting; Sun, Congcong; Wang, Tao; Shen, Yingjie; Cai, Wanhui; Sun, Jia; Chi, Lisha; Wang, Haijun; Song, Na; Niu, Chao; Shen, Jiayi; Cong, Weitao; Zhu, Zhongxin; Xuan, Yuanhu; Li, Xiaokun; Jin, Litai

    2017-01-01

    Skin wound healing is a complex process requiring the coordinated behavior of many cell types, especially in the proliferation and migration of fibroblasts. Basic fibroblast growth factor (bFGF) is a member of the FGF family that promotes fibroblast migration, but the underlying molecular mechanism remains elusive. The present RNA sequencing study showed that the expression levels of several canonical Wnt pathway genes, including Wnt2b, Wnt3, Wnt11, T-cell factor 7 (TCF7), and Frizzled 8 (FZD8) were modified by bFGF stimulation in fibroblasts. Enzyme-linked immunosorbent assay (ELISA) analysis also showed that Wnt pathway was activated under bFGF treatment. Furthermore, treatment of fibroblasts with lithium chloride or IWR-1, an inducer and inhibitor of the Wnt signaling pathway, respectively, promoted and inhibited cell migration. Also, levels of cytosolic glycogen synthase kinase 3 beta phosphorylated at serine(9) (pGSK3β Ser(9)) and nuclear β-catenin were increased upon exposure to bFGF. Molecular and biochemical assays indicated that phosphoinositide 3-kinase (PI3K) signaling activated the GSK3β/β-catenin/Wnt signaling pathway via activation of c-Jun N-terminal kinase (JNK), suggesting that PI3K and JNK act at the upstream of β-catenin. In contrast, knock-down of β-catenin delayed fibroblast cell migration even under bFGF stimulation. RNA sequencing analysis of β-catenin knock-down fibroblasts demonstrated that β-catenin positively regulated the transcription of bFGF and FGF21. Moreover, FGF21 treatment activated AKT and JNK, and accelerated fibroblast migration to a similar extent as bFGF does. In addition, ELISA analysis demonstrated that both of bFGF and FGF21 were auto secretion factor and be regulated by Wnt pathway stimulators. Taken together, our analyses define a feedback regulatory loop between bFGF (FGF21) and Wnt signaling acting through β-catenin in skin fibroblasts.

  9. Biocatalytically Oligomerized Epicatechin with Potent and Specific Anti-proliferative Activity for Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ramaswamy Nagarajan

    2008-11-01

    Full Text Available Catechins, naturally occurring flavonoids derived from wine and green tea, are known to exhibit multiple health benefits. Epigallocatechin gallate (EGCG is one of the most widely investigated catechins, but its efficacy in cancer therapy is still inconsistent and limited. The poor stability of EGCG has contributed to the disparity in the reported anti-cancer activity and other beneficial properties. Here we report an innovative enzymatic strategy for the oligomerization of catechins (specifically epicatechin that yields stable, water-soluble oligomerized epicatechins with enhanced and highly specific anti-proliferative activity for human breast cancer cells. This one-pot oxidative oligomerization is carried out in ambient conditions using Horseradish Peroxidase (HRP as a catalyst yielding water-soluble oligo(epicatechins. The oligomerized epicatechins obtained exhibit excellent growth inhibitory effects against human breast cancer cells with greater specificity towards growth-inhibiting cancer cells as opposed to normal cells, achieving a high therapeutic differential. Our studies indicate that water-soluble oligomeric epicatechins surpass EGCG in stability, selectivity and efficacy at lower doses.

  10. Vegetable-derived isothiocyanates: anti-proliferative activity and mechanism of action.

    Science.gov (United States)

    Zhang, Yuesheng; Yao, Song; Li, Jun

    2006-02-01

    Many isothiocyanates (ITC), which are available to human subjects mainly through consumption of cruciferous vegetables, demonstrate strong cancer-preventive activity in animal models. Human studies also show an inverse association between consumption of ITC and risk of cancer in several organs. Whereas earlier studies primarily focused on the ability of ITC to inhibit carcinogen-activating enzymes and induce carcinogen-detoxifying enzymes, more recent investigations have shown that ITC inhibit the proliferation of tumour cells both in vitro and in vivo by inducing apoptosis and arresting cell cycle progression. ITC cause acute cellular stress, which may be the initiating event for these effects. These findings shed new light on the mechanism of action of ITC and indicate that ITC may be useful both as cancer-preventive and therapeutic agents. ITC activate caspase 9-mediated apoptosis, apparently resulting from mitochondrial damage, and also activate caspase 8, but the mechanism remains to be defined. Cell cycle arrest caused by ITC occurs mainly in the G2/M phase, and both the G2 and M phases are targetted; critical G2-phase regulators, including cyclin B1, cell division cycle (Cdc) 2 and Cdc25C, are down regulated or inhibited, and tubulin polymerization and spindle assembly are disrupted. Moreover, ITC are metabolized in vivo through the mercapturic acid pathway, giving rise to thiol conjugates (dithiocarbamates). Studies show that these dithiocarbamates are similar to their parent ITC in exerting anti-proliferative activity. Taken together, dietary ITC are highly-promising anti-cancer agents, capable of targetting multiple cellular components that are important for tumour cell survival and proliferation.

  11. Separation and purification and in vitro anti-proliferative activity of leukemia cell K562 of Galium aparine L. petroleum

    Directory of Open Access Journals (Sweden)

    Guoqing Shi

    2016-05-01

    Full Text Available To explore material basis of in vitro anti-proliferative activity of leukemia cell K562 of petroleum ether phase of product resulting from Galium aparine L. 60% ethanol extraction, the experiment adopts column chromatography combined with thin layer preparation, isolates and purifies petroleum ether, conducts structural identification of obtained single compound and applies MTT method for viability assay of in vitro anti-proliferative activity of leukemia cell K562. Experimental results show that G. aparine L. petroleum ether contains mainly β-sitosterol, daucosterol and dibutyl phthalate and other substances. Under experimental conditions, the three could inhibit the proliferation of leukemia cell K562 with dose-effect and time-effect relationship, of which dibutyl phthalate has strongest activity. Dibutyl phthalate with excellent activity, β-sitosterol with rich content and moderate effect should be the main contributor to its biological activity.

  12. Cucumarioside A2-2 causes changes in the morphology and proliferative activity in mouse spleen.

    Science.gov (United States)

    Pislyagin, E A; Manzhulo, I V; Dmitrenok, P S; Aminin, D L

    2016-05-01

    The immunomodulatory effect of triterpene glycoside cucumarioside A2-2 (CA2-2), isolated from the Far Eastern sea cucumber Cucumaria japonica, on the mouse spleen was investigated in comparison with lipopolysaccharide (LPS). It has been shown that the intraperitoneal (i.p.) glycoside administration did not influence on splenic weights, while the statistically significant increase in splenic weight was observed after LPS administration. Changes in the ratio of red to white pulp after CA2-2 or LPS administration were observed. The proportion of splenic white pulp after glycoside or LPS administration increased by up to 34% and 36%, respectively. A detailed study of the distribution of the РСNA (Proliferating Cell Nuclear Antigen) marker showed that the proliferative activity in the white pulp under CA2-2 and LPS influence increased 2.07 and 2.24 times, respectively. The localization of PCNA-positive nuclei in the white pulp region, as well as their dimensional characteristics, suggests that a large proportion of the proliferating cell population consisted of B cells. The mass spectrometry profiles of spleen peptide/protein homogenate were obtained using the MALDI-TOF-MS (Matrix -Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) approach. It was found that i.p. stimulation of animals with CA2-2 or LPS leads to marked changes in the intensity of revealed characteristic peaks of peptides/proteins after exposure to immunostimulants.

  13. Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions

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    Shuvalova N. S.

    2015-06-01

    Full Text Available Aim. To optimize the cultivation of Wharton jelly-derived mesenchyma stem cells (WJ-MSCs using physiological oxygen concentrations, and to compare the effect of “hypoxic” gas mixtures, based on nitrogen and argon, on their proliferative activity. Methods. From the first passage, WJ-MSCs were cultivated during five passages in the nitrogen-based gas mixture (3 % oxygen, 4 % carbon dioxide, 93 % nitrogen and argon-based gas mixture (3 % oxygen, 4 % carbon dioxide, 93 % argon, 7 days before replating. At each passage the final cell number was estimated and the number of population doublings was calculated. Results. The proliferation level of WJ-MSCs, cultured in both gas mixtures with 3 % of O2, was significantly higher compared to that under the regular CO2-incubator conditions. In argon-based mixture, the WJ-MSCs proliferation was higher than in the control but lower than in nitrogen-based mixture. Conclusion. Cultivation of human WJ-MSCs under 3 % O2 had a stimulating effect on the cell proliferation potential. The highest intensity of the cell multiplication was observed in the nitrogen-based mixtures.

  14. Angiogenesis, Proliferative Activity and DNA Ploidy in Oral Verrucous Carcinoma: A Comparative Study Including Verrucous Hyperplasia and Squamous Cell Carcinoma.

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    Mallick, Saumyaranjan; Breta, Monika; Gupta, Siddhartha Datta; Dinda, Amit Kumar; Mohanty, Biddhu K; Singh, Manoj K

    2015-09-01

    Verrucous carcinoma (VC) is a rare and distinct clinicopathologic variant of well-differentiated squamous cell carcinoma (SCC). This study aims to evaluate the histomorphology, proliferative activity, level of angiogenesis, and DNA ploidy of these pathological entities. This was a retrospective-prospective study of 18 cases of verrucous hyperplasia (VH), 41 cases of VC, and 44 cases of SCC. Immunohistochemical analysis for Ki-67 (MIB-1) and CD34 were performed. The tumor proliferative index, endothelial proliferative index and microvascular density were calculated. DNA ploidy was determined using image cytometry. The age range and gender ratio were similar in all three groups. The differences in MIB-1 labeling index (p = 0.0001), microvascular density (p = 0.01), and endothelial proliferative index (p = 0.001) between VC and SCC were found to be statistically significant. A non-significant increasing trend was observed in all of these parameters between VH and VC. On ploidy analysis, 100 % of SCC cases were aneuploid, compared to 39 % of VH and 86 % of VC cases. Our study demonstrates a significant difference in tumor proliferation, microvessel density, and ploidy between VC and SCC while increasing trend between VH and VC. These parameters, along with morphological findings, may be useful in differentiating these entities in small mucosal biopsies.

  15. Characterization and anti-proliferative activity of curcumin loaded chitosan nanoparticles in cervical cancer.

    Science.gov (United States)

    Khan, Md Asad; Zafaryab, Md; Mehdi, Syed Hassan; Ahmad, Irfan; Rizvi, M Moshahid A

    2016-12-01

    In the present study the chitosan nanoparticles (CsNPs) and curcumin loaded chitosan nanoparticles (CLCsNPs) were synthesized by tripolyphosphate (TPP) cross-linking method. The nanoparticles were prepared within a zone of appropriate chitosan and TPP concentrations. The average size of CsNPs and CLCsNPs were approximately 189±11.8nm and 197±16.8nm, exhibited a zeta potential of +76±5.6mV and +71±6.4mV respectively and drug entrapment efficiency was ≈85%. The CLCsNPs and CsNPs were further characterized by different physicochemical methods like transmission electron microscopy (TEM), dynamic light scattering (DLS), HPLC, MALDI-TOF, FT-IR, XRD and UV-vis Spectroscopy. In vitro studies revealed a fast release of ≈35% at pH 5 and ≈25% at pH 7.4 of the drug during the first 3h, followed by controlled release of curcumin over a period of 120h and sustained anti-proliferative activity of the drug in a dose and time dependent manner of CLCsNPs and combination with methyl jasmonate. The higher cytotoxicity effect of CLCsNPs may be due to their higher cellular uptake as compared to curcumin. Chitosan nanoparticles were not only stable but also a nontoxic. Our data suggested that curcumin loaded nanoformulations, therefore, might be promising candidates in cancer therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Evaluation of cell proliferative activity after irradiation using immunohistochemical approach and flow cytometry

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    Tamada, Takashi (Okayama Univ. (Japan). School of Medicine)

    1992-06-01

    To evaluate a proliferative activity of post-irradiated malignant cells, we studied the kinetics of HeLa cells using immunohistochemical approach and flow cytometry. HeLa cells were stained with two proliferation-associated monoclonal antibodies, Ki-67 and anti-DNA polymerase {alpha} antibody. Nucleoli of non-irradiated cells were granularly stained with Ki-67. After irradiation, only the center of nuclei was diffusely stained with Ki-67. One hundred forty-four hours after low-dose irradiation, the staining patterns became the same as the control. On the other hand, after high-dose irradiation, the center of nuclei was weakly stained. DNA polymerase {alpha} was diffusely labelled with nuclei of the control. It was located around the border of nuclei of low-dose irradiated cells like a ring. But after high-dose irradiation, it was granularly distributed in the periphery of nuclei. FITC conjugated Ki-67/PI two parameter analysis was done by a single laser flow cytometer. Twenty-four hours after irradiation, DNA-histograms showed the accumulation to G{sub 2}/M phase and the increase of DNA content of G{sub 2}/M cells, as exposure dose was increased. Two parameter analysis showed the increase of FITC uptake of G{sub 2}/M phase as dose increased. These changes of flow cytometry were remarkably observed after 24 hours' incubation. It was shown that the difference of Ki-67 antigen and DNA polymerase {alpha} appearance depended on the irradiation dose. These findings suggest that immunohistochemical staining with Ki-67 or anti-DNA polymerase {alpha} antibody and flow cytometry using Ki-67 are available to evaluate cell damages after irradiation. (author).

  17. Anti-inflammatory and anti-proliferative activities of the wild edible cruciferous: Diplotaxis simplex.

    Science.gov (United States)

    Jdir, Hamida; Khemakham, Bassem; Najjaa, Hanen; Chakroun, Mouna; Jridi, Mourad; Ben Arfa, Abdelkarim; Ben Ali, Yassine; Zouari, Nacim

    2016-10-01

    Context The present study deals with new biological properties of the wild edible Diplotaxis simplex (Viv.) Spreng (Brassicaceae). Objectives The current study evaluates the antioxidant, the anti-inflammatory and the anti-cancer properties of ethyl acetate and ethanol extracts from D. simplex flowers. Materials and methods The anti-proliferative activity of the extracts (10-70 μg/mL) was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) against human colon cancer cell line Caco-2. The anti-inflammatory potential was evaluated by the inhibitory effect of the extracts (1.5-7.5 mg/mL) on phospholipase A2 activity as well as on carrageenan-induced paw oedema in mice. Extracts (200 mg/kg) or indomethacin (50 mg/kg) as positive control were injected intraperitoneally for albino mice prior to the induction of the oedema by carrageenan. Antioxidant activities were investigated using various complementary methods. Results Flower extracts contained a high level of polyphenolics (17.10-52.70 mg GAE/g) and flavonoids (74.20-100.60 mg QE/g), which correlate with its appreciable antioxidant potential in β-carotene peroxidation (IC50 value: 12.50-27.10 μg/mL), DPPH(•) radical-scavenging (IC50 value: 0.20-0.40 mg/mL), Fe(3+ )reducing (EC50 value: 0.10-0.14 mg/mL) and Fe(2+ )chelating (IC50 value: 0.20-0.60 mg/mL) assays. These extracts were effective in inhibiting cancer cell growth (IC50 value: 62.0-63.25 μg/mL). Besides, the ethyl acetate extract inhibited phospholipase A2 activity (IC50 value: 2.97 mg/mL) and reduced the paw oedema in mice (from 0.38 ± 0.01 to 0.24 ± 0.01 cm), 4 h post-carrageenan challenge. Conclusion These data suggest that D. simplex may be useful as a candidate in the treatment of inflammation and the colon cancer.

  18. Activation of neural progenitor cells in human eyes with proliferative vitreoretinopathy.

    Science.gov (United States)

    Johnsen, Erik O; Frøen, Rebecca C; Albert, Réka; Omdal, Bente K; Sarang, Zsolt; Berta, András; Nicolaissen, Bjørn; Petrovski, Goran; Moe, Morten C

    2012-05-01

    In addition to the ability for self-renewal and functional differentiation, neural stem/progenitor cells (NSCs) can respond to CNS injuries by targeted migration. In lower vertebrates, retinal injury is known to activate NSCs in the ciliary marginal zone (CMZ). Cells expressing markers of NSCs are also present in the ciliary body epithelium (CE) and in Müller glia in the peripheral retina (PR) of the adult human eye. However, these cells seem to be quiescent in the adult human eye and recent reports have shown that CE cells have limited properties of NSCs. In order to further clarify whether NSCs exist in the adult human eye, we tested whether NSC-like cells could be activated in eyes with proliferative vitreoretinopathy (PVR). The PR and CE were studied for NSC-associated markers in human enucleated control eyes and eyes with confirmed PVR, as well as in a mouse model of PVR. Furthermore, cells isolated from vitreous samples obtained during vitrectomies for retinal detachment were directly fixed or cultured in a stem cell-promoting medium and compared to cells cultured from the post-mortem retina and CE. In situ characterization of the normal eyes revealed robust expression of markers present in NSCs (Nestin, Sox2, Pax6) only around peripheral cysts of the proximal pars plana region and the PR, the latter population also staining for the glial marker GFAP. Although there were higher numbers of dividing cells in the CE of PVR eyes than in controls, we did not detect NSC-associated markers in the CE except around the proximal pars plana cysts. In the mice PVR eyes, Nestin activation was also found in the CE. In human PVR eyes, proliferation of both non-glial and glial cells co-staining NSC-associated markers was evident around the ora serrata region. Spheres formed in 7/10 vitreous samples from patients with PVR compared to 2/15 samples from patients with no known PVR, and expressed glial - and NSC-associated markers both after direct fixation and repetitive

  19. Signal Intensities in Preoperative MRI Do Not Reflect Proliferative Activity in Meningioma

    Directory of Open Access Journals (Sweden)

    Stefan Schob

    2016-08-01

    representing proliferative activity.

  20. Mitochondrial Oxidative Stress due to Complex I Dysfunction Promotes Fibroblast Activation and Melanoma Cell Invasiveness

    Directory of Open Access Journals (Sweden)

    Maria Letizia Taddei

    2012-01-01

    Full Text Available Increased ROS (cellular reactive oxygen species are characteristic of both fibrosis and tumour development. ROS induce the trans-differentiation to myofibroblasts, the activated form of fibroblasts able to promote cancer progression. Here, we report the role of ROS produced in response to dysfunctions of mitochondrial complex I, in fibroblast activation and in tumour progression. We studied human fibroblasts with mitochondrial dysfunctions of complex I, leading to hyperproduction of ROS. We demonstrated that ROS level produced by the mutated fibroblasts correlates with their activation. The increase of ROS in these cells provides a greater ability to remodel the extracellular matrix leading to an increased motility and invasiveness. Furthermore, we evidentiated that in hypoxic conditions these fibroblasts cause HIF-1α stabilization and promote a proinvasive phenotype of human melanoma cells through secretion of cytokines. These data suggest a possible role of deregulated mitochondrial ROS production in fibrosis evolution as well as in cancer progression and invasion.

  1. Stimulation of fibroblast proliferation by neokyotorphin requires Ca influx and activation of PKA, CaMK II and MAPK/ERK.

    Science.gov (United States)

    Sazonova, Olga V; Blishchenko, Elena Yu; Tolmazova, Anna G; Khachin, Dmitry P; Leontiev, Konstantin V; Karelin, Andrey A; Ivanov, Vadim T

    2007-01-01

    Neokyotorphin [TSKYR, hemoglobin alpha-chain fragment (137-141)] has previously been shown to enhance fibroblast proliferation, its effect depending on cell density and serum level. Here we show the dependence of the effect of neokyotorphin on cell type and its correlation with the effect of protein kinase A (PKA) activator 8-Br-cAMP, but not the PKC activator 4beta-phorbol 12-myristate, 13-acetate (PMA). In L929 fibroblasts, the proliferative effect of neokyotorphin was suppressed by the Ca2+ L-type channel inhibitors verapamil or nifedipine, the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, kinase inhibitors H-89 (PKA), KN-62 (Ca2+/calmodulin-dependent kinase II) and PD98059 (mitogen-activated protein kinase). The proliferative effect of 8-Br-cAMP was also suppressed by KN-62 and PD98059. PKC suppression (downregulation with PMA or inhibition with bisindolylmaleimide XI) did not affect neokyotorphin action. The results obtained point to a cAMP-like action for neokyotorphin.

  2. Endoglin haploinsufficiency promotes fibroblast accumulation during wound healing through Akt activation.

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    Miguel Pericacho

    Full Text Available Accurate regulation of dermal fibroblast function plays a crucial role in wound healing. Many fibrotic diseases are characterized by a failure to conclude normal tissue repair and the persistence of fibroblasts inside lesions. In the present study we demonstrate that endoglin haploinsufficiency promotes fibroblast accumulation during wound healing. Moreover, scars from endoglin-heterozygous (Eng(+/- mice show persisting fibroblasts 12 days after wounding, which could lead to a fibrotic scar. Endoglin haploinsufficiency results in increased proliferation and migration of primary cultured murine dermal fibroblasts (MDFs. Moreover, Eng(+/- MDF have diminished responses to apoptotic signals compared with control cells. Altogether, these modifications could explain the augmented presence of fibroblasts in Eng(+/- mice wounds. We demonstrate that endoglin expression regulates Akt phosphorylation and that PI3K inhibition abolishes the differences in proliferation between endoglin haploinsufficient and control cells. Finally, persistent fibroblasts in Eng(+/- mice wound co-localize with a greater degree of Akt phosphorylation. Thus, endoglin haploinsufficiency seems to promote fibroblast accumulation during wound healing through the activation of the PI3K/Akt pathway. These studies open new non-Smad signaling pathway for endoglin regulating fibroblast cell function during wound healing, as new therapeutic opportunities for the treatment of fibrotic wounds.

  3. Endoglin haploinsufficiency promotes fibroblast accumulation during wound healing through Akt activation.

    Science.gov (United States)

    Pericacho, Miguel; Velasco, Soraya; Prieto, Marta; Llano, Elena; López-Novoa, José M; Rodríguez-Barbero, Alicia

    2013-01-01

    Accurate regulation of dermal fibroblast function plays a crucial role in wound healing. Many fibrotic diseases are characterized by a failure to conclude normal tissue repair and the persistence of fibroblasts inside lesions. In the present study we demonstrate that endoglin haploinsufficiency promotes fibroblast accumulation during wound healing. Moreover, scars from endoglin-heterozygous (Eng(+/-)) mice show persisting fibroblasts 12 days after wounding, which could lead to a fibrotic scar. Endoglin haploinsufficiency results in increased proliferation and migration of primary cultured murine dermal fibroblasts (MDFs). Moreover, Eng(+/-) MDF have diminished responses to apoptotic signals compared with control cells. Altogether, these modifications could explain the augmented presence of fibroblasts in Eng(+/-) mice wounds. We demonstrate that endoglin expression regulates Akt phosphorylation and that PI3K inhibition abolishes the differences in proliferation between endoglin haploinsufficient and control cells. Finally, persistent fibroblasts in Eng(+/-) mice wound co-localize with a greater degree of Akt phosphorylation. Thus, endoglin haploinsufficiency seems to promote fibroblast accumulation during wound healing through the activation of the PI3K/Akt pathway. These studies open new non-Smad signaling pathway for endoglin regulating fibroblast cell function during wound healing, as new therapeutic opportunities for the treatment of fibrotic wounds.

  4. Alkaline phosphatase expression/activity and multilineage differentiation potential are the differences between fibroblasts and orbital fat-derived stem cells--a study in animal serum-free culture conditions.

    Science.gov (United States)

    Martins, Thaís Maria da Mata; de Paula, Ana Cláudia Chagas; Gomes, Dawidson Assis; Goes, Alfredo Miranda

    2014-10-01

    Human orbital fat tissues are a potential source to isolate stem cells for the development of regenerative medicine therapies. For future safe clinical application of these cells, it is critical to establish animal component-free culture conditions as well as to clearly define the stem cell population characteristics differentiating them from other cell types, such as fibroblasts. Therefore, the present study aimed to compare phenotypic and functional characteristics of orbital fat-derived stem cells (OFSCs) and fibroblasts resident in the eyelid skin in donor-matched samples grown in culture medium supplemented with pooled allogeneic human serum (HS) replacing fetal bovine serum (FBS). We first investigated the proliferative effects of OFSCs on HS, and then we compared the alkaline phosphatase (AP) expression and activity, immunophenotypic profile, and in vitro multilineage differentiation potential of OFSCs side-by-side with fibroblasts. The results showed that HS enhanced OFSCs proliferation without compromising their immunophenotype, AP activity, and osteogenic, adipogenic, and chondrogenic differentiation capacities. In contrast to OFSCs, the fibroblasts did not exhibit AP expression and activity and did not have multilineage differentiation potential. The results enabled us to successfully distinguish OFSCs from fibroblasts populations, suggesting that AP expression/activity and multilineage differentiation assays can be used reliably to discriminate mesenchymal stem cells from fibroblasts. Our findings also support the feasibility of pooled allogeneic HS as a safer and more effective alternative to FBS for clinical applications.

  5. Antifibrotic effect by activation of peroxisome proliferator-activated receptor-gamma in corneal fibroblasts.

    Science.gov (United States)

    Pan, Hongwei; Chen, Jiansu; Xu, Jintang; Chen, Miaojiao; Ma, Rong

    2009-11-10

    The transformation of quiescent keratocytes to active phenotypes and the ensuing fibrotic response play important roles in corneal scar formation. This study aims to observe the antifibrotic effect of peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist on corneal fibroblasts cultured in vitro, and to explore the potential application of peroxisome proliferator-activated receptor agonist to the prevention of corneal opacity following wound repair. Rabbit corneal keratocytes were cultured in a medium containing 10% serum to induce their transformation to fibroblasts and myofibroblasts, which are similar to those that repair corneas. After incubation with the PPARgamma agonist pioglitazone at different concentrations, the effect of pioglitazone on the migration, contractility, and viability of corneal fibroblasts was examined. The secretion of matrix metalloproteinase-2 and matrix metalloproteinase-9 was determined by gelatin zymography, and the synthesis of collagen I and fibronectin was investigated by western blotting. Treatment with pioglitazone at concentrations ranging from 1 to 10 mum significantly decreased corneal fibroblast migration, as determined by scrape-wound assay, inhibited corneal fibroblast-induced collagen lattice contraction, and reduced MMP-2 and MMP-9 secretion into the supernatant of cell cultures in a dose-dependent manner. The expression of fibronectin was significantly decreased, while the expression of collagen I was only decreased when treated with 10 mum pioglitazone. Cell viability was not evidently changed compared to the control. This in vitro study demonstrated the anti-fibrotic effect of pioglitazone, suggesting that activation of PPARgamma may be a new approach for the treatment of corneal opacity and scar formation in the corneal wound healing process.

  6. Antinucleosome antibodies as a potential biomarker for the evaluation of renal pathological activity in patients with proliferative lupus nephritis.

    Science.gov (United States)

    Hung, W T; Chen, Y M; Lan, J L; Chen, H H; Chen, Y H; Chen, D Y; Hsieh, C W; Wen, M C

    2011-11-01

    The objective of this study is to evaluate the correlation between antinucleosome antibodies and renal pathological activity in patients with proliferative lupus nephritis (LN). We evaluated 36 patients with proliferative LN, 14 non-renal lupus patients and 10 healthy volunteers. Lupus activity was assessed using the British Isles Lupus Assessment Group 2004 (BILAG 2004) index, serum anti-double stranded DNA (anti-dsDNA) levels, serum complement levels and daily urinary protein levels. All 36 lupus nephritis patients received renal biopsy. Antinucleosome antibodies were detected by enzyme-linked immunosorbent assay (ELISA). Our results showed that levels of serum antinucleosome antibodies were significantly higher in LN patients (median 90.35 units/ml, interquartile range [IQR] 37.38-135.23) than in non-renal SLE patients (median 5.45 units/ml, IQR 2.6-28.93, p antibodies were positively correlated with BILAG index (Spearman's r = 0.645, p antibodies were negatively correlated with serum levels of C3 (r(s) = -0.400, p antibodies were positively correlated with the histological activity index of LN (r(s) = 0.368, p antibodies and the histological chronicity index. In conclusion, the serum level of antinucleosome antibodies is a potential biomarker for early recognition of renal involvement and evaluation of disease activity in SLE. Our preliminary results suggested that serum levels of antinucleosome antibodies might be a potential biomarker in evaluating pathological activity of LN.

  7. GTP depletion synergizes the anti-proliferative activity of chemotherapeutic agents in a cell type-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Tao; Meng, Lingjun [Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A and M Health Science Center, Houston, TX 77030 (United States); Tsai, Robert Y.L., E-mail: rtsai@ibt.tamhsc.edu [Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A and M Health Science Center, Houston, TX 77030 (United States)

    2011-10-22

    Highlights: {yields} Strong synergy between mycophenolic acid (MPA) and 5-FU in MDA-MB-231 cells. {yields} Cell type-dependent synergy between MPA and anti-proliferative agents. {yields} The synergy of MPA on 5-FU is recapitulated by RNA polymerase-I inhibition. {yields} The synergy of MPA on 5-FU requires the expression of nucleostemin. -- Abstract: Mycophenolic acid (MPA) depletes intracellular GTP by blocking de novo guanine nucleotide synthesis. GTP is used ubiquitously for DNA/RNA synthesis and as a signaling molecule. Here, we made a surprising discovery that the anti-proliferative activity of MPA acts synergistically with specific chemotherapeutic agents in a cell type-dependent manner. In MDA-MB-231 cells, MPA shows an extremely potent synergy with 5-FU but not with doxorubicin or etoposide. The synergy between 5-FU and MPA works most effectively against the highly tumorigenic mammary tumor cells compared to the less tumorigenic ones, and does not work in the non-breast cancer cell types that we tested, with the exception of PC3 cells. On the contrary, MPA shows the highest synergy with paclitaxel but not with 5-FU in SCC-25 cells, derived from oral squamous cell carcinomas. Mechanistically, the synergistic effect of MPA on 5-FU in MDA-MB-231 cells can be recapitulated by inhibiting the RNA polymerase-I activity and requires the expression of nucleostemin. This work reveals that the synergy between MPA and anti-proliferative agents is determined by cell type-dependent factors.

  8. Fibroblastic activities post implantation of cobalt chromium alloy and pure germanium in rabbits.

    Science.gov (United States)

    Carter, J M; Natiella, J R; Baier, R E; Natiella, R R

    1984-02-01

    Different preimplantation surface finishes were applied to surgical vitallium discs and germanium prisms implanted for 20 days within the back muscles of adult rabbits. Histopathologic analysis of the numbers of nuclei of active fibroblasts immediately adjacent to the implants was carried out. The mean apparent volume fractions (MAVF) for the subdermal implant sites were found to depend on the surface cleanliness of the implant, the cleanest or highest-surface-energy surfaces giving the highest MAVF values for active fibroblasts.

  9. Stromal fibroblast activation and their potential association with uterine fibroids (Review)

    Science.gov (United States)

    ZHENG, LI-HUA; CAI, FENG-FENG; GE, ISABELL; BISKUP, EWELINA; CHENG, ZHONG-PING

    2014-01-01

    Uterine fibroids are the most common type of benign, gynecologic neoplasm and are the primary indication for performance of a hysterectomy, accounting for >200,000 hysterectomies annually in the USA. At present, females are younger and exhibit larger leiomyomas at the time of diagnosis. Cancer-associated fibroblasts in tumor microenvironments have emerged as an important target for cancer therapy. Repeated stimulation by infectious or non-infectious agents in the uterine tissues, including inflammation, mechanical forces or hypoxia, stimulate the resident fibroblasts to undergo specific activation and, thus, are significant in tumorigenesis. Furthermore, complex signaling pathways regulate the mechanisms of fibroblastic activation. The current review focuses on the molecular mechanisms of fibroblastic activation and the potential association with uterine leiomyoma pathogenesis, enabling an integrated pathogenic analysis for review of the therapeutic options. PMID:25013460

  10. Tumor-produced, active Interleukin-1 {beta} regulates gene expression in carcinoma-associated fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Dudas, Jozsef, E-mail: Jozsef.Dudas@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Fullar, Alexandra, E-mail: fullarsz@gmail.com [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); 1st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Ulloei ut 26, H-1085 Budapest (Hungary); Bitsche, Mario, E-mail: Mario.Bitsche@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Schartinger, Volker, E-mail: Volker.Schartinger@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Kovalszky, Ilona, E-mail: koval@korb1.sote.hu [1st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Ulloei ut 26, H-1085 Budapest (Hungary); Sprinzl, Georg Mathias, E-mail: Georg.Sprinzl@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Riechelmann, Herbert, E-mail: Herbert.Riechelmann@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria)

    2011-09-10

    Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial-mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1{beta} (IL1-{beta}) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-{beta} expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-{beta} processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-{beta}. IL1-{beta} signaling was investigated by western blot and immunocytochemistry. IL1-{beta}-regulated genes were analyzed by real-time qPCR. SCC-25 cells produced 16 kD active IL1-{beta}, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NF{kappa}B{alpha}. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-{beta} reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-{beta}-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-{beta} in the tumor cells leads to IL1-{beta}-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression. -- Graphical abstract: SCC-25 cells produce active, processed IL1-{beta}. PDL fibroblasts possess receptor for IL1-{beta}, and its expression is increased 4.56-times in the

  11. Chemical composition, antioxidant, anti-inflammatory and anti-proliferative activities of essential oils of plants from Burkina Faso.

    Directory of Open Access Journals (Sweden)

    Bagora Bayala

    Full Text Available This research highlights the chemical composition, antioxidant, anti-inflammatory and anti-proliferative activities of essential oils from leaves of Ocimum basilicum, Ocimum americanum, Hyptis spicigera, Lippia multiflora, Ageratum conyzoides, Eucalyptus camaldulensis and Zingiber officinale. Essential oils were analyzed by gas chromatography-mass spectrometry and gas chromatography-flame ionization detector. Major constituents were α-terpineol (59.78% and β-caryophyllene (10.54% for Ocimum basilicum; 1, 8-cineol (31.22%, camphor (12.730%, α-pinene (6.87% and trans α-bergamotene (5.32% for Ocimum americanum; β-caryophyllene (21%, α-pinene (20.11%, sabinene (10.26%, β-pinene (9.22% and α-phellandrene (7.03% for Hyptis spicigera; p-cymene (25.27%, β-caryophyllene (12.70%, thymol (11.88, γ-terpinene (9.17% and thymyle acetate (7.64% for Lippia multiflora; precocene (82.10%for Ageratum conyzoides; eucalyptol (59.55%, α-pinene (9.17% and limonene (8.76% for Eucalyptus camaldulensis; arcurcumene (16.67%, camphene (12.70%, zingiberene (8.40%, β-bisabolene (7.83% and β-sesquiphellandrène (5.34% for Zingiber officinale. Antioxidant activities were examined using 1,1-diphenyl-2-picryl-hydrazyl (DPPH and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS methods. O. basilicum and L. multiflora exhibited the highest antioxidant activity in DPPH and ABTS tests, respectively. Anti-inflammatory properties were evaluated by measuring the inhibition of lipoxygenase activity and essential oil of Z. officinale was the most active. Anti-proliferative effect was assayed by the measurement of MTT on LNCaP and PC-3 prostate cancer cell lines, and SF-763 and SF-767 glioblastoma cell lines. Essential oils from A. conyzoides and L. multiflora were the most active on LNCaP and PC-3 cell lines, respectively. The SF-767 glioblastoma cell line was the most sensitive to O. basilicum and L. multiflora EOs while essential oil of A. conyzoides showed the

  12. Chemical composition, antioxidant, anti-inflammatory and anti-proliferative activities of essential oils of plants from Burkina Faso.

    Science.gov (United States)

    Bayala, Bagora; Bassole, Imaël Henri Nestor; Gnoula, Charlemagne; Nebie, Roger; Yonli, Albert; Morel, Laurent; Figueredo, Gilles; Nikiema, Jean-Baptiste; Lobaccaro, Jean-Marc A; Simpore, Jacques

    2014-01-01

    This research highlights the chemical composition, antioxidant, anti-inflammatory and anti-proliferative activities of essential oils from leaves of Ocimum basilicum, Ocimum americanum, Hyptis spicigera, Lippia multiflora, Ageratum conyzoides, Eucalyptus camaldulensis and Zingiber officinale. Essential oils were analyzed by gas chromatography-mass spectrometry and gas chromatography-flame ionization detector. Major constituents were α-terpineol (59.78%) and β-caryophyllene (10.54%) for Ocimum basilicum; 1, 8-cineol (31.22%), camphor (12.730%), α-pinene (6.87%) and trans α-bergamotene (5.32%) for Ocimum americanum; β-caryophyllene (21%), α-pinene (20.11%), sabinene (10.26%), β-pinene (9.22%) and α-phellandrene (7.03%) for Hyptis spicigera; p-cymene (25.27%), β-caryophyllene (12.70%), thymol (11.88), γ-terpinene (9.17%) and thymyle acetate (7.64%) for Lippia multiflora; precocene (82.10%)for Ageratum conyzoides; eucalyptol (59.55%), α-pinene (9.17%) and limonene (8.76%) for Eucalyptus camaldulensis; arcurcumene (16.67%), camphene (12.70%), zingiberene (8.40%), β-bisabolene (7.83%) and β-sesquiphellandrène (5.34%) for Zingiber officinale. Antioxidant activities were examined using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) methods. O. basilicum and L. multiflora exhibited the highest antioxidant activity in DPPH and ABTS tests, respectively. Anti-inflammatory properties were evaluated by measuring the inhibition of lipoxygenase activity and essential oil of Z. officinale was the most active. Anti-proliferative effect was assayed by the measurement of MTT on LNCaP and PC-3 prostate cancer cell lines, and SF-763 and SF-767 glioblastoma cell lines. Essential oils from A. conyzoides and L. multiflora were the most active on LNCaP and PC-3 cell lines, respectively. The SF-767 glioblastoma cell line was the most sensitive to O. basilicum and L. multiflora EOs while essential oil of A. conyzoides

  13. Chemical Composition, Antioxidant, Anti-Inflammatory and Anti-Proliferative Activities of Essential Oils of Plants from Burkina Faso

    Science.gov (United States)

    Bayala, Bagora; Bassole, Imaël Henri Nestor; Gnoula, Charlemagne; Nebie, Roger; Yonli, Albert; Morel, Laurent; Figueredo, Gilles; Nikiema, Jean-Baptiste; Lobaccaro, Jean-Marc A.; Simpore, Jacques

    2014-01-01

    This research highlights the chemical composition, antioxidant, anti-inflammatory and anti-proliferative activities of essential oils from leaves of Ocimum basilicum, Ocimum americanum, Hyptis spicigera, Lippia multiflora, Ageratum conyzoides, Eucalyptus camaldulensis and Zingiber officinale. Essential oils were analyzed by gas chromatography–mass spectrometry and gas chromatography–flame ionization detector. Major constituents were α-terpineol (59.78%) and β-caryophyllene (10.54%) for Ocimum basilicum; 1, 8-cineol (31.22%), camphor (12.730%), α-pinene (6.87%) and trans α-bergamotene (5.32%) for Ocimum americanum; β-caryophyllene (21%), α-pinene (20.11%), sabinene (10.26%), β-pinene (9.22%) and α-phellandrene (7.03%) for Hyptis spicigera; p-cymene (25.27%), β-caryophyllene (12.70%), thymol (11.88), γ-terpinene (9.17%) and thymyle acetate (7.64%) for Lippia multiflora; precocene (82.10%)for Ageratum conyzoides; eucalyptol (59.55%), α-pinene (9.17%) and limonene (8.76%) for Eucalyptus camaldulensis; arcurcumene (16.67%), camphene (12.70%), zingiberene (8.40%), β-bisabolene (7.83%) and β-sesquiphellandrène (5.34%) for Zingiber officinale. Antioxidant activities were examined using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) methods. O. basilicum and L. multiflora exhibited the highest antioxidant activity in DPPH and ABTS tests, respectively. Anti-inflammatory properties were evaluated by measuring the inhibition of lipoxygenase activity and essential oil of Z. officinale was the most active. Anti-proliferative effect was assayed by the measurement of MTT on LNCaP and PC-3 prostate cancer cell lines, and SF-763 and SF-767 glioblastoma cell lines. Essential oils from A. conyzoides and L. multiflora were the most active on LNCaP and PC-3 cell lines, respectively. The SF-767 glioblastoma cell line was the most sensitive to O. basilicum and L. multiflora EOs while essential oil of A. conyzoides

  14. Acyl sulfonamide anti-proliferatives: benzene substituent structure-activity relationships for a novel class of antitumor agents.

    Science.gov (United States)

    Lobb, Karen L; Hipskind, Philip A; Aikins, James A; Alvarez, Enrique; Cheung, Yiu-Yin; Considine, Eileen L; De Dios, Alfonso; Durst, Gregory L; Ferritto, Rafael; Grossman, Cora Sue; Giera, Deborah D; Hollister, Beth A; Huang, Zhongping; Iversen, Philip W; Law, Kevin L; Li, Tiechao; Lin, Ho-Shen; Lopez, Beatriz; Lopez, Jose E; Cabrejas, Luisa M Martin; McCann, Denis J; Molero, Victoriano; Reilly, John E; Richett, Michael E; Shih, Chuan; Teicher, Beverly; Wikel, James H; White, Wesley T; Mader, Mary M

    2004-10-21

    Two closely related diaryl acylsulfonamides were recently reported as potent antitumor agents against a broad spectrum of human tumor xenografts (colon, lung, breast, ovary, and prostate) in nude mice. Especially intriguing was their activity against colorectal cancer xenografts. In this paper, rapid parallel synthesis along with traditional medicinal chemistry techniques were used to quickly delineate the structure-activity relationships of the substitution patterns in both phenyl rings of the acylsufonamide anti-proliferative scaffold. Although the molecular target of the compounds remains unclear, we determined that the vascular endothelial growth factor-dependent human umbilical vein endothelial cells assay in combination with a soft agar disk diffusion assay allowed for optimization of potency in the series. The pharmacokinetic properties and in vivo activity in an HCT116 xenograft model are reported for representative compounds.

  15. Endothelin-1 induces proliferation of human lung fibroblasts and IL-11 secretion through an ET(A) receptor-dependent activation of MAP kinases.

    Science.gov (United States)

    Gallelli, Luca; Pelaia, Girolamo; D'Agostino, Bruno; Cuda, Giovanni; Vatrella, Alessandro; Fratto, Donatella; Gioffrè, Vincenza; Galderisi, Umberto; De Nardo, Marilisa; Mastruzzo, Claudio; Salinaro, Elisa Trovato; Maniscalco, Mauro; Sofia, Matteo; Crimi, Nunzio; Rossi, Francesco; Caputi, Mario; Costanzo, Francesco S; Maselli, Rosario; Marsico, Serafino A; Vancheri, Carlo

    2005-11-01

    Endothelin-1 (ET-1) is implicated in the fibrotic responses characterizing interstitial lung diseases, as well as in the airway remodeling process occurring in asthma. Within such a context, the aim of our study was to investigate, in primary cultures of normal human lung fibroblasts (NHLFs), the ET-1 receptor subtypes, and the intracellular signal transduction pathways involved in the proliferative effects of this peptide. Therefore, cells were exposed to ET-1 in the presence or absence of an overnight pre-treatment with either ET(A) or ET(B) selective receptor antagonists. After cell lysis, immunoblotting was performed using monoclonal antibodies against the phosphorylated, active forms of mitogen-activated protein kinases (MAPK). ET-1 induced a significant increase in MAPK phosphorylation pattern, and also stimulated fibroblast proliferation and IL-6/IL-11 release into cell culture supernatants. All these effects were inhibited by the selective ET(A) antagonist BQ-123, but not by the specific ET(B) antagonist BQ-788. The stimulatory influence of ET-1 on IL-11, but not on IL-6 secretion, was prevented by MAPK inhibitors. Therefore, such results suggest that in human lung fibroblasts ET-1 exerts a profibrogenic action via an ET(A) receptor-dependent, MAPK-mediated induction of IL-11 release and cell proliferation.

  16. N-acetyl-L-cysteine increases MnSOD activity and enhances the recruitment of quiescent human fibroblasts to the proliferation cycle during wound healing.

    Science.gov (United States)

    Mao, Gaowei; Goswami, Monali; Kalen, Amanda L; Goswami, Prabhat C; Sarsour, Ehab H

    2016-01-01

    The rebuilding of the connective tissue during wound healing requires the recruitment of fibroblasts to the wound area as well as reentry of quiescent fibroblasts to the proliferative cycle. Whether this process can be modulated by a small molecular weight thiol antioxidant N-acetyl-L-cysteine (NAC) was tested in normal human skin fibroblasts (NHFs) using a uni-directional wound healing assay. NAC treated cells demonstrated a decreased migration rate but increased number of proliferating cells recruited into the wound area post wounding. Fifteen day quiescent control and NAC treated NHFs were re-plated at a lower density and cell numbers counted at different days post-plating. Interestingly, NAC treated cells exhibited increased cellular proliferation indicated by both decreased cell population doubling time and increased S phase cells. NAC treated cells demonstrated decreased steady state levels of reactive oxygen species as well as increased protein and activity levels of manganese superoxide dismutase (MnSOD). NAC treatment failed to induce proliferation in quiescent cells lacking MnSOD expression. These results demonstrate that NAC enhanced the recruitment of quiescent NHFs into proliferation cycle during wound healing. Our results also suggest that the wound healing properties of NAC might be due to its ability to induce and enhance MnSOD expression and activity. Altogether, these findings suggest NAC might be potentially developed as a dietary intervention to improve tissue injury in animals and humans.

  17. The potential of a niacinamide dominated cosmeceutical formulation on fibroblast activity and wound healing in vitro.

    Science.gov (United States)

    Wessels, Quenton; Pretorius, Etheresia; Smith, Celeste M; Nel, Hugo

    2014-04-01

    Knowledge on the intrinsic mechanisms involved in wound healing provides opportunity for various therapeutic strategies. The manipulation of dermal fibroblast proliferation and differentiation might prove to beneficially augment wound healing. This study evaluated the combined effects of niacinamide, L-carnosine, hesperidin and Biofactor HSP(®) on fibroblast activity. The effects on fibroblast collagen production, cellular proliferation, migration and terminal differentiation were assessed. In addition, the authors determined the effects on in vitro wound healing. The optimal concentrations of actives were determined in vitro. Testing parameters included microscopic morphological cell analysis, cell viability and proliferation determination, calorimetric collagen detection and in vitro wound healing dynamics. Results show that 0·31 mg/ml niacinamide, 0·10 mg/ml L-carnosine, 0·05 mg/ml hesperidin and 5·18 µg/ml Biofactor HSP® proved optimal in vitro. The results show that fibroblast collagen synthesis was increased alongside with cellular migration and proliferation.

  18. Anti-proliferative and antioxidative activities of Thai noni/Yor (Morinda citrifolia Linn.) leaf extract.

    Science.gov (United States)

    Thani, Wasina; Vallisuta, Omboon; Siripong, Pongpan; Ruangwises, Nongluck

    2010-03-01

    In this study the leaves of the Thai noni/Yor, (Morinda citrifolia Linn.) were extracted by several methods and evaluated against human cancer cell lines: KB (human epidermoid carcinoma), HeLa (human cervical carcinoma), MCF-7 (human breast carcinoma) and HepG2 (human hepatocellular carcinoma) cell lines as well as a Vero (African green monkey kidney) cell line, employing the MTT colorimetric method, comparing it to damnacanthal, rutin, and scopoletin. The dichloromethane extract of the fresh leaf showed a better inhibitory effect against KB and HeLa cells with IC50 values of 21.67 and 68.50 microg/ml, respectively. The dichloromethane extract of dried leaves revealed cytotoxicity against the KB cell line with an IC50 value of 39.00 microg/ml. Other extracts, as well as rutin and scopoletin, showed reduced anti-proliferative effects on all cancer cell lines (IC50 103 to over 600 microg/ml). Interestingly, the damnacanthal had potent cytotoxicity against all cancer cell lines and Vero cell lines. These results suggest Thai noni extracts may be safer than the pure compounds, due to their higher safety ratios, which is a good indicator for possible cancer treatment. Several non-aqueous extracts from the leaves showed antioxidant properties, giving IC50 values of 0.20-0.35 mg/ml. It can be concluded the leaves of M. citrifolia may have benefit as a food supplement for chemoprevention against epidermoid and cervical cancers.

  19. Proliferative index activity in oral squamous cell carcinoma: indication for postoperative radiotherapy?

    Science.gov (United States)

    Gontarz, M; Wyszyńska-Pawelec, G; Zapała, J; Czopek, J; Lazar, A; Tomaszewska, R

    2014-10-01

    The predictive value of the Ki-67 labelling index and its relationship with radiosensitivity in oral squamous cell carcinoma (SCC) remains controversial. We sought to evaluate whether the expression of Ki-67 antigen found in SCC of the tongue and the floor of the mouth is an indication for postoperative radiotherapy (PORT). The first study group included 34 patients who were treated only with primary surgery, while the second group included 26 patients who underwent primary surgery combined with PORT. The correlation between Ki-67 expression and loco-regional recurrence, as well as the 5-year disease-specific survival, was assessed in the two groups. Cases of high-proliferative tumours showed a significantly higher risk of loco-regional recurrence (P=0.018) and a poorer prognosis (P=0.001) only in the 34 patients treated with surgery alone. In multivariate Cox regression analysis, high Ki-67 expression was an independent predictor of loco-regional recurrence (HR 5.42, P=0.029) and disease-specific survival (HR 9.02, P=0.004). The correlation between Ki-67 expression and the risk of loco-regional recurrence in SCC of the tongue and the floor of the mouth may be useful in the selection of patients at a higher risk of recurrence who would benefit from PORT, despite adequate margins of resection and early stage of the disease.

  20. Mitogenic and anti-proliferative activity of a lectin from the tubers of Voodoo lily (Sauromatum venosum).

    Science.gov (United States)

    Singh Bains, Jagmohan; Singh, Jatinder; Kamboj, Sukhdev Singh; Nijjar, Kamaljeet Kaur; Agrewala, Javed N; Kumar, Vinod; Kumar, Ashok; Saxena, A K

    2005-05-25

    A new lectin with the potent mitogenic and in vitro anti-proliferative activity was isolated from the tubers of a wild monocotyledonous plant Sauromatum venosum (Schott), from the family Araceae, by affinity chromatography on the asialofetuin linked amino-activated silica beads. The apparent native molecular mass of S. venosum lectin (SVL), as determined by gel filtration chromatography, was 54 kDa. In HPLC, size exclusion and cation exchange chromatography, SVL gave a single peak and also a single band of 13.5 kDa in SDS-PAGE, pH 8.3, under reducing and non-reducing conditions, indicating that the lectin is composed of four identical subunits. S. venosum lectin agglutinated rabbit, rat, sheep and guinea pig erythrocytes but reacted with goat erythrocytes after the neuraminidase treatment. However, SVL was unable to agglutinate human ABO blood group erythrocytes even after treatment with neuraminidase. SVL was inhibited by N-acetyl-D-Lactosamine (LacNAc), which is an important marker in various carcinomas and a complex desialylated glycoprotein, asialofetuin. The amino acid composition showed that lectin contained a high amount of aspartic acid and glycine but totally devoid of cysteine. However, trace amounts of methionine was present. The lectin showed a potent mitogenic response towards BALB/c splenocytes and human lymphocytes. As the mitogenic stimulation was more than that of Con A, a standard well-known plant mitogen and the response of this lectin was almost double than that of Con A. This lectin is endowed with proliferation of T cells as revealed by IL-2 bioassay but showed no production of immunoglobulins thus indicating the non-stimulation of B cells. SVL significantly inhibited the proliferation of murine cancer cell-lines, i.e., WEHI-279 to 84.6%, J774 to 81%, P388D1 to 74% and A-20 to 47%. In addition, the in vitro anti-proliferative activity of SVL was also evaluated against nine human cancer cell lines representing different organs and tissues

  1. Increased Stiffness in Aged Skeletal Muscle Impairs Muscle Progenitor Cell Proliferative Activity.

    Directory of Open Access Journals (Sweden)

    Grégory Lacraz

    Full Text Available Skeletal muscle aging is associated with a decreased regenerative potential due to the loss of function of endogenous stem cells or myogenic progenitor cells (MPCs. Aged skeletal muscle is characterized by the deposition of extracellular matrix (ECM, which in turn influences the biomechanical properties of myofibers by increasing their stiffness. Since the stiffness of the MPC microenvironment directly impacts MPC function, we hypothesized that the increase in muscle stiffness that occurs with aging impairs the behavior of MPCs, ultimately leading to a decrease in regenerative potential.We showed that freshly isolated individual myofibers from aged mouse muscles contain fewer MPCs overall than myofibers from adult muscles, with fewer quiescent MPCs and more proliferative and differentiating MPCs. We observed alterations in cultured MPC behavior in aged animals, where the proliferation and differentiation of MPCs were lower and higher, respectively. These alterations were not linked to the intrinsic properties of aged myofibers, as shown by the similar values for the cumulative population-doubling values and fusion indexes. However, atomic force microscopy (AFM indentation experiments revealed a nearly 4-fold increase in the stiffness of the MPC microenvironment. We further showed that the increase in stiffness is associated with alterations to muscle ECM, including the accumulation of collagen, which was correlated with higher hydroxyproline and advanced glycation end-product content. Lastly, we recapitulated the impaired MPC behavior observed in aging using a hydrogel substrate that mimics the stiffness of myofibers.These findings provide novel evidence that the low regenerative potential of aged skeletal muscle is independent of intrinsic MPC properties but is related to the increase in the stiffness of the MPC microenvironment.

  2. Evaluation of the proliferative activity of immunocompetent cells in the jejunal and iliac lymph nodes of prepubertal female wild boars diagnosed with mixed mycotoxicosis

    Directory of Open Access Journals (Sweden)

    Zielonka Łukasz

    2015-06-01

    Full Text Available The study evaluated the proliferative activity of immunocompetent cells in the jejunal and iliac lymph nodes of prepubertal female wild boars exposed to deoxynivalenol and zearalenone in naturally contaminated feed. The evaluation was performed with the use of the MTT assay and 2 mitogens: lipopolysaccharide (LPS and concanavalin A. Intensified proliferative processes in T and B lymphocytes were revealed. The mitogenic activity of LPS was more expressed in the lymphocytes of both iliac and jejunal lymph nodes in comparison with the control group. Proliferative activity was higher in iliac lymph nodes than in jejunal lymph nodes. A reverse trend was observed in the percentage of live cells, which was higher in jejunal lymph nodes during the evaluation of lymphocyte proliferation.

  3. Proliferative and anti-proliferative effects of dietary levels of phytoestrogens in rat pituitary GH3/B6/F10 cells - the involvement of rapidly activated kinases and caspases

    Directory of Open Access Journals (Sweden)

    Watson Cheryl S

    2009-09-01

    Full Text Available Abstract Background Phytoestogens are a group of lipophillic plant compounds that can have estrogenic effects in animals; both tumorigenic and anti-tumorigenic effects have been reported. Prolactin-secreting adenomas are the most prevalent form of pituitary tumors in humans and have been linked to estrogen exposures. We examined the proliferative effects of phytoestrogens on a rat pituitary tumor cell line, GH3/B6/F10, originally subcloned from GH3 cells based on its ability to express high levels of the membrane estrogen receptor-α. Methods We measured the proliferative effects of these phytoestrogens using crystal violet staining, the activation of several mitogen-activated protein kinases (MAPKs and their downstream targets via a quantitative plate immunoassay, and caspase enzymatic activities. Results Four phytoestrogens (coumestrol, daidzein, genistein, and trans-resveratrol were studied over wide concentration ranges. Except trans-resveratrol, all phytoestrogens increased GH3/B6/F10 cell proliferation at some concentration relevant to dietary levels. All four phytoestrogens attenuated the proliferative effects of estradiol when administered simultaneously. All phytoestrogens elicited MAPK and downstream target activations, but with time course patterns that often differed from that of estradiol and each other. Using selective antagonists, we determined that MAPKs play a role in the ability of these phytoestrogens to elicit these responses. In addition, except for trans-resveratrol, a serum removal-induced extrinsic apoptotic pathway was blocked by these phytoestrogens. Conclusion Phytoestrogens can block physiological estrogen-induced tumor cell growth in vitro and can also stimulate growth at high dietary concentrations in the absence of endogenous estrogens; these actions are correlated with slightly different signaling response patterns. Consumption of these compounds should be considered in strategies to control endocrine tumor cell

  4. Investigation of cell proliferative activity on the surface of the nanocomposite material produced by laser radiation

    Science.gov (United States)

    Zhurbina, N. N.; Kurilova, U. E.; Ickitidze, L. P.; Podgaetsky, V. M.; Selishchev, S. V.; Suetina, I. A.; Mezentseva, M. V.; Eganova, E. M.; Pavlov, A. A.; Gerasimenko, A. Y.

    2016-04-01

    A new method for the formation of composite nanomaterials based on multi-walled and single-walled carbon nanotubes (CNT) on a silicon substrate has been developed. Formation is carried out by ultrasound coating of a silicon substrate by homogenous dispersion of CNTs in the albumin matrix and further irradiation with the continuous laser beam with a wavelength of 810 nm and power of 5.5 watts. The high electrical conductivity of CNTs provides its structuring under the influence of the laser radiation electric field. The result is a scaffold that provides high mechanical strength of nanocomposite material (250 MPa). For in vitro studies of materials biocompatibility a method of cell growth microscopic analysis was developed. Human embryonic fibroblasts (EPP) were used as biological cells. Investigation of the interaction between nanocomposite material and cells was carried out by optical and atomic force microscopy depending on the time of cells incubation. The study showed that after 3 hours incubation EPP were fixed on the substrate surface, avoiding the surface of the composite material. However, after 24 hours of incubation EPP fix on the sample surface and then begin to grow and divide. After 72 hours of incubation, the cells completely fill the sample surface of nanocomposite material. Thus, a nanocomposite material based on CNTs in albumin matrix does not inhibit cell growth on its surface, and favours their growth. The nanocomposite material can be used for creating soft tissue implants

  5. EGb-761 Attenuates the Anti-proliferative Activity of Fluoride via DDK1 in PC-12 Cells.

    Science.gov (United States)

    Zhang, Cai-Yi; Chen, Rui; Wang, Fen; Ren, Chao; Zhang, Peng; Li, Qian; Li, Hui-Hua; Guo, Ke-Tai; Geng, De-Qin; Liu, Chun-Feng

    2017-02-01

    EGb-761 is commonly used as a treatment for ischemic brain injury, neurodegenerative diseases and some types of tumors (Christen and Maixent, in Cell Mol Biol 48(6):601-611, 2002). However, it is unclear whether EGb-761 affects the proliferation of cells exposed to fluoride. In this study, the proliferation and apoptosis of PC-12 cells exposed to fluoride were investigated and EGb-761 was used to protect PC-12 cells against the effects of fluoride. We found that the canonical Wnt signaling pathway was involved in the anti-proliferation of PC-12 cells exposed to fluoride. Furthermore, the results also showed that EGb-761 could attenuate the anti-proliferative activity of fluoride via DDK1 in PC-12 cells. This study may provide a new method for protecting against the inhibition of cell proliferation induced by fluoride.

  6. Amniocar as a proliferative medium for mesenchymal cells

    Directory of Open Access Journals (Sweden)

    V. V. Chestkov

    2014-01-01

    Full Text Available Objectives. To develop the Amniocar nutrient medium that contains fetal calf serum (FCS and growth factors cocktail for mass cultivation of human fibroblasts. To study proliferative activity of the medium on cultures of HUVEC cells of mesenchymal origin and mesenchymal stromal cells, as well as on cell culture of human amniotic fluid.Materials and methods. Determination of the rate of accumulation of the cellular mass and cell morphology in the course of cultivation of cells of various histogenesis in the Amniocar medium and nutrient medium that contains 10 % of FCS.Results. It has been demonstrated that the Amniocar medium is prevalent as compared to the standard DMEM medium with 10 % of FCS by 2 to 5 times for cultivation of skin fibroblasts, HUVEC, and mesenchymal stem cells. The Amniocar medium increased the quantity of endothelial cells that enter mitosis and maintained the culture of HUVEC cells with prolonged passaging in vitro. Clonal cultivation of human amniotic fluid cells in the Amniocar medium secured development of colonies of both fibroblast and epithelial type.Conclusions. Proliferative Amniocar medium is efficient for mass cultivation of various cells of mesenchymal origin and can be used for diagnostic purposes in medical genetics, oncology, etc.

  7. Relationship between proliferative activity of cancer cells and clinicopathological factors in patients with esophageal squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Jun-Xing Huang; Wei Yan; Zheng-Xiang Song; Rong-Yu Qian; Ping Chen; Eeva Salminen; Jorma Toppari

    2005-01-01

    AIM: To assess whether the molecular markers of malignant tumors could improve the understanding of tumor characteristics, and to observe the characteristics of expression of cell cycle markers Ki-67 and cydin A in esophageal carcinoma and to analyze the relationship between proliferative activity of cancer cells and clinicopathological factors.METHODS: Seventy of surgically resected esophageal squamous cell carcinoma (SCC) were examined by immunohistochemistry utilizing commercially available antibodies. Nuclear staining was regarded as a positive result. At least 50 fields in each tumor and non-tumor section were evaluated at a medium power (x200) to determine the proportion of tumor cells and the staining intensity of nuclei in the entire sections.RESULTS: Ki-67 and cyclin A were only expressed in base cells of normal esophageal mucosa. The positive immunostaining of nuclei of SCC was significantly higher than that in normal esophageal mucosa (t= 13.32 and t= 7.52,respectively, P<0.01). The distribution of positively stained was more diffuse and stronger in poorly differentiated SCC. Both Ki-67 and cyclin A expressions were related to histological grades of tumors (t = 3.5675 and t = 3.916; t= 2.13, respectively, P<0.05) but not to the sex and age of the patients, tumor size, lymphatic invasion, location, or stage grouping.CONCLUSION: The proliferative activity of cancer cells may be understood by immunohistochemistry of Ki-67 and cyclin A in Chinese patients with esophageal SCC. These cell cycle markers may serve as an indicator of cancer cell proliferation rate. The overexpression of cell cycle markers Ki-67 and cyclin A suggests the poor SCC differentiation in patients with esophageal carcinoma.

  8. Oleic, Linoleic and Linolenic Acids Increase ROS Production by Fibroblasts via NADPH Oxidase Activation

    Science.gov (United States)

    Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurélio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui

    2013-01-01

    The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47phox mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts. PMID:23579616

  9. Centrifugal force induces human ligamentum flavum fibroblasts inflammation through activation of JNK and p38 pathways.

    Science.gov (United States)

    Chao, Yuan-Hung; Tsuang, Yang-Hwei; Sun, Jui-Sheng; Sun, Man-Ger; Chen, Ming-Hong

    2012-01-01

    Inflammation has been proposed to be an important causative factor in ligamentum flavum hypertrophy. However, the mechanisms of mechanical load on inflammation of ligamentum flavum remain unclear. In this study, we used an in vitro model of human ligamentum flavum fibroblasts subjected to centrifugal force to elucidate the effects of mechanical load on cultured human ligamentum flavum fibroblasts; we further studied its molecular and biochemical mechanisms. Human ligamentum flavum fibroblasts were obtained from six patients undergoing lumbar spine surgery. Monolayer cultures of human ligamentum flavum fibroblasts were subjected to different magnitudes of centrifugal forces. Cell viability, cell death, biochemical response, and molecular response to centrifugal forces were analyzed. It was found that centrifugal stress significantly suppressed cell viability without inducing cell death. Centrifugal force at 67.1 g/cm(2) for 60 min significantly increases the production of prostaglandin E2 and nitric oxide as well as gene expression of proinflammatory cytokines, including interleukin (IL)-1α, IL-1β and IL-6, showed that centrifugal force-dependent induction of cyclooxygense-2 and inducible NO synthase required JNK and p38 mitogen-activated protein kinase, but not ERK 1/2 activities. This study suggested that centrifugal force does induce inflammatory responses in human ligamentum flavum fibroblasts. The activation of both JNK and p38 mitogen-activated protein kinase mechanotransduction cascades is a crucial intracellular mechanism that mediates cyclooxygense-2/prostaglandin E2 and inducible NO synthase/nitric oxide production.

  10. Proliferative activity, lectin-dependent and natural cytotoxicity in blood, lymph node and spleen from patients with Hodgkin's disease.

    Science.gov (United States)

    Bykovskaya, S N; Blochina, N G; Charabadze, M V; Agaphonov, V A; Kupriyanova, T A

    1990-01-01

    Mononuclear cells and T-lymphocytes of the blood, spleen and lymph nodes from 48 patients with Hodgkin disease (HD) and blood donors were tested in assays for lectin-dependent (LD) and natural killer (NK) cytotoxic activity. On average, peripheral blood T cell lectin-dependent cytotoxicity differs from that of the donors. However, cytotoxic activity appears to be dependent on the stage of disease; in the IY stage LD cytotoxicity was decreased 2-fold. The lectin-dependent cytotoxicity was also dependent on the histological type of disease and the lowest level (50% of the control level) was associated with the lymphoid depletion type. The cytotoxic activity of T-lymphocytes from the affected areas of the patients' spleen was more marked than that of the unaffected areas. Spleen cell cytotoxicity showed no other correlations. Cytotoxicity of lymphocytes from the affected lymph nodes was drastically lower than activity of blood and spleen lymphocytes. NK activity of the patients' blood and spleen lymphocytes was twice as low as the control level (healthy donors) and did not correlate with stage and/or histological type of disease. The proliferative activity of lymphocytes from 33 HD patients was tested in vitro using allogeneic mononuclear cells from healthy donors or HD patients and/or PHA as stimulators. The response of patients' lymphocytes to alloantigens appeared to be much less affected than response to polyclonal mitogen. Thus, the results obtained by us demonstrate signs of stimulation of the lymphoid system against a background of general immunosuppression in HD.

  11. Diabetic Nephropathy: The Role of Inflammation in Fibroblasts Activation and Kidney Fibrosis

    Directory of Open Access Journals (Sweden)

    Keizo eKanasaki

    2013-02-01

    Full Text Available Kidney disease associated with diabetes mellitus is a major health problem worldwide. Although established therapeutic strategies, such as appropriate blood glucose control, blood pressure control with renin-angiotensin system blockade and lipid lowering with statins, are used to treat diabetes, the contribution of diabetic end-stage kidney disease to the total number of cases requiring hemodialysis has increased tremendously in the past two decades. Once renal function starts declining, it can result in a higher frequency of renal and extra-renal events, including cardiovascular events. Therefore, slowing renal function decline is one of the main areas of focus in diabetic nephropathy research, and novel strategies are urgently needed to prevent diabetic kidney disease progression. Regardless of the type of injury and etiology, kidney fibrosis is the commonly the final outcome of progressive kidney diseases, and it results in significant destruction of normal kidney structure and accompanying functional deterioration. Kidney fibrosis is caused by prolonged injury and dysregulation of the normal wound-healing process in association with excess extracellular matrix deposition. Kidney fibroblasts play an important role in the fibrotic process, but the origin of the fibroblasts remains elusive. In addition to the activation of residential fibroblasts, other important sources of fibroblasts have been proposed, such as pericytes, fibrocytes and fibroblasts originating from epithelial and endothelial mesenchymal transition. Inflammatory cells and cytokines play a vital role In the process of fibroblast activation. In this review, we will analyze the contribution of inflammation to the process of tissue fibrosis, the type of fibroblast activation and the therapeutic strategies targeting the inflammatory pathways in an effort to slow the progression of diabetic kidney disease.

  12. Diabetic nephropathy: the role of inflammation in fibroblast activation and kidney fibrosis.

    Science.gov (United States)

    Kanasaki, Keizo; Taduri, Gangadhar; Koya, Daisuke

    2013-01-01

    Kidney disease associated with diabetes mellitus is a major health problem worldwide. Although established therapeutic strategies, such as appropriate blood glucose control, blood pressure control with renin-angiotensin system blockade, and lipid lowering with statins, are used to treat diabetes, the contribution of diabetic end-stage kidney disease to the total number of cases requiring hemodialysis has increased tremendously in the past two decades. Once renal function starts declining, it can result in a higher frequency of renal and extra-renal events, including cardiovascular events. Therefore, slowing renal function decline is one of the main areas of focus in diabetic nephropathy research, and novel strategies are urgently needed to prevent diabetic kidney disease progression. Regardless of the type of injury and etiology, kidney fibrosis is the commonly the final outcome of progressive kidney diseases, and it results in significant destruction of normal kidney structure and accompanying functional deterioration. Kidney fibrosis is caused by prolonged injury and dysregulation of the normal wound-healing process in association with excess extracellular matrix deposition. Kidney fibroblasts play an important role in the fibrotic process, but the origin of the fibroblasts remains elusive. In addition to the activation of residential fibroblasts, other important sources of fibroblasts have been proposed, such as pericytes, fibrocytes, and fibroblasts originating from epithelial-to-mesenchymal and endothelial-to-mesenchymal transition. Inflammatory cells and cytokines play a vital role In the process of fibroblast activation. In this review, we will analyze the contribution of inflammation to the process of tissue fibrosis, the type of fibroblast activation and the therapeutic strategies targeting the inflammatory pathways in an effort to slow the progression of diabetic kidney disease.

  13. Anti-proliferative and apoptosis-inducing activity of lycopene against three subtypes of human breast cancer cell lines.

    Science.gov (United States)

    Takeshima, Mikako; Ono, Misaki; Higuchi, Takako; Chen, Chen; Hara, Takayuki; Nakano, Shuji

    2014-03-01

    Although lycopene, a major carotenoid component of tomatoes, has been suggested to attenuate the risk of breast cancer, the underlying preventive mechanism remains to be determined. Moreover, it is not known whether there are any differences in lycopene activity among different subtypes of human breast cancer cells. Using ER/PR positive MCF-7, HER2-positive SK-BR-3 and triple-negative MDA-MB-468 cell lines, we investigated the cellular and molecular mechanism of the anticancer activity of lycopene. Lycopene treatment for 168 consecutive hours exhibited a time-dependent and dose-dependent anti-proliferative activity against these cell lines by arresting the cell cycle at the G0 /G1 phase at physiologically achievable concentrations found in human plasma. The greatest growth inhibition was observed in MDA-MB-468 where the sub-G0 /G1 apoptotic population was significantly increased, with demonstrable cleavage of PARP. Lycopene induced strong and sustained activation of the ERK1/2, with concomitant cyclin D1 suppression and p21 upregulation in these three cell lines. In triple negative cells, lycopene inhibited the phosphorylation of Akt and its downstream molecule mTOR, followed by subsequent upregulation of proapoptotic Bax without affecting anti-apoptotic Bcl-xL. Taken together, these data indicate that the predominant anticancer activity of lycopene in MDA-MB-468 cells suggests a potential role of lycopene for the prevention of triple negative breast cancer.

  14. Phytochemical profile of Rosmarinus officinalis and Salvia officinalis extracts and correlation to their antioxidant and anti-proliferative activity.

    Science.gov (United States)

    Kontogianni, Vassiliki G; Tomic, Goran; Nikolic, Ivana; Nerantzaki, Alexandra A; Sayyad, Nisar; Stosic-Grujicic, Stanislava; Stojanovic, Ivana; Gerothanassis, Ioannis P; Tzakos, Andreas G

    2013-01-01

    The goal of this study was to monitor the anti-proliferative activity of Rosmarinus officinalis and Salvia officinalis extracts against cancer cells and to correlate this activity with their phytochemical profiles using liquid chromatography/diode array detection/electrospray ion trap tandem mass spectrometry (LC/DAD/ESI-MS(n)). For the quantitative estimation of triterpenic acids in the crude extracts an NMR based methodology was used and compared with the HPLC measurements, both applied for the first time, for the case of betulinic acid. Both extracts exerted cytotoxic activity through dose-dependent impairment of viability and mitochondrial activity of rat insulinoma m5F (RINm5F) cells. Decrease of RINm5F viability was mediated by nitric oxide (NO)-induced apoptosis. Importantly, these extracts potentiated NO and TNF-α release from macrophages therefore enhancing their cytocidal action. The rosemary extract developed more pronounced antioxidant, cytotoxic and immunomodifying activities, probably due to the presence of betulinic acid and a higher concentration of carnosic acid in its phytochemical profile.

  15. Biological Characterization of Cynara cardunculus L. Methanolic Extracts: Antioxidant, Anti-proliferative, Anti-migratory and Anti-angiogenic Activities

    Directory of Open Access Journals (Sweden)

    Maria Duarte

    2012-12-01

    Full Text Available Cynara cardunculus (Cc is a multipurpose species; beyond its use in southwestern European cuisine, it is also used for the production of solid biofuel, seed oil, biodiesel, paper pulp and cheese, as well as animal feed. In addition, Cc has a long tradition of use in folk medicine as a diuretic and liver protector. The value of this species as a source of bioactive compounds is known; however, pharmacological use would further increase its cultivation. The main goal of the current work was to evaluate the potential of Cc as source of anti-carcinogenic phytochemicals. Different methanolic extracts obtained from wild and cultivated plants were tested for antioxidant activity and effect on breast tumor cell viability. The most effective extract, both as antioxidant and inhibition of tumor cell viability, was tested for effects on angiogenesis and tumor cell migration capacity. All the extracts tested had high antioxidant activity; however, only green leaves and dry head extracts exhibit anti-proliferative activity. Green cultivated leaves (GCL were the most effective extract both as antioxidant and inhibiting the proliferation of tumor cells; it is equally active inhibiting tumor cell migration and in vivo angiogenesis. GCL extract is an effective inhibitor of several key points in tumor development and thus a promising source of anti-carcinogenic phytochemicals.

  16. Helium generated cold plasma finely regulates activation of human fibroblast-like primary cells.

    Directory of Open Access Journals (Sweden)

    Paola Brun

    Full Text Available Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2',7'-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and

  17. Anti-Oxidative and Anti-Proliferative Activity on Human Prostate Cancer Cells Lines of the Phenolic Compounds from Corylopsis coreana Uyeki

    Directory of Open Access Journals (Sweden)

    So Ra Kim

    2013-04-01

    Full Text Available Fifteen phenolic compounds, including three caffeoyl derivatives, four gallotannins, three ellagitannins and five flavonoids, were isolated from an 80% MeOH extract of the leaves of Corylopsis coreana Uyeki (Korean winter hazel; CL. The anti-oxidative activities [1,1-diphenyl-2-picrylhydrazyl (DPPH radical scavenging activity and xanthine oxidase superoxide scavenging activities (NBT] and the anti-proliferative activity on human prostate cancer cell lines (DU145 and LNCaP were also evaluated.

  18. An interspecies conserved motif of the mouse immune system-released activating agent (ISRAA) induces proliferative effects on human cells.

    Science.gov (United States)

    Taha, Safa; Fathallah, Mohamed Dahmani; Bakhiet, Moiz

    2014-07-01

    We have recently described an immune system-released activating agent (ISRAA) as a nervous system-induced factor that stimulates immune responses in the mouse spleen. However, the human ISRAA has not yet been identified. In this study, we examined the effects of the mouse ISRAA protein on human peripheral blood mononuclear cells (PBMCs), to observe if the biological activity of this molecule is consistent between the two different species. Mouse ISRAA demonstrated dose-dependent dualistic effects on human cells, as 5 µg exhibited positive apoptosis and 50 pg exhibited significant proliferation (P<0.05). Furthermore, immunosuppressed cells from patients undergoing immunosuppressive therapy demonstrated significant proliferation to 50 pg ISRAA (P<0.05). Studies to compare sequences in different species revealed a preserved motif, exhibiting 72% similarity with the interspecies conserved signal peptide motif of tumor necrosis factor receptor 1 (TNFR1). A mutant ISRAA lacking this motif was produced and tested for its biological effects. The mutant ISRAA demonstrated neither apoptotic nor proliferative effects compared with wild type. Therefore, an interspecies conserved domain of ISRAA constitutes the active site of the molecule, and its effects on immunocompromised cells should be investigated for future therapies in the treatment of immunosuppressive disorders.

  19. The prognostic value of oncogenic antigen 519 (OA-519) expression and proliferative activity detected by antibody MIB-1 in node-negative breast cancer

    DEFF Research Database (Denmark)

    Jensen, V; Ladekarl, M; Holm-Nielsen, P

    1995-01-01

    of invasion of skin or deep fascia (= T1N0M0 and T2N0M0). The median follow-up time was 104 months (range 5-143 months). Immunohistochemical analysis of OA-519 expression was performed on formalin-fixed, paraffin-embedded tissue. The proliferative activity was estimated using a Ki-67 equivalent monoclonal...

  20. Immunomodulator CD200 promotes neurotrophic activity by interacting with and activating the fibroblast growth factor receptor

    DEFF Research Database (Denmark)

    Pankratova, Stanislava; Bjornsdottir, Halla; Christensen, Claus;

    2016-01-01

    in the suppression of microglia activation. We for the first time demonstrated that CD200 can interact with and transduce signaling through activation of the fibroblast growth factor receptor (FGFR), thereby inducing neuritogenesis and promoting neuronal survival in primary neurons. CD200-induced FGFR...... phosphorylation was abrogated by CD200R, whereas FGF2-induced FGFR activation was inhibited by CD200. We also identified a sequence motif located in the first Ig-like module of CD200, likely representing the minimal CD200 binding site for FGFR. The FGFR binding motif overlaps with the CD200R binding site......, suggesting that they can compete for CD200 binding in cells that express both receptors. We propose that CD200 in neurons functions as a ligand of FGFR....

  1. Prolonged effect of fluid flow stress on the proliferative activity of mesothelial cells after abrupt discontinuation of fluid streaming

    Energy Technology Data Exchange (ETDEWEB)

    Aoki, Shigehisa, E-mail: aokis@cc.saga-u.ac.jp [Department of Pathology and Microbiology, Faculty of Medicine, Saga University, Saga (Japan); Ikeda, Satoshi [Department of Pathology and Microbiology, Faculty of Medicine, Saga University, Saga (Japan); Takezawa, Toshiaki [Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Ibaraki (Japan); Kishi, Tomoya [Department of Internal Medicine, Saga University, Saga (Japan); Makino, Junichi [Makino Clinic, Saga (Japan); Uchihashi, Kazuyoshi; Matsunobu, Aki [Department of Pathology and Microbiology, Faculty of Medicine, Saga University, Saga (Japan); Noguchi, Mitsuru [Department of Urology, Faculty of Medicine, Saga University, Saga (Japan); Sugihara, Hajime [Department of Physical Therapy, International University of Health and Welfare, Fukuoka (Japan); Toda, Shuji [Department of Pathology and Microbiology, Faculty of Medicine, Saga University, Saga (Japan)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Late-onset peritoneal fibrosis leading to EPS remains to be elucidated. Black-Right-Pointing-Pointer Fluid streaming is a potent factor for peritoneal fibrosis in PD. Black-Right-Pointing-Pointer We focused on the prolonged effect of fluid streaming on mesothelial cell kinetics. Black-Right-Pointing-Pointer A history of fluid streaming exposure promoted mesothelial proliferative activity. Black-Right-Pointing-Pointer We have thus identified a potent new factor for late-onset peritoneal fibrosis. -- Abstract: Encapsulating peritoneal sclerosis (EPS) often develops after transfer to hemodialysis and transplantation. Both termination of peritoneal dialysis (PD) and transplantation-related factors are risks implicated in post-PD development of EPS, but the precise mechanism of this late-onset peritoneal fibrosis remains to be elucidated. We previously demonstrated that fluid flow stress induced mesothelial proliferation and epithelial-mesenchymal transition via mitogen-activated protein kinase (MAPK) signaling. Therefore, we speculated that the prolonged bioactive effect of fluid flow stress may affect mesothelial cell kinetics after cessation of fluid streaming. To investigate how long mesothelial cells stay under the bioactive effect brought on by fluid flow stress after removal of the stress, we initially cultured mesothelial cells under fluid flow stress and then cultured the cells under static conditions. Mesothelial cells exposed to fluid flow stress for a certain time showed significantly high proliferative activity compared with static conditions after stoppage of fluid streaming. The expression levels of protein phosphatase 2A, which dephosphorylates MAPK, in mesothelial cells changed with time and showed a biphasic pattern that was dependent on the duration of exposure to fluid flow stress. There were no differences in the fluid flow stress-related bioactive effects on mesothelial cells once a certain time had passed

  2. Fibroblasts in myocardial infarction: a role in inflammation and repair

    Science.gov (United States)

    Shinde, Arti V.; Frangogiannis, Nikolaos G.

    2014-01-01

    Fibroblasts do not only serve as matrix-producing reparative cells, but exhibit a wide range of functions in inflammatory and immune responses, angiogenesis and neoplasia. The adult mammalian myocardium contains abundant fibroblasts enmeshed within the interstitial and perivascular extracellular matrix. The current review manuscript discusses the dynamic phenotypic and functional alterations of cardiac fibroblasts following myocardial infarction. Extensive necrosis of cardiomyocytes in the infarcted heart triggers an intense inflammatory reaction. In the early stages of infarct healing, fibroblasts become pro-inflammatory cells, activating the inflammasome and producing cytokines, chemokines and proteases. Pro-inflammatory cytokines (such as Interleukin-1) delay myofibroblast transformation, until the wound is cleared from dead cells and matrix debris. Resolution of the inflammatory infiltrate is associated with fibroblast migration, proliferation, matrix protein synthesis and myofibroblast conversion. Growth factors and matricellular proteins play an important role in myofibroblast activation during the proliferative phase of healing. Formation of a mature cross-linked scar is associated with clearance of fibroblasts, as poorly-understood inhibitory signals restrain the fibrotic response. However, in the non-infarcted remodeling myocardium, local fibroblasts may remain activated in response to volume and pressure overload and may promote interstitial fibrosis. Considering their abundance, their crucial role in cardiac inflammation and repair, and their involvement in myocardial dysfunction and arrhythmogenesis, cardiac fibroblasts may be key therapeutic targets in cardiac remodeling. PMID:24321195

  3. Pulsed Electromagnetic Field Stimulation Promotes Anti-cell Proliferative Activity in Doxorubicin-treated Mouse Osteosarcoma Cells.

    Science.gov (United States)

    Muramatsu, Yoshitaka; Matsui, Takuya; Deie, Masataka; Sato, Keiji

    2017-01-02

    We aimed to investigate the synergistic effects of pulsed electromagnetic field (PEMF) and doxorubicin therapy in a mouse osteosarcoma cell line (LM8 cells) in vitro. The effects of PEMF (5 mT, 200 Hz) of different durations and doxorubicin on the proliferative activity of LM8 cells were measured by the MTT assay. Apoptotic-related factors such as cell-cycle phase, mitochondrial membrane potential, and caspase 3/7 activity were investigated using 4',6-diamidino-2-phenylindole staining and apoptosis kits. Identification of intracellular signaling molecules induced by the combination was comprehensively explored using a stress and apoptosis-related protein array kit. PEMF enhanced the inhibition of cell proliferation mediated by doxorubicin but did not affect the cell cycle, mitochondrial membrane potential, or doxorubicin-induced G2/M arrest. The combination of PEMF and doxorubicin altered a few signaling molecules. PEMF tended to reduce the doxorubicin-induced decrease of phosphorylated BAD, while reducing the increased expression of total IĸB and phosphorylated-CHK1 induced by doxorubicin. Our results indicate that combination of PEMF and doxorubicin could be a novel chemotherapeutic strategy. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  4. A novel microtubule inhibitor 4SC-207 with anti-proliferative activity in taxane-resistant cells.

    Directory of Open Access Journals (Sweden)

    Elena Bausch

    Full Text Available Microtubule inhibitors are invaluable tools in cancer chemotherapy: taxanes and vinca alkaloids have been successfully used in the clinic over the past thirty years against a broad range of tumors. However, two factors have limited the effectiveness of microtubule inhibitors: toxicity and resistance. In particular, the latter is highly unpredictable, variable from patient to patient and is believed to be the cause of treatment failure in most cases of metastatic cancers. For these reasons, there is an increasing demand for new microtubule inhibitors that can overcome resistance mechanisms and that, at the same time, have reduced side effects. Here we present a novel microtubule inhibitor, 4SC-207, which shows strong anti-proliferative activity in a large panel of tumor cell lines with an average GI50 of 11 nM. In particular, 4SC-207 is active in multi-drug resistant cell lines, such as HCT-15 and ACHN, suggesting that it is a poor substrate for drug efflux pumps. 4SC-207 inhibits microtubule growth in vitro and in vivo and promotes, in a dose dependent manner, a mitotic delay/arrest, followed by apoptosis or aberrant divisions due to chromosome alignment defects and formation of multi-polar spindles. Furthermore, preliminary data from preclinical studies suggest low propensity towards bone marrow toxicities at concentrations that inhibit tumor growth in paclitaxel-resistant xenograft models. In summary, our results suggest that 4SC-207 may be a potential anti-cancer agent.

  5. Local induction of pacemaking activity in a monolayer of electrically coupled quiescent NRK fibroblasts

    NARCIS (Netherlands)

    Dernison, M.M.; Kusters, J.M.A.M.; Peters, P.H.J.; Meerwijk, W.P. van; Ypey, D.L.; Gielen, C.C.A.M.; Zoelen, E.J.J. van; Theuvenet, A.P.R.

    2008-01-01

    Cultures of normal rat kidney (NRK) fibroblasts may display spontaneous calcium action potentials which propagate throughout the cellular monolayer. Pacemaking activity of NRK cells was studied by patch clamp electrophysiology and vital calcium imaging, using a new experimental approach in which a r

  6. Resveratrol Alters Proliferative Responses and Apoptosis in Human Activated B Lymphocytes In Vitro

    Science.gov (United States)

    We hypothesized that resveratrol, a polyphenol found in grapes, peanuts, and berries would modulate B lymphocyte proliferation, immunoglobulin synthesis, and apoptosis after activation with T-cell dependent pokeweed mitogen. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of ...

  7. Association of Pro12Ala polymorphism in peroxisome proliferator activated receptor gamma with proliferative diabetic retinopathy

    NARCIS (Netherlands)

    Tariq, K.; Malik, S.B.; Ali, S.H.; Maqsood, S.E.; Azam, A.; Muslim, I.; Khan, M.S.; Azam, M.; Waheed, N.K.; Qamar, R.

    2013-01-01

    PURPOSE: The association of non-synonymous substitution polymorphism rs1801282 (c.34C>G, p.Pro12Ala) in exon 4 of the peroxisome proliferator activated receptor gamma gene with diabetic retinopathy (DR) has been reported inconsistently. Therefore, the purpose of the present study was to

  8. A Low-Level Carbon Dioxide Laser Promotes Fibroblast Proliferation and Migration through Activation of Akt, ERK, and JNK

    Science.gov (United States)

    Shingyochi, Yoshiaki; Kanazawa, Shigeyuki; Tajima, Satoshi; Tanaka, Rica; Mizuno, Hiroshi; Tobita, Morikuni

    2017-01-01

    Background Low-level laser therapy (LLLT) with various types of lasers promotes fibroblast proliferation and migration during the process of wound healing. Although LLLT with a carbon dioxide (CO2) laser was also reported to promote wound healing, the underlying mechanisms at the cellular level have not been previously described. Herein, we investigated the effect of LLLT with a CO2 laser on fibroblast proliferation and migration. Materials and Methods Cultured human dermal fibroblasts were prepared. MTS and cell migration assays were performed with fibroblasts after LLLT with a CO2 laser at various doses (0.1, 0.5, 1.0, 2.0, or 5.0 J/cm2) to observe the effects of LLLT with a CO2 laser on the proliferation and migration of fibroblasts. The non-irradiated group served as the control. Moreover, western blot analysis was performed using fibroblasts after LLLT with a CO2 laser to analyze changes in the activities of Akt, extracellular signal-regulated kinase (ERK), and Jun N-terminal kinase (JNK), which are signaling molecules associated with cell proliferation and migration. Finally, the MTS assay, a cell migration assay, and western blot analysis were performed using fibroblasts treated with inhibitors of Akt, ERK, or JNK before LLLT with a CO2 laser. Results In MTS and cell migration assays, fibroblast proliferation and migration were promoted after LLLT with a CO2 laser at 1.0 J/cm2. Western blot analysis revealed that Akt, ERK, and JNK activities were promoted in fibroblasts after LLLT with a CO2 laser at 1.0 J/cm2. Moreover, inhibition of Akt, ERK, or JNK significantly blocked fibroblast proliferation and migration. Conclusions These findings suggested that LLLT with a CO2 laser would accelerate wound healing by promoting the proliferation and migration of fibroblasts. Activation of Akt, ERK, and JNK was essential for CO2 laser-induced proliferation and migration of fibroblasts. PMID:28045948

  9. In vitro cytotoxic activity of equine lymphocytes on equine herpesvirus-1 infected allogenic fibroblasts

    OpenAIRE

    Edens, Lucy Marie

    1994-01-01

    The objectives of this study were to: 1) develop a technique to analyze the in vitro cytotoxic activity of lymphocytes from adult horses against equine herpes virus-1 (EHV-1) infected allogenic equine dermal fibroblasts (EDF); 2) evaluate the ability of a 72 hour in vitro incubation with interleukin-2 (I L-2) to enhance the lymphocytic cytolytic activity against EHV-1 infected EDF; 3) compare the cytotoxic activity among lymphocytes isolated from pregnant mares and non-pregnant...

  10. Anti-Proliferative Activity of Meroditerpenoids Isolated from the Brown Alga Stypopodium flabelliforme against Several Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Patricia Valentao

    2011-05-01

    Full Text Available The sea constitutes one of the most promising sources of novel compounds with potential application in human therapeutics. In particular, algae have proved to be an interesting source of new bioactive compounds. In this work, six meroditerpenoids (epitaondiol, epitaondiol diacetate, epitaondiol monoacetate, stypotriol triacetate, 14-ketostypodiol diacetate and stypodiol isolated from the brown alga Stypopodium flabelliforme were tested for their cell proliferation inhibitory activity in five cell lines. Cell lines tested included human colon adenocarcinoma (Caco-2, human neuroblastoma (SH-SY5Y, rat basophilic leukemia (RBL-2H3, murine macrophages (RAW.267 and Chinese hamster fibroblasts (V79. Antimicrobial activity of the compounds was also evaluated against Staphylococcus aureus, Salmonella typhimurium, Proteus mirabilis, Bacillus cereus, Enterococcus faecalis and Micrococcus luteus. Overall, the compounds showed good activity against all cell lines, with SH-SY5Y and RAW.267 being the most susceptible. Antimicrobial capacity was observed for epitaondiol monoacetate, stypotriol triacetate and stypodiol, with the first being the most active. The results suggest that these molecules deserve further studies in order to evaluate their potential as therapeutic agents.

  11. Inflammation and N-formyl peptide receptors mediate the angiogenic activity of human vitreous humour in proliferative diabetic retinopathy.

    Science.gov (United States)

    Rezzola, Sara; Corsini, Michela; Chiodelli, Paola; Cancarini, Anna; Nawaz, Imtiaz M; Coltrini, Daniela; Mitola, Stefania; Ronca, Roberto; Belleri, Mirella; Lista, Liliana; Rusciano, Dario; De Rosa, Mario; Pavone, Vincenzo; Semeraro, Francesco; Presta, Marco

    2017-04-01

    Angiogenesis and inflammation characterise proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus. However, the impact of inflammation on the pathogenesis of PDR neovascularisation has not been elucidated. Here, we assessed the capacity of PDR vitreous fluid to induce pro-angiogenic/proinflammatory responses in endothelium and the contribution of the inflammation-related pattern recognition N-formyl peptide receptors (FPRs) in mediating these responses. Pooled and individual pars plana vitrectomy-derived PDR vitreous fluid ('PDR vitreous') samples were assessed in endothelial cell proliferation, motility, sprouting and morphogenesis assays, and for the capacity to induce proinflammatory transcription factor activation, reactive oxygen species production, intercellular junction disruption and leucocyte-adhesion molecule upregulation in these cells. In vivo, the pro-angiogenic/proinflammatory activity of PDR vitreous was tested in murine Matrigel plug and chick embryo chorioallantoic membrane (CAM) assays. Finally, the FPR inhibitors Boc-Phe-Leu-Phe-Leu-Phe (Boc-FLFLF) and Ac-L-Arg-Aib-L-Arg-L-Cα(Me)Phe-NH2 tetrapeptide (UPARANT) were evaluated for their capacity to affect the biological responses elicited by PDR vitreous. PDR vitreous activates a pro-angiogenic/proinflammatory phenotype in endothelial cells. Accordingly, PDR vitreous triggers a potent angiogenic/inflammatory response in vivo. Notably, the different capacity of individual PDR vitreous samples to induce neovessel formation in the CAM correlates with their ability to recruit infiltrating CD45(+) cells. Finally, the FPR inhibitor Boc-FLFLF and the novel FPR antagonist UPARANT inhibit neovessel formation and inflammatory responses triggered by PDR vitreous in the CAM assay. This study provides evidence that inflammation mediates the angiogenic activity of PDR vitreous and paves the way for the development of FPR-targeting anti-inflammatory/anti-angiogenic approaches for PDR

  12. The calcium channel blocker amlodipine exerts its anti-proliferative action via p21(Waf1/Cip1) gene activation.

    Science.gov (United States)

    Ziesche, Rolf; Petkov, Ventzislav; Lambers, Christopher; Erne, Paul; Block, Lutz-Henning

    2004-10-01

    Proliferation of vascular smooth muscle cells (VSMC) contributes to the progression of atherosclerotic plaques. Calcium channel blockers have been shown to reduce VSMC proliferation, but the underlying molecular mechanism remains unclear. p21(Waf1/Cip1) is a potent inhibitor of cell cycle progression. Here, we demonstrate that amlodipine (10(-6) to 10(-8) M) activates de novo synthesis of p21(Waf1/Cip1) in vitro. We show that amlodipine-dependent activation of p21(Waf1/Cip1) involves the action of the glucocorticoid receptor (GR) and C/EBP-alpha. The underlying pathway apparently involves the action of mitogen-activated protein kinase or protein kinase C, but not of extracellular signal-related kinase or changes of intracellular calcium. Amlodipine-induced p21(Waf1/Cip1) promoter activity and expression were abrogated by C/EBP-alpha antisense oligonucleotide or by the GR antagonist RU486. Amlodipine-dependent inhibition of cell proliferation was partially reversed by RU486 at 10(-8) M (58%+/-29%), antisense oligonucleotides targeting C/EBP-alpha (91%+/-26%), or antisense mRNAs targeting p21(Waf1/Cip1) (96%+/-32%, n=6); scrambled antisense oligonucleotides or those directed against C/EBP-beta were ineffective. The data suggest that the anti-proliferative action of amlodipine is achieved by induction of the p21 (Waf1/Cip1) gene, which may explain beneficial covert effects of this widely used cardiovascular therapeutic drug beyond a more limited role as a vascular relaxant.

  13. Malignant Trigeminal Nerve Sheath Tumor and Anaplastic Astrocytoma Collision Tumor with High Proliferative Activity and Tumor Suppressor P53 Expression

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    Maher Kurdi

    2014-01-01

    Full Text Available Background. The synchronous development of two primary brain tumors of distinct cell of origin in close proximity or in contact with each other is extremely rare. We present the first case of collision tumor with two histological distinct tumors. Case Presentation. A 54-year-old woman presented with progressive atypical left facial pain and numbness for 8 months. MRI of the brain showed left middle cranial fossa heterogeneous mass extending into the infratemporal fossa. At surgery, a distinct but intermingled intra- and extradural tumor was demonstrated which was completely removed through left orbitozygomatic-temporal craniotomy. Histopathological examination showed that the tumor had two distinct components: malignant nerve sheath tumor of the trigeminal nerve and temporal lobe anaplastic astrocytoma. Proliferative activity and expressed tumor protein 53 (TP53 gene mutations were demonstrated in both tumors. Conclusions. We describe the first case of malignant trigeminal nerve sheath tumor (MTNST and anaplastic astrocytoma in collision and discuss the possible hypothesis of this rare occurrence. We propose that MTNST, with TP53 mutation, have participated in the formation of anaplastic astrocytoma, or vice versa.

  14. Plasma Plasminogen Activator Inhibitor-1 Is Associated with End-Stage Proliferative Diabetic Retinopathy in the Northern Chinese Han Population

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    Ze-Long Zhong

    2012-01-01

    Full Text Available Objective. To identify predictors of end-stage proliferative diabetic retinopathy (PDR in a cohort of individuals with type 2 diabetes mellitus (T2DM from the Northern Chinese Han population. Methods. We investigated characteristics of 153 consecutive diabetic patients with end-stage PDR (62 males, 91 females, 123 consecutive PDR patients without end-stage PDR (48 males, 75 females, and 151 normal subjects (63 males, 88 females. Only one eye of each patient or healthy subject was included in this study. Univariate logistic regression models and multivariate logistic regression models were constructed to evaluate the predictors of end-stage PDR. Results. In univariate analysis, systolic blood pressure, diastolic blood pressure, duration of diabetes, family history of T2DM, and plasminogen activator inhibitor-1 (PAI-1 were significently associated with end-stage PDR. After multivariate analysis, family history of T2DM, plasma PAI-1 levels, smoking, and duration of diabetes were four positive predictors associated with end-stage PDR. Conclusions. Higher plasma levels of PAI-1 were associated with end-stage PDR in the Northern Chinese Han population with T2DM.

  15. Patterns of proliferative activity in the colonic crypt determine crypt stability and rates of somatic evolution.

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    Rui Zhao

    Full Text Available Epithelial cells in the colon are arranged in cylindrical structures called crypts in which cellular proliferation and migration are tightly regulated. We hypothesized that the proliferation patterns of cells may determine the stability of crypts as well as the rates of somatic evolution towards colorectal tumorigenesis. Here, we propose a linear process model of colonic epithelial cells that explicitly takes into account the proliferation kinetics of cells as a function of cell position within the crypt. Our results indicate that proliferation kinetics has significant influence on the speed of cell movement, kinetics of mutation propagation, and sensitivity of the system to selective effects of mutated cells. We found that, of all proliferation curves tested, those with mitotic activities concentrated near the stem cell, including the actual proliferation kinetics determined in in vivo labeling experiments, have a greater ability of delaying the rate of mutation accumulation in colonic stem cells compared to hypothetical proliferation curves with mitotic activities focused near the top of the crypt column. Our model can be used to investigate the dynamics of proliferation and mutation accumulation in spatially arranged tissues.

  16. [Use of colchicine in studying the proliferative activity of chicken embryos].

    Science.gov (United States)

    Efremov, V I; El Zajat, M

    1977-07-01

    Manifestation of mitotic activity of colchicin in 4- and 5-day-old chick embryos was studied under different modes of colchicin injection into the eggs. Three methods were tested: colchicin solution was injected into the serous-amniotic cavity (1) and into the yolk sac (2) and also by dripping on the serous membrane over the enbryo (3). Perfect metastatic effect was observed only when colchicin was used in concentration of 1 X 10(-4) g/ml and injected by the third mehtod. Increase in the solution volume over 0.1 ml resulted in a greater percentage of embryo death. Lack of a definite inhibitory action after colchicin injection into the serous-amniotic cavity might be explained by decrease of the substance concentration as a result of its dilution by the cavity fluid. A complete lack of blocked mitoses in the embryo tissues after colchicin injection into the yolk sac can be explained, according to the authors, by the presence, in the yolk, of a great number of ovoflavins capable to inhibit mitotic activity of colchicin.

  17. Cathepsin B Regulates Collagen Expression by Fibroblasts via Prolonging TLR2/NF-κB Activation

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    Xue Li

    2016-01-01

    Full Text Available Fibroblasts are essential for tissue repair due to producing collagens, and lysosomal proteinase cathepsin B (CatB is involved in promoting chronic inflammation. We herein report that CatB regulates the expression of collagens III and IV by fibroblasts in response to a TLR2 agonist, lipopolysaccharide from Porphyromonas gingivalis (P.g. LPS. In cultured human BJ fibroblasts, mRNA expression of CatB was significantly increased, while that of collagens III and IV was significantly decreased at 24 h after challenge with P.g. LPS (1 μg/mL. The P.g. LPS-decreased collagen expression was completely inhibited by CA-074Me, the specific inhibitor of CatB. Surprisingly, expression of collagens III and IV was significantly increased in the primary fibroblasts from CatB-deficient mice after challenge with P.g. LPS. The increase of CatB was accompanied with an increase of 8-hydroxy-2′-deoxyguanosine (8-OHdG and a decrease of IκBα. Furthermore, the P.g. LPS-increased 8-OHdG and decreased IκBα were restored by CA-074Me. Moreover, 87% of CatB and 86% of 8-OHdG were colocalized with gingival fibroblasts of chronic periodontitis patients. The findings indicate the critical role of CatB in regulating the expression of collagens III and IV by fibroblasts via prolonging TLR2/NF-κB activation and oxidative stress. CatB-specific inhibitors may therefore improve chronic inflammation-delayed tissue repair.

  18. Differentiation-inducing and anti-proliferative activities of lupeol on canine melanoma cells.

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    Ogihara, Kikumi; Naya, Yuko; Okamoto, Yoshiharu; Hata, Keishi

    2014-01-01

    Canine melanoma is the most common oral malignant tumor reported in the field of veterinary medicine. We found that lupeol, a lupine triterpene, inhibited mouse melanoma cell growth in vitro and in vivo by inducing cell differentiation. In the present study, we examined the differentiation-inducing activities of lupeol on 4 canine melanoma cells in vitro and in vivo. The induction of canine melanoma cell differentiation by lupeol was confirmed by evaluating some differentiation markers such as tyrosinase with real-time RT-PCR. Furthermore, we transplanted canine melanoma cells into a severe combined immunodeficiency mouse, and studied the anti-progressive effects of lupeol on tumor tissue. The gene expression of microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related protein-2, which are markers of pigment cell differentiation, was induced in 4 canine oral malignant melanoma cells by lupeol, and the agent markedly inhibited tumor progression in canine melanoma-bearing mice.

  19. Chalcone derivatives from the fern Cyclosorus parasiticus and their anti-proliferative activity.

    Science.gov (United States)

    Wei, Han; Zhang, Xuenong; Wu, Guanghua; Yang, Xian; Pan, Songwei; Wang, Yanyan; Ruan, Jinlan

    2013-10-01

    Three new chalcone derivatives, named parasiticins A-C (1-3), were isolated from the leaves of Cyclosorus parasiticus, together with four known chalcones, 5,7-dihydroxy-4-phenyl-8-(3-phenyl-trans-acryloyl)-3,4-dihydro-1-benzopyran-2-one (4), 2'-hydroxy-4',6'-dimethoxychalcone (5), 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (6), 2',4'-dihydroxy-6'-methoxy-3'-methylchalcone (7). The chemical structures of the new isolated compounds were elucidated unambiguously by spectroscopic data analysis. The cytotoxic activities of compounds 1-7 were evaluated against six human cancer cell lines in vitro. Compounds 3 and 6 exhibited substantial cytotoxicity against all six cell lines, especially toward HepG2 with the IC₅₀ values of 1.60 and 2.82 μM, respectively. Furthermore, we demonstrated that compounds 3 and 6 could induce apoptosis in the HepG2 cell line, which may contribute significantly to their cytotoxicity.

  20. Ets-1 targeted by microrna-221 regulates angiotensin II-induced renal fibroblast activation and fibrosis.

    Science.gov (United States)

    Di, Jia; Jiang, Lei; Zhou, Yang; Cao, Hongdi; Fang, Li; Wen, Ping; Li, Xiurong; Dai, Chunsun; Yang, Junwei

    2014-01-01

    Fibroblast activation is one of the most important mechanisms for Angiotensin II (Ang II) in promoting renal fibrosis. Transcription factor Ets-1 is recognized to play a key role in kidney diseases. However, the role and mechanisms of Ets-1 in Ang-II induced fibroblast activation and kidney fibrosis are not fully understood. Mice were treated with Ang II via osmotic mini-pumps or Ang II expression plasmid (pAng II). Cultured normal rat kidney interstitial fibroblast (NRK-49F) cells were incubated with Ang II. Role of Ets-1 in renal fibrosis and fibroblast activation were assessed by Western blot, Immunohistochemical staining'MTT, Boyden chamber and Immunofluorescence staining. Effects of miR-221 on Ets-1 and fibroblast activation were investigated by MTT, Boyden chamber, Western blot and Q-PCR. We found that Ets-1 was up-regulated in fibrotic kidneys. Similarly, Ang II could activate NRK-49F cells as demonstrated by up-regulated α-SMA and fibronectin(FN) expression and enhanced cell proliferation and migration. Ang II also induced Ets-1 expression in NRK-49F cells in a dose and time dependent manner. Knock-down of Ets-1 by RNA interference attenuated Ang II-induced activation of NRK-49F cells. Ets-1 was previously reported as a target of microRNA-221 (miR-221). In Ang II-induced fibrotic kidney, miR-221 was down-regulated. Similar results were observed in Ang II treated NRK-49F cells. Ectopic expression of miR-221 mimic attenuated the up-regulation of Ets-1 by Ang II in NRK-49F cells, which further prevented the activation of NRK-49F cells. However, the inhibitor of miR-221 aggravated Ang II induced Ets-1 expression and NRK-49F cells activation. Our study suggests that miR-221/Ets-1 axis takes an important role in mediating AngII induced interstitial fibroblast activation and renal fibrosis. © 2014 S. Karger AG, Basel.

  1. Fibroblasts from phenotypically normal palmar fascia exhibit molecular profiles highly similar to fibroblasts from active disease in Dupuytren's Contracture

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    Satish Latha

    2012-05-01

    Full Text Available Abstract Background Dupuytren's contracture (DC is a fibroproliferative disorder characterized by the progressive development of a scar-like collagen-rich cord that affects the palmar fascia of the hand and leads to digital flexion contractures. DC is most commonly treated by surgical resection of the diseased tissue, but has a high reported recurrence rate ranging from 27% to 80%. We sought to determine if the transcriptomic profiles of fibroblasts derived from DC-affected palmar fascia, adjacent phenotypically normal palmar fascia, and non-DC palmar fascial tissues might provide mechanistic clues to understanding the puzzle of disease predisposition and recurrence in DC. Methods To achieve this, total RNA was obtained from fibroblasts derived from primary DC-affected palmar fascia, patient-matched unaffected palmar fascia, and palmar fascia from non-DC patients undergoing carpal tunnel release (6 patients in each group. These cells were grown on a type-1 collagen substrate (to better mimic their in vivo environments. Microarray analyses were subsequently performed using Illumina BeadChip arrays to compare the transcriptomic profiles of these three cell populations. Data were analyzed using Significance Analysis of Microarrays (SAM v3.02, hierarchical clustering, concordance mapping and Venn diagram. Results We found that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected fascia of DC patients exhibited a much greater overlap than fibroblasts derived from the palmar fascia of patients undergoing carpal tunnel release. Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses. These data are consistent with the hypothesis that predisposition and recurrence in DC may stem, at least in part, from intrinsic similarities in the basal gene expression of diseased and phenotypically unaffected palmar fascia fibroblasts. These data also demonstrate that

  2. Fibroblasts from phenotypically normal palmar fascia exhibit molecular profiles highly similar to fibroblasts from active disease in Dupuytren's Contracture

    Science.gov (United States)

    2012-01-01

    Background Dupuytren's contracture (DC) is a fibroproliferative disorder characterized by the progressive development of a scar-like collagen-rich cord that affects the palmar fascia of the hand and leads to digital flexion contractures. DC is most commonly treated by surgical resection of the diseased tissue, but has a high reported recurrence rate ranging from 27% to 80%. We sought to determine if the transcriptomic profiles of fibroblasts derived from DC-affected palmar fascia, adjacent phenotypically normal palmar fascia, and non-DC palmar fascial tissues might provide mechanistic clues to understanding the puzzle of disease predisposition and recurrence in DC. Methods To achieve this, total RNA was obtained from fibroblasts derived from primary DC-affected palmar fascia, patient-matched unaffected palmar fascia, and palmar fascia from non-DC patients undergoing carpal tunnel release (6 patients in each group). These cells were grown on a type-1 collagen substrate (to better mimic their in vivo environments). Microarray analyses were subsequently performed using Illumina BeadChip arrays to compare the transcriptomic profiles of these three cell populations. Data were analyzed using Significance Analysis of Microarrays (SAM v3.02), hierarchical clustering, concordance mapping and Venn diagram. Results We found that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected fascia of DC patients exhibited a much greater overlap than fibroblasts derived from the palmar fascia of patients undergoing carpal tunnel release. Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses. These data are consistent with the hypothesis that predisposition and recurrence in DC may stem, at least in part, from intrinsic similarities in the basal gene expression of diseased and phenotypically unaffected palmar fascia fibroblasts. These data also demonstrate that a collagen

  3. Adiponectin attenuates lung fibroblasts activation and pulmonary fibrosis induced by paraquat.

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    Rong Yao

    dose-dependent manner, via suppression of lung fibroblast activation. Functional AdipoR1 are expressed by human WI-38 lung fibroblasts, suggesting potential future clinical applicability of APN against pulmonary fibrosis.

  4. Evaluation of the proliferative activity of methanol extracts from six medicinal plants in murine spleen cells

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    Rodrigo Hermes Zandonai

    2010-06-01

    Full Text Available A number of natural compounds have been used as immunomodulatory agents, enabling the function of the immune system to be modified by stimulating or suppressing it. There has been increasing interest in the study of therapeutic action of plant extracts regarding their immunomodulatory activity. The aim of this study was to identify and evaluate the action of extracts of the medicinal plants Calophyllum brasiliense, Ipomoea pes-caprae, Matayba elaeagnoides, Maytenus robusta, Rubus imperialis and Vernonia scorpioides on the development of spleen cells from mice, using the in vitro cellular proliferation assay. The cells, obtained by mechanical rupture of mice spleen (5x10(4 cells/mL, were incubated with methanol extracts (10, 50, 100 and 200 µg/mL and phytohemagglutinin (PHA, 5 µg/mL. The basal control for proliferation consisted of cells alone, while the positive control consisted of cells and PHA. The cell culture was kept at 37 ºC in 5% CO2 for 72 hours, and cell proliferation was revealed by the blue tetrazolium reduction assay (MTT. The results were expressed as percentage of growth and were analyzed using the Kruskal-Wallis and Mann-Whitney tests. The C. brasiliense, I. pes-caprae and M. elaeagnoides extracts showed dose-dependent induction of cell proliferation, with a significant increase in cell proliferation (pVárias substâncias de origem natural têm sido utilizadas como agentes imunomoduladores, permitindo modificar a função do sistema imune e propiciando o estudo de atividades terapêuticas de extratos de plantas. Este trabalho objetivou identificar a atividade imunomodulatória dos extratos de seis plantas medicinais da flora brasileira, Calophyllum brasiliense, Ipomoea pes-caprae, Matayba elaeagnoides, Maytenus robusta, Rubus imperialis e Vernonia scorpioides, sobre a proliferação de células esplênicas de camundongos. As células esplênicas murinas obtidas por ruptura mecânica do baço (5x14³ células/mL foram

  5. The Proliferative Activity of Mammary Epithelial Cells in Normal Tissue Predicts Breast Cancer Risk in Premenopausal Women.

    Science.gov (United States)

    Huh, Sung Jin; Oh, Hannah; Peterson, Michael A; Almendro, Vanessa; Hu, Rong; Bowden, Michaela; Lis, Rosina L; Cotter, Maura B; Loda, Massimo; Barry, William T; Polyak, Kornelia; Tamimi, Rulla M

    2016-04-01

    The frequency and proliferative activity of tissue-specific stem and progenitor cells are suggested to correlate with cancer risk. In this study, we investigated the association between breast cancer risk and the frequency of mammary epithelial cells expressing p27, estrogen receptor (ER), and Ki67 in normal breast tissue. We performed a nested case-control study of 302 women (69 breast cancer cases, 233 controls) who had been initially diagnosed with benign breast disease according to the Nurses' Health Studies. Immunofluorescence for p27, ER, and Ki67 was performed on tissue microarrays constructed from benign biopsies containing normal mammary epithelium and scored by computational image analysis. We found that the frequency of Ki67(+) cells was positively associated with breast cancer risk among premenopausal women [OR = 10.1, 95% confidence interval (CI) = 2.12-48.0]. Conversely, the frequency of ER(+) or p27(+) cells was inversely, but not significantly, associated with subsequent breast cancer risk (ER(+): OR = 0.70, 95% CI, 0.33-1.50; p27(+): OR = 0.89, 95% CI, 0.45-1.75). Notably, high Ki67(+)/low p27(+) and high Ki67(+)/low ER(+) cell frequencies were significantly associated with a 5-fold higher risk of breast cancer compared with low Ki67(+)/low p27(+) and low Ki67(+)/low ER(+) cell frequencies, respectively, among premenopausal women (Ki67(hi)/p27(lo): OR = 5.08, 95% CI, 1.43-18.1; Ki67(hi)/ER(lo): OR = 4.68, 95% CI, 1.63-13.5). Taken together, our data suggest that the fraction of actively cycling cells in normal breast tissue may represent a marker for breast cancer risk assessment, which may therefore impact the frequency of screening procedures in at-risk women. Cancer Res; 76(7); 1926-34. ©2016 AACR.

  6. Senescence-associated β-galactosidase activity in the in vitro ovarian stromal fibroblasts

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    Lilian Chuaire-Noack

    2011-04-01

    Full Text Available A growing biological research field is the cellular senescence, a mechanism that has been associated, under certain circumstances, withmalignant transformation. Given the high incidence of ovarian cancerand its main origin from the ovarian surface epithelium, as well asthe possibility that an epithelial-mesenchymal transition occurs, weevaluated both the in vitro growth of stromal fibroblasts from the ovarian cortex and their β-galactosidase activity at pH 6,enzyme whose expression is considered as a marker of replicativesenescence. Methods: 48 samples of ovarian cortical fibroblasts fromdonors without a history of cancer were serially cultured untilthe end of their replicative life. β-galactosidase activity at pH 6was quantified in each passage by the chemiluminiscent method. Ascontrol, we used ovarian epithelial cell cultures from the samedonors. The enzyme activity was also evaluated in fibroblastspreviously induced to senescence by exposure to hydrogen peroxide.Results: The analysis of the enzyme activity and the replicativecapacity taken together showed that the fibroblast cultures reachedthe senescent state at passages 4-5, as what happened with the control epithelial cells. Fibroblasts induced to senescence showed high variability in the values of enzymatic activity. Conclusions:The similarity between both types of cells in reaching the senescent state deserves to be taken into account in relation to theepithelialmesenchymal transition that has been proposed to explaintheir behavior in the genesis of cancer arising from ovarian surfaceepithelium. Low β-galactosidase activity values at pH 6 would suggestpossible inactivation of the response pathways to oxidative stress.

  7. Korean Ginseng Berry Fermented by Mycotoxin Non-producing Aspergillus niger and Aspergillus oryzae: Ginsenoside Analyses and Anti-proliferative Activities.

    Science.gov (United States)

    Li, Zhipeng; Ahn, Hyung Jin; Kim, Nam Yeon; Lee, Yu Na; Ji, Geun Eog

    2016-01-01

    To transform ginsenosides, Korean ginseng berry (KGB) was fermented by mycotoxin non-producing Aspergillus niger and Aspergillus oryzae. Changes of ginsenoside profile and anti-proliferative activities were observed. Results showed that A. niger tended to efficiently transform protopanaxadiol (PPD) type ginsenosides such as Rb1, Rb2, Rd to compound K while A. oryzae tended to efficiently transform protopanaxatriol (PPT) type ginsenoside Re to Rh1 via Rg1. Butanol extracts of fermented KGB showed high cytotoxicity on human adenocarcinoma HT-29 cell line and hepatocellular carcinoma HepG2 cell line while that of unfermented KGB showed little. The minimum effective concentration of niger-fermented KGB was less than 2.5 µg/mL while that of oryzae-fermented KGB was about 5 µg/mL. As A. niger is more inclined to transform PPD type ginsenosides, niger-fermented KGB showed stronger anti-proliferative activity than oryzae-fermented KGB.

  8. Bifunctional Therapeutic High-Valence Silver-Pyridoxine Nanoparticles with Proliferative and Antibacterial Wound-Healing Activities.

    Science.gov (United States)

    Rangasamy, Sabarinathan; Tak, Yu Kyung; Kim, Sunhee; Paul, Avijit; Song, Joon Myong

    2016-01-01

    The antibacterial and moisturizing effects inherent to silver nanoparticles contribute greatly to their use as a topical antibacterial agent. The antibacterial property of silver nanoparticles provides topical wounds with an indirect environment for healing through the prevention of pathogenic infection. However, the direct wound-healing effects of silver nanoparticles have not been previously explored. In this work, we report a bimodal therapeutic silver nanoparticle that possesses both direct wound-healing and antibacterial properties. The nanoparticles consist of high-valence silver-pyridoxine complexes. The wound-healing efficacy was verified in diabetic mice, as well as in vitro assays. A MAPK pathway study demonstrated that silver-pyridoxine nanoparticles induced the proliferation and migration of keratinocyte and fibroblast cells. Antibacterial activities in 8 different pathogenic bacteria responsible for the infection of burn wounds were tested. The rapid wound healing occurring on skin wounds of diabetic mice attests to the utility of bimodal therapeutic silver nanoparticles as a next-generation topical therapeutic agent.

  9. Elevated transforming growth factor β and mitogen-activated protein kinase pathways mediate fibrotic traits of Dupuytren's disease fibroblasts

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    Krause Carola

    2011-06-01

    Full Text Available Abstract Background Dupuytren's disease is a fibroproliferative disorder of the palmar fascia. The treatment used to date has mostly been surgery, but there is a high recurrence rate. Transforming growth factor β (TGF-β has been implicated as a key stimulator of myofibroblast activity and fascial contraction in Dupuytren's disease. Results We studied Dupuytren's fibroblasts in tissues ex vivo and in cells cultured in vitro and found increased TGF-β expression compared to control fibroblasts. This correlated not only with elevated expression and activation of downstream Smad effectors but also with overactive extracellular signal-regulated kinase 1/2 (ERK1/2/mitogen-activated protein (MAP kinase signalling. Treatment with the TGF-β type I receptor kinase inhibitor SB-431542 and bone morphogenetic protein 6 (BMP6 led to inhibition of elevated Smad and ERK1/2/MAP kinase signalling as well as to inhibition of the increased contractility of Dupuytren's fibroblasts. BMP6 attenuated TGF-β expression in Dupuytren's fibroblasts, but not in control fibroblasts. Platelet-derived growth factor (PDGF expression was strongly promoted by TGF-β in Dupuytren's fibroblasts and was curbed by SB-431542 or BMP6 treatment. High basal expression of phosphorylated ERK1/2 MAP kinase and fibroproliferative markers was attenuated in Dupuytren's fibroblasts by a selective PDGF receptor kinase inhibitor. Cotreatment of Dupuytren's fibroblasts with SB-431542 and the mitogen-activated protein kinase kinase 1 inhibitor PD98059 was sufficient to abrogate proliferation and contraction of Dupuytren's fibroblasts. Conclusions Both TGF-β and ERK1/2 MAP kinase pathways cooperated in mediating the enhanced proliferation and high spontaneous contraction of Dupuytren's fibroblasts. Our data indicate that both signalling pathways are prime targets for the development of nonsurgical intervention strategies to treat Dupuytren's disease.

  10. Adiponectin attenuates lung fibroblasts activation and pulmonary fibrosis induced by paraquat.

    Science.gov (United States)

    Yao, Rong; Cao, Yu; He, Ya-rong; Lau, Wayne Bond; Zeng, Zhi; Liang, Zong-an

    2015-01-01

    Pulmonary fibrosis is one of the most common complications of paraquat (PQ) poisoning, which demands for more effective therapies. Accumulating evidence suggests adiponectin (APN) may be a promising therapy against fibrotic diseases. In the current study, we determine whether the exogenous globular APN isoform protects against pulmonary fibrosis in PQ-treated mice and human lung fibroblasts, and dissect the responsible underlying mechanisms. BALB/C mice were divided into control group, PQ group, PQ + low-dose APN group, and PQ + high-dose APN group. Mice were sacrificed 3, 7, 14, and 21 days after PQ treatment. We compared pulmonary histopathological changes among different groups on the basis of fibrosis scores, TGF-β1, CTGF and α-SMA pulmonary content via Western blot and real-time quantitative fluorescence-PCR (RT-PCR). Blood levels of MMP-9 and TIMP-1 were determined by ELISA. Human lung fibroblasts WI-38 were divided into control group, PQ group, APN group, and APN receptor (AdipoR) 1 small-interfering RNA (siRNA) group. Fibroblasts were collected 24, 48, and 72 hours after PQ exposure for assay. Cell viability and apoptosis were determined via Kit-8 (CCK-8) and fluorescein Annexin V-FITC/PI double labeling. The protein and mRNA expression level of collagen type III, AdipoR1, and AdipoR2 were measured by Western blot and RT-PCR. APN treatment significantly decreased the lung fibrosis scores, protein and mRNA expression of pulmonary TGF-β1, CTGF and α-SMA content, and blood MMP-9 and TIMP-1 in a dose-dependent manner (ppulmonary fibrosis in a dose-dependent manner, via suppression of lung fibroblast activation. Functional AdipoR1 are expressed by human WI-38 lung fibroblasts, suggesting potential future clinical applicability of APN against pulmonary fibrosis.

  11. Impairment of the transition from proliferative stage to prehypertrophic stage in chondrogenic differentiation of human induced pluripotent stem cells harboring the causative mutation of achondroplasia in fibroblast growth factor receptor 3

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    Naohiro Horie

    2017-06-01

    Conclusions: These results suggested that chondrocyte maturation was impaired between the proliferative stage and prehypertrophic stage in the chondrocytes of ACH. The development of chemical compounds which affect the specific maturation stage of chondrocytes is expected to contribute to the ACH treatment, and FGFR3 genome-edited hiPSCs will be a valuable tool in such research studies.

  12. Cytosol cathepsin-D content and proliferative activity of human breast cancer. The Comitato Italiano per il Controllo di Qualita del Laboratorio in Oncologia.

    Science.gov (United States)

    Paradiso, A; Mangia, A; Correale, M; Abbate, I; Ferri, G; Piffanelli, A; Catozzi, L; Amadori, D; Riccobon, A; De Lena, M

    1992-01-01

    Mitogenic properties have been demonstrated in vitro for the lysosomal acidic protease cathepsin-D (cath-D). We investigated possible relationships between cath-D cytosol cell content and tumor proliferative activity in a series of 129 operable breast cancer patients. For total cytosol cath-D evaluation, a solid phase two-site immunoradiometric assay was utilized on tumor cell cytosol obtained for hormone receptor assay (DCC method). The percentage of S-phase cells was analyzed by 3H-thymidine autoradiographic assay. Median 3H-thymidine Labeling Index (3H-Tdr-LI) of the series was 2.7%; median cath-D content resulted 57 pmol/mg of protein cytosol and was significantly higher in node-positive with respect to the node-negative subgroup (p < 0.03). When classified in low, intermediate or high tumor cath-D content and slow or fast proliferative activity (cut-off: median values of the series), no significant agreement was found between the two variables. Statistical analysis, however, showed that a significant inverse correlation existed in node positive tumors between cath-D and 3H-Tdr-LI values which was even more evident in N-positive high estrogen receptor-positive (ER+) cases (coefficient of correlation = 0.6828; p = 0.0001). Cytosol cath-D content cannot be generally proposed as a direct marker of proliferative activity for operable breast cancer.

  13. Acidic environment activates inflammatory programs in fibroblasts via a cAMP-MAPK pathway.

    Science.gov (United States)

    Riemann, A; Ihling, A; Thomas, J; Schneider, B; Thews, O; Gekle, M

    2015-02-01

    The tissue micromilieu in disorders (inflammation, ischemia, tumor) often shows pronounced metabolic acidosis that may alter signaling and transcriptional activity in resident cells which can be of special importance for omnipresent fibroblasts. In the present study we investigated the impact of metabolic acidosis on rat fibroblasts with special emphasis on their role in inflammation by regulation of TNF-α, MCP-1, COX-2 and iNOS expression and the signaling pathways involved. Extracellular acidosis led to an enhanced expression of TNF-α, COX-2 and iNOS in parallel to an activation of p38 and ERK1/2 kinases that was not observed by sole intracellular acidosis. Accordingly, the protein amounts of TNF-α and COX-2 as well as the production of nitrate and nitrite were elevated. Acidosis-induced expression of COX-2 and iNOS depended on p38 kinase, but not on ERK1/2. In contrast acidosis-induced TNF-α expression was independent of both kinases. Although GPR4, GPR68 and GPR132 are expressed in fibroblasts, the involvement of these potential candidate pH sensors could be ruled out since no acidosis-induced elevation in intracellular cAMP or free calcium content was observed. Furthermore our data show that MAPK activation by an acidic micromilieu depends on Ser/Thr phosphatase activity, but not on the production of reactive oxygen species and is sensitive to cAMP antagonism by Rp-cAMPS. In conclusion, our results show that an acidic microenvironment induces a differential transcriptional program of pathological relevant genes in fibroblasts via the cAMP-phosphatase-MAPK pathway and thereby generates a parainflammatory situation that can result in tissue remodeling.

  14. Stability and biological activity evaluations of PEGylated human basic fibroblast growth factor

    OpenAIRE

    Hadadian, Shahin; Shamassebi, Dariush Norouzian; Mirzahoseini, Hasan; Shokrgozar, Mohamad Ali; Bouzari, Saeid; Sepahi, Mina

    2015-01-01

    Background: Human basic fibroblast growth factor (hBFGF) is a heparin-binding growth factor and stimulates the proliferation of a wide variety of cells and tissues causing survival properties and its stability and biological activity improvements have received much attention. Materials and Methods: In the present work, hBFGF produced by engineered Escherichia coli and purified by cation exchange and heparin affinity chromatography, was PEGylated under appropriate condition employing 10 kD pol...

  15. GLD-4-mediated translational activation regulates the size of the proliferative germ cell pool in the adult C. elegans germ line.

    Science.gov (United States)

    Millonigg, Sophia; Minasaki, Ryuji; Nousch, Marco; Novak, Jakub; Eckmann, Christian R

    2014-09-01

    To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult Caenorhabditis elegans reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A) polymerase (cytoPAP) GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that gld-4 activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with glp-1 mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of glp-1 mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant translational circuit with

  16. GLD-4-mediated translational activation regulates the size of the proliferative germ cell pool in the adult C. elegans germ line.

    Directory of Open Access Journals (Sweden)

    Sophia Millonigg

    2014-09-01

    Full Text Available To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult Caenorhabditis elegans reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A polymerase (cytoPAP GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that gld-4 activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with glp-1 mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of glp-1 mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant

  17. Cytoprotective effects of mild plasma-activated medium against oxidative stress in human skin fibroblasts

    Science.gov (United States)

    Horiba, Minori; Kamiya, Tetsuro; Hara, Hirokazu; Adachi, Tetsuo

    2017-01-01

    Non-thermal atmospheric pressure plasma (NTAPP) has recently been applied to living cells and tissues and has emerged as a novel technology for medical applications. NTAPP affects cells not only directly, but also indirectly with previously prepared plasma-activated medium (PAM). The objective of this study was to demonstrate the preconditioning effects of “mild PAM” which was prepared under relatively mild conditions, on fibroblasts against cellular injury generated by a high dose of hydrogen peroxide (H2O2). We observed the preconditioning effects of mild PAM containing approximately 50 μM H2O2. Hydrogen peroxide needs to be the main active species in mild PAM for it to exert preconditioning effects because the addition of catalase to mild PAM eliminated these effects. The nuclear translocation and recruitment of nuclear factor erythroid 2-related factor 2 (Nrf2) to antioxidant response elements (ARE) in heme oxygenase 1 (HO-1) promoters and the up-regulation of HO-1 were detected in fibroblasts treated with mild PAM. The addition of ZnPP, a HO-1-specific inhibitor, or the knockdown of Nrf2 completely abrogated the preconditioning effects. Our results demonstrate that mild PAM protects fibroblasts from oxidative stress by up-regulating HO-1, and the H2O2-induced activation of the Nrf2-ARE pathway needs to be involved in this reaction. PMID:28169359

  18. Adenosine monophosphate-activated protein kinase activation and suppression of inflammatory response by cell stretching in rabbit synovial fibroblasts.

    Science.gov (United States)

    Kunanusornchai, Wanlop; Muanprasat, Chatchai; Chatsudthipong, Varanuj

    2016-12-01

    Joint mobilization is known to be beneficial in osteoarthritis (OA) patients. This study aimed to investigate the effect of stretching on adenosine monophosphate-activated protein kinase (AMPK) activity and its role in modulating inflammation in rabbit synovial fibroblasts. Uniaxial stretching of isolated rabbit synovial fibroblasts for ten min was performed. Stretching-induced AMPK activation, its underlying mechanism, and its anti-inflammatory effect were investigated using Western blot. Static stretching at 20 % of initial length resulted in AMPK activation characterized by expression of phosphorylated AMPK and phosphorylated acetyl-Co A carboxylase. AMP-activated protein kinase phosphorylation peaked 1 h after stretching and declined toward resting activity. Using cell viability assays, static stretching did not appear to cause cellular damage. Activation of AMPK involves Ca(2+) influx via a mechanosensitive L-type Ca(2+) channel, which subsequently raises intracellular Ca(2+) and activates AMPK via Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ). Interestingly, stretching suppressed TNFα-induced expression of COX-2, iNOS, and phosphorylated NF-κB. These effects were prevented by pretreatment with compound C, an AMPK inhibitor. These results suggest that mechanical stretching suppressed inflammatory responses in synovial fibroblasts via a L-type Ca(2+)-channel-CaMKKβ-AMPK-dependent pathway which may underlie joint mobilization's ability to alleviate OA symptoms.

  19. A new feature of Mpl receptor: ligand-induced transforming activity in FRE rat fibroblasts.

    Science.gov (United States)

    Challier, C; Cocault, L; Flon, M; Pauchard, M; Porteu, F; Gisselbrecht, S; Souyri, M

    2000-04-13

    Mpl is the receptor for thrombopoietin, the primary regulator of platelet production by megakaryocytes. Upon stimulation by its ligand, Mpl receptor induces proliferation and differentiation of hematopoietic cell lines of various origins. In this paper, we show that Mpl is also able to transform FRE rat fibroblasts in the presence of MGDF (pegylated Megakaryocyte Growth and Development Factor), a modified form of its ligand. We also demonstrate that upon MGDF stimulation Mpl receptor activates the classical transduction pathways described for hematopoietic cell lines in FRE cells. Introduction of Mpl deletion mutants in FRE cells allowed us to demonstrate that the C-terminal region of the Mpl intracytoplasmic domain, which is involved in hematopoietic differentiation, is necessary for the transformation process. Within that region, site-directed mutagenesis showed that the Y112 residue, which is required for Shc phosphorylation, is essential for rat fibroblast transformation by Mpl/MGDF, suggesting the involvement of Shc in Mpl-mediated transformation. Interestingly, we showed that transformation correlated with strong and sustained MAPK activation. Neither Jak2, Stat3 nor Stat5 phosphorylation was sufficient to induce the transformation process. Taken altogether, our results suggest the oncogenicity of Mpl in fibroblastic cells in the presence of its ligand.

  20. Omega-3 Polyunsaturated Fatty Acids Attenuate Fibroblast Activation and Kidney Fibrosis Involving MTORC2 Signaling Suppression

    Science.gov (United States)

    Zeng, Zhifeng; Yang, Haiyuan; Wang, Ying; Ren, Jiafa; Dai, Yifan; Dai, Chunsun

    2017-01-01

    Epidemiologic studies showed the correlation between the deficiency of omega-3 polyunsaturated fatty acids (n-3 PUFAs) and the progression of chronic kidney diseases (CKD), however, the role and mechanisms for n-3 PUFAs in protecting against kidney fibrosis remain obscure. In this study, NRK-49F cells, a rat kidney interstitial fibroblast cell line, were stimulated with TGFβ1. A Caenorhabditis elegans fat-1 transgenic mouse model in which n-3 PUFAs are endogenously produced from n-6 PUFAs owing to the expression of n-3 fatty acid desaturase were deployed. Docosahexaenoic acid (DHA), one member of n-3 PUFAs family, could suppress TGFβ1-induced fibroblast activation at a dose and time dependent manner. Additionally, DHA could largely inhibit TGFβ1-stimulated Akt but not S6 or Smad3 phosphorylation at a time dependent manner. To decipher the role for n-3 PUFAs in protecting against kidney fibrosis, fat-1 transgenic mice were operated with unilateral ureter obstruction (UUO). Compared to the wild types, fat-1 transgenics developed much less kidney fibrosis and inflammatory cell accumulation accompanied by less p-Akt (Ser473), p-Akt (Thr308), p-S6 and p-Smad3 in kidney tissues at day 7 after UUO. Thus, n-3 PUFAs can attenuate fibroblast activation and kidney fibrosis, which may be associated with the inhibition of mTORC2 signaling. PMID:28393852

  1. Activation of pluripotency genes in human fibroblast cells by a novel mRNA based approach.

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    Jordan R Plews

    Full Text Available BACKGROUND: Several methods have been used to induce somatic cells to re-enter the pluripotent state. Viral transduction of reprogramming genes yields higher efficiency but involves random insertions of viral sequences into the human genome. Although induced pluripotent stem (iPS cells can be obtained with the removable PiggyBac transposon system or an episomal system, both approaches still use DNA constructs so that resulting cell lines need to be thoroughly analyzed to confirm they are free of harmful genetic modification. Thus a method to change cell fate without using DNA will be very useful in regenerative medicine. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we synthesized mRNAs encoding OCT4, SOX2, cMYC, KLF4 and SV40 large T (LT and electroporated them into human fibroblast cells. Upon transfection, fibroblasts expressed these factors at levels comparable to, or higher than those in human embryonic stem (ES cells. Ectopically expressed OCT4 localized to the cell nucleus within 4 hours after mRNA introduction. Transfecting fibroblasts with a mixture of mRNAs encoding all five factors significantly increased the expression of endogenous OCT4, NANOG, DNMT3β, REX1 and SALL4. When such transfected fibroblasts were also exposed to several small molecules (valproic acid, BIX01294 and 5'-aza-2'-deoxycytidine and cultured in human embryonic stem cell (ES medium they formed small aggregates positive for alkaline phosphatase activity and OCT4 protein within 30 days. CONCLUSION/SIGNIFICANCE: Our results demonstrate that mRNA transfection can be a useful approach to precisely control the protein expression level and short-term expression of reprogramming factors is sufficient to activate pluripotency genes in differentiated cells.

  2. Hypoxic regulation of plasminogen activator inhibitor-1 expression in human buccal mucosa fibroblasts stimulated with arecoline.

    Science.gov (United States)

    Tsai, Chung-Hung; Lee, Shiuan-Shinn; Chang, Yu-Chao

    2015-10-01

    Oral submucous fibrosis (OSF) is regarded as a pre-cancerous condition with fibrosis in oral subepithelial connective tissue. Hypoxia-inducible factor (HIF)-1α regulates a wide variety of profibrogenic genes, which are closely associated with tissue fibrosis. The aim of this study was to compare HIF-1α expression in normal buccal mucosa tissues and OSF specimens and further explore the potential mechanisms that may lead to the induction of HIF-1α expression. Twenty-five OSF specimens and six normal buccal mucosa were examined by immunohistochemistry. The expression of HIF-1α from fibroblasts cultured from OSF and normal buccal mucosa was measured by Western blot. Arecoline, a major areca nut alkaloid, was challenged to normal buccal mucosa fibroblasts (BMFs) to elucidate whether HIF-1α expression could affect by arecoline. In addition, the effects of arecoline on plasminogen activator inhibitor (PAI)-1 expression were evaluated in environmental hypoxia. HIF-1α expression was significantly higher in OSF specimens and expressed mainly by fibroblasts, epithelial cells, and inflammatory cells. Fibroblasts derived from OSF were found to exhibit higher HIF-1α protein expression than BMFs (P Arecoline was found to upregulate HIF-1α protein in a dose-dependent manner (P arecoline-induced PAI-1 protein expression than normoxic conditions (P < 0.05). These results suggest that HIF-1α expression is significantly upregulated in OSF tissues from areca quid chewers, implying a potential role as a biomarker for local tissue hypoxia. The activation of HIF-1α may promote fibrogenesis by an increase of PAI-1 expression and a subsequent elevation of extracellular matrix production in oral submucosa leading to fibrosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Targeting Inhibition of Fibroblast Activation Protein-α and Prolyl Oligopeptidase Activities on Cells Common to Metastatic Tumor Microenvironments

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    Victoria J. Christiansen

    2013-04-01

    Full Text Available Fibroblast activation protein (FAP, a membrane prolyl-specific proteinase with both dipeptidase and endopeptidase activities, is overexpressed by reactive stromal fibroblasts during epithelial-derived cancer growth. FAP digests extracellular matrix as tissue is remodeled during cancer expansion and may also promote an immunotolerant tumor microenvironment. Recent studies suggest that nonspecific FAP inhibitors suppress human cancer xenografts in mouse models. Prolyl oligopeptidase (POP, another prolyl-specific serine proteinase, is also elevated in many cancers and may have a regulatory role in angiogenesis promotion. FAP and POP cell-associated activities may be targets for diagnosis and treatment of various cancers, but their accessibilities to highly effective specific inhibitors have not been shown for cells important to cancer growth. Despite their frequent simultaneous expression in many cancers and their overlapping activities toward commonly used substrates, precise, separate measurement of FAP or POP activity has largely been ignored. To distinguish each of the two activities, we synthesized highly specific substrates and inhibitors for FAP or POP based on amino acid sequences surrounding the scissile bonds of their respective putative substrates. We found varying amounts of FAP and POP protein and activities on activated fibroblasts, mesenchymal cells, normal breast cells, and one breast cancer cell line, with some cells exhibiting more POP than FAP activity. Replicating endothelial cells (ECs expressed POP but not FAP until tubulogenesis began. Targeting FAP-positive cells, especially mesenchymal stem cells and cancer-associated fibroblasts for inactivation or destruction, and inhibiting POP-producing EC may abrogate stromal invasion and angiogenesis simultaneously and thereby diminish cancer growth.

  4. Sp3 controls fibroblast growth factor receptor 4 gene activity during myogenic differentiation.

    Science.gov (United States)

    Cavanaugh, Eric; DiMario, Joseph X

    2017-03-27

    Fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling is a critical component in the regulation of myoblast proliferation and differentiation. The transient FGFR4 gene expression during the transition from proliferating myoblasts to differentiated myotubes indicates that FGFR4 regulates this critical phase of myogenesis. The Specificity Protein (SP) family of transcription factors controls FGFR family member gene activity. We sought to determine if members of the Sp family regulate mouse FGFR4 gene activity during myogenic differentiation. RT-PCR and western blot analysis of FGFR4 mRNA and protein revealed transient expression over 72h, with peak expression between 24 and 36h after addition of differentiation medium to C2C12 myogenic cultures. Sp3 also displayed a transient expression pattern with peak expression occurring after 6h of differentiation. We cloned a 1527bp fragment of the mouse FGFR4 promoter into a luciferase reporter. This FGFR4 promoter contains eight putative Sp binding sites and directed luciferase gene activity comparable to native FGFR4 expression. Overexpression of Sp1 and Sp3 showed that Sp1 repressed FGFR4 gene activity, and Sp3 activated FGFR4 gene activity during myogenic differentiation. Mutational analyses of multiple Sp binding sites within the FGFR4 promoter revealed that three of these sites were transcriptionally active. Electromobility shift assays and chromatin immunoprecipitation of the area containing the activator sites showed that Sp3 bound to this promoter location.

  5. Phenolic composition, antioxidant and anti-proliferative activities of edible and medicinal plants from the Peruvian Amazon

    Directory of Open Access Journals (Sweden)

    Jan Tauchen

    Full Text Available ABSTRACT Among 23 extracts of medicinal and edible plants tested, Mauritia flexuosa L.f., Arecaceae, showed significant antioxidant ability (DPPH and ORAC = 1062.9 and 645.9 ± 51.4 µg TE/mg extract, respectively, while Annona montana Macfad., Annonaceae, demonstrated the most promising anti-proliferative effect (IC50 for Hep-G2 and HT-29 = 2.7 and 9.0 µg/ml, respectively. However, combinatory antioxidant/anti-proliferative effect was only detected in Oenocarpus bataua Mart., Arecaceae (DPPH = 903.8 and ORAC = 1024 µg TE/mg extract; IC50 for Hep-G2 and HT-29 at 102.6 and 38.8 µg/ml, respectively and Inga edulis Mart., Fabaceae (DPPH = 337.0 and ORAC = 795.7 µg TE/mg extract; IC50 for Hep-G2 and HT-29 at 36.3 and 57.9 µg/ml, respectively. Phenolic content was positively correlated with antioxidant potential, however not with anti-proliferative effect. None of these extracts possessed toxicity towards normal foetal lung cells, suggesting their possible use in development of novel plant-based agents with preventive and/or therapeutic action against oxidative stress-related diseases.

  6. Sialidase activities of cultured human fibroblasts and the metabolism of GM3 ganglioside

    Energy Technology Data Exchange (ETDEWEB)

    Usuki, S.; Lyu, S.C.; Sweeley, C.C.

    1988-05-15

    Free sialic acid has been found in the cell-conditioned medium of human foreskin fibroblasts. It is proposed that the accumulation of extracellular sialic acid may result from the hydrolysis of GM3 ganglioside on the cell surface of these fibroblasts. Sialidase activities with GM3 ganglioside and sialyllactitol as substrates were demonstrated in cell-conditioned medium, and the levels of their activities correlated positively with cell density. The GM3 sialidase activity at pH 4.5 was 4.1 and 38 pmol/h/ml of medium at sparse and confluent densities, respectively; the corresponding activities with sialyllactitol as the substrate were 12 and 75 pmol/h/ml of medium (pH 4.5). The pH versus activity profiles with GM3 as the substrate suggested the presence of a second sialidase with an optimal activity at pH 6.5 in the conditioned medium of preconfluent cells. This activity was virtually absent in the medium of contact-inhibited cells and could not be assayed with sialyllactitol as the substrate. The turnover of cell surface GM3 was assessed by pulse labeling human foreskin fibroblasts with a radioactive precursor of sialic acid ((1-14C)N-acetylmannosamine) and a radioactive precursor of ceramide ((3,3-3H2)serine). During a chase period of 24 h turnover of the doubly labeled cellular GM3 was observed; there was a loss of about 35% of the 14C-labeled sialic acid without any measureable loss of 3H-labeled ceramide from GM3. We have speculated that the enzyme-catalyzed removal of sialic acid from the GM3 ganglioside on the extracellular aspect of the plasma membrane may be a necessary event involved in the modulation of cell growth.

  7. Dihydrotestosterone Potentiates EGF-Induced ERK Activation by Inducing SRC in Fetal Lung Fibroblasts

    Science.gov (United States)

    Smith, Susan M.; Murray, Sandy; Pham, Lucia D.; Minoo, Parviz; Nielsen, Heber C.

    2014-01-01

    Lung maturation is regulated by interactions between mesenchymal and epithelial cells, and is delayed by androgens. Fibroblast–Type II cell communications are dependent on extracellular signal-regulated kinases (ERK) 1/2 activation by the ErbB receptor ligands epidermal growth factor (EGF), transforming growth factor (TGF)-α, and neuregulin (Nrg). In other tissues, dihydrotestosterone (DHT) has been shown to activate SRC by a novel nontranscriptional mechanism, which phosphorylates EGF receptors to potentiate EGF-induced ERK1/2 activation. This study sought to determine if DHT potentiates EGFR signaling by a nontranscriptional mechanism. Embryonic day (E)17 fetal lung cells were isolated from dams treated with or without DHT since E12. Cells were exposed to 30 ng/ml DHT for periods of 30 minutes to 3 days before being stimulated with 100 ng/ml EGF, TGF-α, or Nrg for up to 30 minutes. Lysates were immunoblotted for ErbB and SRC pathway signaling intermediates. DHT increased ERK1/2 activation by EGF, TGF-α, and Nrg in fibroblasts and Type II cells. Characterization in fibroblasts showed that potentiation of the EGF pathway was significant after 60 minutes of DHT exposure and persisted in the presence of the translational inhibitor cycloheximide. SRC and EGF receptor phosphorylation was increased by DHT, as was EGF-induced SHC1 phosphorylation and subsequent association with GRB2. Finally, SRC silencing, SRC inhibition with PP2, and overexpression of a dominant-negative SRC each prevented DHT from increasing EGF-induced ERK1/2 phosphorylation. These results suggest that DHT activates SRC to potentiate the signaling pathway leading from the EGF receptor to ERK activation in primary fetal lung fibroblasts. PMID:24484548

  8. TAZ contributes to pulmonary fibrosis by activating profibrotic functions of lung fibroblasts

    Science.gov (United States)

    Noguchi, Satoshi; Saito, Akira; Mikami, Yu; Urushiyama, Hirokazu; Horie, Masafumi; Matsuzaki, Hirotaka; Takeshima, Hideyuki; Makita, Kosuke; Miyashita, Naoya; Mitani, Akihisa; Jo, Taisuke; Yamauchi, Yasuhiro; Terasaki, Yasuhiro; Nagase, Takahide

    2017-01-01

    Transcriptional coactivator with PDZ-binding motif (TAZ) regulates a variety of biological processes. Nuclear translocation and activation of TAZ are regulated by multiple mechanisms, including actin cytoskeleton and mechanical forces. TAZ is involved in lung alveolarization during lung development and Taz-heterozygous mice are resistant to bleomycin-induced lung fibrosis. In this study, we explored the roles of TAZ in the pathogenesis of idiopathic pulmonary fibrosis (IPF) through histological analyses of human lung tissues and cell culture experiments. TAZ was highly expressed in the fibroblastic foci of lungs from patients with IPF. TAZ controlled myofibroblast marker expression, proliferation, migration, and matrix contraction in cultured lung fibroblasts. Importantly, actin stress fibers and nuclear accumulation of TAZ were more evident when cultured on a stiff matrix, suggesting a feedback mechanism to accelerate fibrotic responses. Gene expression profiling revealed TAZ-mediated regulation of connective tissue growth factor (CTGF) and type I collagen. Clinical relevance of TAZ-regulated gene signature was further assessed using publicly available transcriptome data. These findings suggest that TAZ is involved in the pathogenesis of IPF through multifaceted effects on lung fibroblasts. PMID:28195168

  9. Immunization of stromal cell targeting fibroblast activation protein providing immunotherapy to breast cancer mouse model.

    Science.gov (United States)

    Meng, Mingyao; Wang, Wenju; Yan, Jun; Tan, Jing; Liao, Liwei; Shi, Jianlin; Wei, Chuanyu; Xie, Yanhua; Jin, Xingfang; Yang, Li; Jin, Qing; Zhu, Huirong; Tan, Weiwei; Yang, Fang; Hou, Zongliu

    2016-08-01

    Unlike heterogeneous tumor cells, cancer-associated fibroblasts (CAF) are genetically more stable which serve as a reliable target for tumor immunotherapy. Fibroblast activation protein (FAP) which is restrictively expressed in tumor cells and CAF in vivo and plays a prominent role in tumor initiation, progression, and metastasis can function as a tumor rejection antigen. In the current study, we have constructed artificial FAP(+) stromal cells which mimicked the FAP(+) CAF in vivo. We immunized a breast cancer mouse model with FAP(+) stromal cells to perform immunotherapy against FAP(+) cells in the tumor microenvironment. By forced expression of FAP, we have obtained FAP(+) stromal cells whose phenotype was CD11b(+)/CD34(+)/Sca-1(+)/FSP-1(+)/MHC class I(+). Interestingly, proliferation capacity of the fibroblasts was significantly enhanced by FAP. In the breast cancer-bearing mouse model, vaccination with FAP(+) stromal cells has significantly inhibited the growth of allograft tumor and reduced lung metastasis indeed. Depletion of T cell assays has suggested that both CD4(+) and CD8(+) T cells were involved in the tumor cytotoxic immune response. Furthermore, tumor tissue from FAP-immunized mice revealed that targeting FAP(+) CAF has induced apoptosis and decreased collagen type I and CD31 expression in the tumor microenvironment. These results implicated that immunization with FAP(+) stromal cells led to the disruption of the tumor microenvironment. Our study may provide a novel strategy for immunotherapy of a broad range of cancer.

  10. Cyclic mechanical deformation stimulates human lung fibroblast proliferation and autocrine growth factor activity.

    Science.gov (United States)

    Bishop, J E; Mitchell, J J; Absher, P M; Baldor, L; Geller, H A; Woodcock-Mitchell, J; Hamblin, M J; Vacek, P; Low, R B

    1993-08-01

    Cellular hypertrophy and hyperplasia and increased extracellular matrix deposition are features of tissue hypertrophy resulting from increased work load. It is known, for example, that mechanical forces play a critical role in lung development, cardiovascular remodeling following pressure overload, and skeletal muscle growth. The mechanisms involved in these processes, however, remain unclear. Here we examined the effect of mechanical deformation on fibroblast function in vitro. IMR-90 human fetal lung fibroblasts grown on collagen-coated silastic membranes were subjected to cyclical mechanical deformation (10% increase in culture surface area; 1 Hz) for up to 5 days. Cell number was increased by 39% after 2 days of deformation (1.43 +/- .01 x 10(5) cells/membrane compared with control, 1.03 +/- 0.02 x 10(5) cells; mean +/- SEM; P < 0.02) increasing to 163% above control by 4 days (2.16 +/- 0.16 x 10(5) cells compared with 0.82 +/- 0.03 x 10(5) cells; P < 0.001). The medium from mechanically deformed cells was mitogenic for IMR-90 cells, with maximal activity in the medium from cells mechanically deformed for 2 days (stimulating cell replication by 35% compared with media control; P < 0.002). These data suggest that mechanical deformation stimulates human lung fibroblast replication and that this effect is mediated by the release of autocrine growth factors.

  11. Effect of charge at an amino acid of basic fibroblast growth factor on its mitogenic activity

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The amino acid at the 119th position of human basic fibroblast growth factor(hbFGF),lysine(K119),is a critical component for its mitogenic activity.However,little is known about the effects of the characteristics of this residue including charge on the mitogenic activity of hbFGF.Herein,this basic residue was replaced with neutral glutamine residue and acidic glutamic acid residue to construct mutants hbFGF~(K119Q) and hbFGF~(K119E),respectively.The mutants were produced by BL21(DE3)/pET3c expression sys...

  12. P190B RhoGAP overexpression in the developing mammary epithelium induces TGFβ-dependent fibroblast activation.

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    Melissa Gillette

    Full Text Available Rho GTPases mediate stromal-epithelial interactions that are important for mammary epithelial cell (MEC morphogenesis. Increased extracellular matrix (ECM deposition and reorganization affect MEC morphogenesis in a Rho GTPase-dependent manner. Although the effects of altered ECM on MEC morphogenesis have been described, how MECs regulate stromal deposition is not well understood. Previously, we showed that p190B RhoGAP overexpression disrupts mammary gland morphogenesis by inducing hyperbranching in association with stromal alterations. We therefore hypothesized that MEC overexpression of p190B regulates paracrine interactions to impact fibroblast activation. Using a combination of in vivo morphometric and immunohistochemical analyses and primary cell culture assays, we found that p190B overexpression in MECs activates fibroblasts leading to increased collagen, fibronectin, and laminin production and elevated expression of the collagen crosslinking enzyme lysyl oxidase. Phosphorylation of the TGF-β effector SMAD2 and expression of the TGF-β target gene αSma were increased in p190B-associated fibroblasts, suggesting that elevated TGF-β signaling promoted fibroblast activation. Mechanical tension and TGF-β cooperate to activate fibroblasts. Interestingly, active TGF-β was elevated in conditioned medium from p190B overexpressing MECs compared to control MECs, and p190B overexpressing MECs exhibited increased contractility in a collagen gel contraction assay. These data suggest that paracrine signaling from the p190B overexpressing MECs may activate TGF-β signaling in adjacent fibroblasts. In support of this, transfer of conditioned medium from p190B overexpressing MECs onto wildtype fibroblasts or co-culture of p190B overexpressing MECs with wildtype fibroblasts increased SMAD2 phosphorylation and mRNA expression of ECM genes in the fibroblasts when compared to fibroblasts treated with control CM or co-cultured with control MECs. The

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

    Directory of Open Access Journals (Sweden)

    Yau Sang Chan

    Full Text Available A dimeric 64-kDa glucosamine-specific lectin was purified from seeds of Phaseolus vulgaris cv. "brown kidney bean." The simple 2-step purification protocol involved affinity chromatography on Affi-gel blue gel and gel filtration by FPLC on Superdex 75. The lectin was absorbed on Affi-gel blue gel and desorbed using 1M NaCl in the starting buffer. Gel filtration on Superdex 75 yielded a major absorbance peak that gave a single 32-kDa band in SDS-PAGE. Hemagglutinating activity was completely preserved when the ambient temperature was in the range of 20 °C-60 °C. However, drastic reduction of the activity occurred at temperatures above 65 °C. Full hemagglutinating activity of the lectin was observed at an ambient pH of 3 to 12. About 50% activity remained at pH 0-2, and only residual activity was observed at pH 13-14. Hemagglutinating activity of the lectin was inhibited by glucosamine. The brown kidney bean lectin elicited maximum mitogenic activity toward murine splenocytes at 2.5 µM. The mitogenic activity was nearly completely eliminated in the presence of 250 mM glucosamine. The lectin also increased mRNA expression of the cytokines IL-2, TNF-α and IFN-γ. The lectin exhibited antiproliferative activity toward human breast cancer (MCF7 cells, hepatoma (HepG2 cells and nasopharyngeal carcinoma (CNE1 and CNE2 cells with IC(50 of 5.12 µM, 32.85 µM, 3.12 µM and 40.12 µM respectively after treatment for 24 hours. Flow cytometry with Annexin V and propidum iodide staining indicated apoptosis of MCF7 cells. Hoechst 33342 staining also indicated formation of apoptotic bodies in MCF7 cells after exposure to brown kidney bean lectin. Western blotting revealed that the lectin-induced apoptosis involved ER stress and unfolded protein response.

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

    Science.gov (United States)

    Chan, Yau Sang; Wong, Jack Ho; Fang, Evandro Fei; Pan, Wenliang; Ng, Tzi Bun

    2012-01-01

    A dimeric 64-kDa glucosamine-specific lectin was purified from seeds of Phaseolus vulgaris cv. "brown kidney bean." The simple 2-step purification protocol involved affinity chromatography on Affi-gel blue gel and gel filtration by FPLC on Superdex 75. The lectin was absorbed on Affi-gel blue gel and desorbed using 1M NaCl in the starting buffer. Gel filtration on Superdex 75 yielded a major absorbance peak that gave a single 32-kDa band in SDS-PAGE. Hemagglutinating activity was completely preserved when the ambient temperature was in the range of 20 °C-60 °C. However, drastic reduction of the activity occurred at temperatures above 65 °C. Full hemagglutinating activity of the lectin was observed at an ambient pH of 3 to 12. About 50% activity remained at pH 0-2, and only residual activity was observed at pH 13-14. Hemagglutinating activity of the lectin was inhibited by glucosamine. The brown kidney bean lectin elicited maximum mitogenic activity toward murine splenocytes at 2.5 µM. The mitogenic activity was nearly completely eliminated in the presence of 250 mM glucosamine. The lectin also increased mRNA expression of the cytokines IL-2, TNF-α and IFN-γ. The lectin exhibited antiproliferative activity toward human breast cancer (MCF7) cells, hepatoma (HepG2) cells and nasopharyngeal carcinoma (CNE1 and CNE2) cells with IC(50) of 5.12 µM, 32.85 µM, 3.12 µM and 40.12 µM respectively after treatment for 24 hours. Flow cytometry with Annexin V and propidum iodide staining indicated apoptosis of MCF7 cells. Hoechst 33342 staining also indicated formation of apoptotic bodies in MCF7 cells after exposure to brown kidney bean lectin. Western blotting revealed that the lectin-induced apoptosis involved ER stress and unfolded protein response.

  15. Distinct DNA-based epigenetic switches trigger transcriptional activation of silent genes in human dermal fibroblasts.

    Science.gov (United States)

    Pandian, Ganesh N; Taniguchi, Junichi; Junetha, Syed; Sato, Shinsuke; Han, Le; Saha, Abhijit; AnandhaKumar, Chandran; Bando, Toshikazu; Nagase, Hiroki; Vaijayanthi, Thangavel; Taylor, Rhys D; Sugiyama, Hiroshi

    2014-01-24

    The influential role of the epigenome in orchestrating genome-wide transcriptional activation instigates the demand for the artificial genetic switches with distinct DNA sequence recognition. Recently, we developed a novel class of epigenetically active small molecules called SAHA-PIPs by conjugating selective DNA binding pyrrole-imidazole polyamides (PIPs) with the histone deacetylase inhibitor SAHA. Screening studies revealed that certain SAHA-PIPs trigger targeted transcriptional activation of pluripotency and germ cell genes in mouse and human fibroblasts, respectively. Through microarray studies and functional analysis, here we demonstrate for the first time the remarkable ability of thirty-two different SAHA-PIPs to trigger the transcriptional activation of exclusive clusters of genes and noncoding RNAs. QRT-PCR validated the microarray data, and some SAHA-PIPs activated therapeutically significant genes like KSR2. Based on the aforementioned results, we propose the potential use of SAHA-PIPs as reagents capable of targeted transcriptional activation.

  16. Fatty Acid Accumulation and Resulting PPARα Activation in Fibroblasts due to Trifunctional Protein Deficiency

    Directory of Open Access Journals (Sweden)

    Masato Wakabayashi

    2012-01-01

    Full Text Available To examine fatty acid accumulation and its toxic effects in cells, we analyzed skin fibroblasts from six patients with mitochondrial trifunctional protein deficiency, who had abnormalities in the second through fourth reactions in fatty acid β-oxidation system. We found free fatty acid accumulation, enhanced three acyl-CoA dehydrogenases, catalyzing the first reaction in the β-oxidation system and being assumed to have normal activities in these patients, and PPARα activation that was confirmed in the experiments using MK886, a PPARα specific antagonist and fenofibrate, a PPARα specific agonist. These novel findings suggest that the fatty acid accumulation and the resulting PPARα activation are major causes of the increase in the β-oxidation ability as probable compensation for fatty acid metabolism in the patients’ fibroblasts, and that enhanced cell proliferation and increased oxidative stress due to the PPARα activation relate to the development of specific clinical features such as hypertrophic cardiomyopathy, slight hepatomegaly, and skeletal myopathy. Additionally, significant suppression of the PPARα activation by means of MK886 treatment is assumed to provide a new method of treating this deficiency.

  17. Testicular histomorphometry and the proliferative and apoptotic activities of the seminiferous epithelium in Syrian hamster (Mesocricetus auratus) during regression owing to short photoperiod.

    Science.gov (United States)

    Seco-Rovira, V; Beltrán-Frutos, E; Ferrer, C; Saez, F J; Madrid, J F; Canteras, M; Pastor, L M

    2015-05-01

    During the non-breeding season some animals exhibit testicular atrophy, decreased testicular weight and reduced seminiferous tubule diameter accompanied by depletion of the seminiferous epithelium. Some cellular factors involved in this depletion are changes in germ cell proliferation and apoptosis. In the Syrian hamster this depletion has been studied histologically and in terms of the involvement of proliferation and apoptosis in the seminiferous epithelium of fully regressed testes. The objectives of this study included the histomorphometrical characterization of the testis and the determination of the proliferative and apoptotic activity of germ cells in the seminiferous epithelium during testicular regression owing to short photoperiod. The study was performed using conventional light microscopy (hematoxylin and eosin), proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end labelling staining, image analysis software, and transmission electron microscopy in three established regression groups: mild regression (MR), strong regression (SR), and total regression (TR). Morphometrically a gradual decrease in total tubular area and in the testicular, tubular, and epithelial volumes was observed during testicular regression. Interstitial and luminal volumes decreased from the MR group onwards. The tubular length decreased from MR to SR. As regards spermatogonial proliferation, only an initial decrease in proliferative activity was observed, whereas apoptotic germ cell activity increased throughout regression. The number of germ cells studied decreased throughout the process of testicular regression. In conclusion, testicular regression in Syrian hamster comprises two histomorphometrical phases, the first involving a decrease in seminiferous tubular diameter and volume and the second involving shortening of the seminiferous tubule and a decrease in interstitial volume. At the cellular level, there is an

  18. Inhibition of Intermediate-Conductance Calcium-Activated K Channel (KCa3.1) and Fibroblast Mitogenesis by α-Linolenic Acid and Alterations of Channel Expression in the Lysosomal Storage Disorders, Fabry Disease, and Niemann Pick C

    Science.gov (United States)

    Oliván-Viguera, Aida; Lozano-Gerona, Javier; López de Frutos, Laura; Cebolla, Jorge J.; Irún, Pilar; Abarca-Lachen, Edgar; García-Malinis, Ana J.; García-Otín, Ángel Luis; Gilaberte, Yolanda; Giraldo, Pilar; Köhler, Ralf

    2017-01-01

    The calcium/calmodulin-gated KCa3.1 channel regulates normal and abnormal mitogenesis by controlling K+-efflux, cell volume, and membrane hyperpolarization-driven calcium-entry. Recent studies suggest modulation of KCa3.1 by omega-3 fatty acids as negative modulators and impaired KCa3.1 functions in the inherited lysosomal storage disorder (LSD), Fabry disease (FD). In the first part of present study, we characterize KCa3.1 in murine and human fibroblasts and test the impact of omega-3 fatty acids on fibroblast proliferation. In the second, we study whether KCa3.1 is altered in the LSDs, FD, and Niemann-Pick disease type C (NPC). Our patch-clamp and mRNA-expression studies on murine and human fibroblasts show functional expression of KCa3.1. KCa currents display the typical pharmacological fingerprint of KCa3.1: Ca2+-activation, potentiation by the positive-gating modulators, SKA-31 and SKA-121, and inhibition by TRAM-34, Senicapoc (ICA-17043), and the negative-gating modulator, 13b. Considering modulation by omega-3 fatty acids we found that α-linolenic acid (α-LA) and docosahexanenoic acid (DHA) inhibit KCa3.1 currents and strongly reduce fibroblast growth. The α-LA-rich linseed oil and γ-LA-rich borage oil at 0.5% produce channel inhibition while α-LA/γ-LA-low oils has no anti-proliferative effect. Concerning KCa3.1 in LSD, mRNA expression studies, and patch-clamp on primary fibroblasts from FD and NPC patients reveal lower KCa3.1-gene expression and membrane expression than in control fibroblasts. In conclusion, the omega-3 fatty acid, α-LA, and α-LA/γ-LA-rich plant oils, inhibit fibroblast KCa3.1 channels and mitogenesis. Reduced fibroblast KCa3.1 functions are a feature and possible biomarker of cell dysfunction in FD and NPC and supports the concept that biased lipid metabolism is capable of negatively modulating KCa3.1 expression. PMID:28197106

  19. Single Exposure of Human Oral Mucosa Fibroblasts to Ultraviolet B Radiation Reduces Proliferation and Induces COX-2 Expression and Activation

    Directory of Open Access Journals (Sweden)

    Y Boza

    2010-12-01

    Full Text Available The lip vermillion constitutes a transition tissue, between oral mucosa and skin, where oral mucosal cells from epithelial and connective tissue compartments are exposed to ultraviolet (UV sunlight. Fibroblasts are abundant resident cells of the connective tissue which are key regulators of extracellular matrix composition, as well as, epithelial and endothelial cell function. UVB light, an inherent component of sunlight, causes several alterations in skin fibroblasts, including premature senescence and increased cyclooxygenase (COX-2 expression. To assess if UVB irradiation had similar effects on fibroblasts derived from human oral mucosa (HOM, primary cultures of HOM fibroblasts were irradiated with a single dose of 30 or 60 mJ/cm²of UVB light or sham-irradiated. Fibroblast proliferation was assessed from 3 to 48 hrs after UVB-irradiation utilizing [³H]-thymidine incorporation and MTT assays. In addition, COX-2 mRNA expression was detected by RT-PCR, and PGE2 production was assessed using enzyme immunoassay from 0.5 to 24 hrs after UVB-irradiation. The results showed a significant decrease in proliferation of UVB-irradiated HOM fibroblasts as compared to controls as measured by both [³H]-thymidine incorporation and MTT assays (p<0.001. HOM fibroblasts had increased COX-2 mRNA expression at 0.5 and 12 hrs after irradiation, and PGE2 production was elevated at 12 and 24 hrs post-irradiation as compared to controls (p<0.05. The results showed an inhibitory effect of a single dose of UVB irradiation on HOM fibroblast proliferation with an increase in COX-2 expression and activation. Therefore, photodamaged fibroblasts may play and important role in the pathogenesis of UV-induced lesions of the lip.

  20. c-Ski activates cancer-associated fibroblasts to regulate breast cancer cell invasion.

    Science.gov (United States)

    Wang, Liyang; Hou, Yixuan; Sun, Yan; Zhao, Liuyang; Tang, Xi; Hu, Ping; Yang, Jiajia; Zeng, Zongyue; Yang, Guanglun; Cui, Xiaojiang; Liu, Manran

    2013-12-01

    Aberrant expression of c-Ski oncoprotein in some tumor cells has been shown to be associated with cancer development. However, the role of c-Ski in cancer-associated fibroblasts (CAFs) of tumor microenvironment has not been characterized. In the current study, we found that c-Ski is highly expressed in CAFs derived from breast carcinoma microenvironment and this CAF-associated c-Ski expression is associated with invasion and metastasis of human breast tumors. We showed that c-Ski overexpression in immortalized breast normal fibroblasts (NFs) induces conversion to breast CAFs by repressing p53 and thereby upregulating SDF-1 in NFs. SDF-1 treatment or p53 knockdown in NFs had similar effects on the activation of NFs as c-Ski overexpression. The c-Ski-activated CAFs show increased proliferation, migration, invasion and contraction compared with NFs. Furthermore, c-Ski-activated CAFs facilitated the migration and invasion of MDA-MB-231 breast cancer cells. Our data suggest that c-Ski is an important regulator in the activation of CAFs and may serve as a potential therapeutic target to block breast cancer progression.

  1. Relationship between proliferative activity of bone marrow micrometastasis and plasmatic level of a new cancer marker: lipid associated sialic acid (LASA) in human breast cancer.

    Science.gov (United States)

    Ginsbourg, M; Musset, M; Misset, J L; Mathé, G

    1986-01-01

    The correlation between elevated level of a new plasma cancer marker, Lipid Associated Sialic Acid (LASA) and detection of bone marrow heterotopic epithelial cells by monoclonal antibodies using an immunocytologic technique, associated with the study of the proliferative activity of these heterotopic cells by measurement of their labelling index (LI) was analyzed in 158 samples obtained from 94 breast cancer patients. Six different groups of patients in complete remission of breast cancer, after radical treatment of the primary (minimal residual disease (MRD] were defined, according to the presence with or without proliferative activity of heterotopic cells in the bone marrow or the absence of such cells and to the level, normal or elevated of LASA in the serum of the same patient at the same time. 115 samples (73%) of the patients had an elevated LASA level at the time of the study among which 71 (45%) came from patients in which heterotopic cells were detected in the bone marrow, 32 (20%) of which with proliferative activity (LI+) 39 (25%) without (LI-). In 44 (28%) samples, no heterotopic cells were detected in the B.M. 43 samples (27%) of the patients had a normal LASA level. In 29 (18%) no heterotopic cells could be detected in the B.M. In only 14 could such cells be found, 5 with LI+, 9 with LI-. Both of these cytological and biological parameters can be useful markers of minimal residual disease and help to determine the prognosis and define optimal therapeutic strategy for curable breast cancer. Their prognostic value in predicting relapse awaits further observation with longer follow-up.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Effects of free amino acids on cytokine secretion and proliferative activity of feline T cells in an in vitro study using the cell line MYA-1.

    Science.gov (United States)

    Paßlack, Nadine; Doherr, Marcus G; Zentek, Jürgen

    2016-10-01

    In vitro studies might be an interesting screening method for targeted in vivo studies in the field of immunonutrition and help to reduce and refine animal studies. As the role of amino acids for immune function of cats has not been evaluated in detail so far, the present study aimed at investigating the effects of eight different amino acids (arginine, leucine, isoleucine, valine, glutamine, lysine, threonine and tryptophan) in six concentrations each (0, 0.25, 0.5, 1, 2 and 8x the cat blood level) on cytokine secretion and proliferative activity of feline T cells (MYA-1) in vitro. The results demonstrated that high doses of arginine increased IL-4, IL-10 and TNF-α secretion of T cells, while increasing concentrations of lysine increased IL-10 secretion and proliferative activity of the T cells. High doses of leucine enhanced GM-CSF and IL-10 secretion, while concentrations of threonine in the cell culture media greater than blood concentration also increased GM-CSF and additionally TNF-α secretion of the cells. The effects of glutamine and isoleucine on T cell function were only small. In conclusion, the present in vitro study could evaluate the immunomodulating potential of specific amino acids for feline T cell function. High doses of arginine, lysine, leucine and threonine had a significant impact on cytokine secretion and proliferative activity of the T cells. Targeted in vivo studies should investigate the clinical relevance of dietary supplementation of those amino acids in healthy and diseased cats as a next step.

  3. Glycosides from Stevia rebaudiana Bertoni Possess Insulin-Mimetic and Antioxidant Activities in Rat Cardiac Fibroblasts

    Directory of Open Access Journals (Sweden)

    Cecilia Prata

    2017-01-01

    Full Text Available Stevia rebaudiana Bertoni is a shrub having a high content of sweet diterpenoid glycosides in its leaves, mainly stevioside and rebaudioside A, which are used as noncaloric, natural sweeteners. The aim of this study was to deepen the knowledge about the insulin-mimetic effect exerted by four different mixtures of steviol glycosides, rich in stevioside and rebaudioside A, in neonatal rat cardiac fibroblasts. The potential antioxidant activity of these steviol glycosides was also assessed, as oxidative stress is associated with diabetes. Likewise the insulin effect, steviol glycosides caused an increase in glucose uptake into rat fibroblasts by activating the PI3K/Akt pathway, thus inducing Glut4 translocation to the plasma membrane. The presence of S961, an insulin antagonist, completely abolished these effects, allowing to hypothesize that steviol glycosides could act as ligands of the same receptor engaged by insulin. Moreover, steviol glycosides counteracted oxidative stress by increasing reduced glutathione intracellular levels and upregulating expression and activity of the two antioxidant enzymes superoxide dismutase and catalase. The present work unravels the insulin-mimetic effect and the antioxidant property exerted by steviol glycosides, suggesting their potential beneficial role in the cotreatment of diabetes and in health maintenance.

  4. Glycosides from Stevia rebaudiana Bertoni Possess Insulin-Mimetic and Antioxidant Activities in Rat Cardiac Fibroblasts

    Science.gov (United States)

    Prata, Cecilia; Zambonin, Laura; Rizzo, Benedetta; Vieceli Dalla Sega, Francesco

    2017-01-01

    Stevia rebaudiana Bertoni is a shrub having a high content of sweet diterpenoid glycosides in its leaves, mainly stevioside and rebaudioside A, which are used as noncaloric, natural sweeteners. The aim of this study was to deepen the knowledge about the insulin-mimetic effect exerted by four different mixtures of steviol glycosides, rich in stevioside and rebaudioside A, in neonatal rat cardiac fibroblasts. The potential antioxidant activity of these steviol glycosides was also assessed, as oxidative stress is associated with diabetes. Likewise the insulin effect, steviol glycosides caused an increase in glucose uptake into rat fibroblasts by activating the PI3K/Akt pathway, thus inducing Glut4 translocation to the plasma membrane. The presence of S961, an insulin antagonist, completely abolished these effects, allowing to hypothesize that steviol glycosides could act as ligands of the same receptor engaged by insulin. Moreover, steviol glycosides counteracted oxidative stress by increasing reduced glutathione intracellular levels and upregulating expression and activity of the two antioxidant enzymes superoxide dismutase and catalase. The present work unravels the insulin-mimetic effect and the antioxidant property exerted by steviol glycosides, suggesting their potential beneficial role in the cotreatment of diabetes and in health maintenance.

  5. PGE2 reduces MMP-14 and increases plasminogen activator inhibitor-1 in cardiac fibroblasts.

    Science.gov (United States)

    Kassem, Kamal M; Clevenger, Margarette H; Szandzik, David L; Peterson, Edward; Harding, Pamela

    2014-10-01

    Prostaglandin E2 (PGE2) is elevated during cardiac injury and we have previously shown that mice lacking the PGE2 EP4 receptor display dilated cardiomyopathy (DCM) with increased expression of the membrane type matrix metalloproteinase, MMP-14. We thus hypothesized that PGE2 regulates expression of MMP-14 and also affects fibroblast migration. Primary cultures of neonatal rat ventricular fibroblasts (NVFs) were used to test the effects of PGE2. Gene and protein expression was assessed by real time RT-PCR and Western blot, MMP activity was determined by zymography and migration of NVF was assessed by motility in a transwell system. PGE2 reduced expression of MMP-14 and these effects were antagonized by an EP4 antagonist. An EP4 agonist mimicked the effect of PGE2. PGE2 also increased mRNA and protein levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of MMP activation. However, PGE2-stimulation of PAI-1 was mediated by the EP1/EP3 receptor and not EP4. Migration of NVF was assessed by motility in a transwell system. Treatment of NVFs with PGE2 reduced the number of cells migrating toward 10% FCS. Treatment with the EP2 agonist also reduced migration but did not affect MMP-14 expression or PAI-1. Our results suggest that PGE2 utilizes different receptors and mechanisms to ultimately decrease MMP expression and NVF migration. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol

    Science.gov (United States)

    Donejko, Magdalena; Rysiak, Edyta; Galicka, Elżbieta; Terlikowski, Robert; Głażewska, Edyta Katarzyna; Przylipiak, Andrzej

    2017-01-01

    Aim The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA) on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK), and the influence of HA on those processes. Materials and methods Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT) assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Results Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. Conclusion This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol. PMID:28293103

  7. Deregulated MAPK activity prevents adipocyte differentiation of fibroblasts lacking the retinoblastoma protein

    DEFF Research Database (Denmark)

    Hansen, Jacob B; Petersen, Rasmus K; Jørgensen, Claus;

    2002-01-01

    A functional retinoblastoma protein (pRB) is required for adipose conversion of preadipocyte cell lines and primary mouse embryo fibroblasts (MEFs) in response to treatment with standard adipogenic inducers. Interestingly, lack of functional pRB in MEFs was recently linked to elevated Ras activity....../Akt are significantly increased in pRB-deficient MEFs both before and after the addition of adipogenic inducers. Consistently, we detected higher levels of activated Ras in MEFs lacking pRB. Suppression of ERK1/2 activation by the MEK inhibitor UO126 restored the ability of pRB-deficient MEFs to undergo adipocyte...... differentiation, as manifested by expression of adipocyte marker genes and lipid accumulation. Furthermore and reflecting the elevated levels of activated PKB/Akt in the pRB-deficient MEFs, differentiation proceeded in an insulin-independent manner. In conclusion, we suggest that pRB plays a pivotal role...

  8. Uric acid stimulates proliferative pathways in vascular smooth muscle cells through the activation of p38 MAPK, p44/42 MAPK and PDGFRβ.

    Science.gov (United States)

    Kırça, M; Oğuz, N; Çetin, A; Uzuner, F; Yeşilkaya, A

    2017-04-01

    Hyperuricemia and angiotensin II (Ang II) may have a pathogenetic role in the development of hypertension and atherosclerosis as well as cardiovascular disease (CVD) and its prognosis. The purpose of this study was to investigate whether uric acid can induce proliferative pathways of vascular smooth muscle cell (VSMC) that are thought to be responsible for the development of CVD. The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), p44/42 mitogen-activated protein kinase (p44/42 MAPK) and platelet-derived growth factor receptor β (PDGFRβ) was measured by Elisa and Western blot techniques to determine the activation of proliferative pathways in primary cultured VSMCs from rat aorta. Results demonstrated that uric acid can stimulate p38 MAPK, p44/42 MAPK and PDGFRβ phosphorylation in a time- and concentration-dependent manner. Furthermore, treatment of VSMCs with the angiotensin II type I receptor (AT1R) inhibitor losartan suppressed p38 MAPK and p44/42 MAPK induction by uric acid. The stimulatory effect of uric acid on p38 MAPK was higher compared to that of Ang II. The results of this study show for the first time that uric acid-induced PDGFRβ phosphorylation plays a crucial role in the development of CVDs and that elevated uric acid levels could be a potential therapeutical target in CVD patients.

  9. Basic Fibroblast Growth Factor Contributes to a Shift in the Angioregulatory Activity of Retinal Glial (Müller) Cells

    Science.gov (United States)

    Yafai, Yousef; Iandiev, Ianors; Lange, Johannes; Yang, Xiu Mei; Wiedemann, Peter; Bringmann, Andreas; Eichler, Wolfram

    2013-01-01

    Basic fibroblast growth factor (bFGF) is a pleiotropic cytokine with pro-angiogenic and neurotrophic effects. The angioregulatory role of this molecule may become especially significant in retinal neovascularization, which is a hallmark of a number of ischemic eye diseases. This study was undertaken to reveal expression characteristics of bFGF, produced by retinal glial (Müller) cells, and to determine conditions under which glial bFGF may stimulate the proliferation of retinal microvascular endothelial cells. Immunofluorescence labeling detected bFGF in Müller cells of the rat retina and in acutely isolated Müller cells with bFGF levels, which increased after ischemia-reperfusion in postischemic retinas. In patients with proliferative diabetic retinopathy or myopia, the immunoreactivity of bFGF co-localized to glial fibrillary acidic protein (GFAP)-positive cells in surgically excised retinal tissues. RT-PCR and ELISA analyses indicated that cultured Müller cells produce bFGF, which is elevated under hypoxia or oxidative stress, as well as under stimulation with various growth factors and cytokines, including pro-inflammatory factors. When retinal endothelial cells were cultured in the presence of media from hypoxia (0.2%)-conditioned Müller cells, a distinct picture of endothelial cell proliferation emerged. Media from 24-h cultured Müller cells inhibited proliferation, whereas 72-h conditioned media elicited a stimulatory effect. BFGF-neutralizing antibodies suppressed the enhanced endothelial cell proliferation to a similar extent as anti-VEGF antibodies. Furthermore, phosphorylation of extracellular signal-regulated kinases (ERK−1/−2) in retinal endothelial cells was increased when the cells were cultured in 72-h conditioned media, while neutralizing bFGF attenuated the activation of this signaling pathway. These data provide evidence that retinal (glial) Müller cells are major sources of bFGF in the ischemic retina. Müller cells under

  10. Basic fibroblast growth factor contributes to a shift in the angioregulatory activity of retinal glial (Muller cells.

    Directory of Open Access Journals (Sweden)

    Yousef Yafai

    Full Text Available Basic fibroblast growth factor (bFGF is a pleiotropic cytokine with pro-angiogenic and neurotrophic effects. The angioregulatory role of this molecule may become especially significant in retinal neovascularization, which is a hallmark of a number of ischemic eye diseases. This study was undertaken to reveal expression characteristics of bFGF, produced by retinal glial (Müller cells, and to determine conditions under which glial bFGF may stimulate the proliferation of retinal microvascular endothelial cells. Immunofluorescence labeling detected bFGF in Müller cells of the rat retina and in acutely isolated Müller cells with bFGF levels, which increased after ischemia-reperfusion in postischemic retinas. In patients with proliferative diabetic retinopathy or myopia, the immunoreactivity of bFGF co-localized to glial fibrillary acidic protein (GFAP-positive cells in surgically excised retinal tissues. RT-PCR and ELISA analyses indicated that cultured Müller cells produce bFGF, which is elevated under hypoxia or oxidative stress, as well as under stimulation with various growth factors and cytokines, including pro-inflammatory factors. When retinal endothelial cells were cultured in the presence of media from hypoxia (0.2%-conditioned Müller cells, a distinct picture of endothelial cell proliferation emerged. Media from 24-h cultured Müller cells inhibited proliferation, whereas 72-h conditioned media elicited a stimulatory effect. BFGF-neutralizing antibodies suppressed the enhanced endothelial cell proliferation to a similar extent as anti-VEGF antibodies. Furthermore, phosphorylation of extracellular signal-regulated kinases (ERK-1/-2 in retinal endothelial cells was increased when the cells were cultured in 72-h conditioned media, while neutralizing bFGF attenuated the activation of this signaling pathway. These data provide evidence that retinal (glial Müller cells are major sources of bFGF in the ischemic retina. Müller cells under

  11. Current perspectives on the role of orbital fibroblasts in the pathogenesis of Graves' ophthalmopathy.

    Science.gov (United States)

    Dik, Willem A; Virakul, Sita; van Steensel, Leendert

    2016-01-01

    Graves' ophthalmopathy (GO) is an extra-thyroidal complication of Graves' disease (GD; Graves' hyperthyroidism) characterized by orbital tissue inflammation, expansion, remodeling and fibrosis. Although the initiating trigger of GO is still indistinct, excessive orbital fibroblast activity is at the heart of its pathogenesis. Orbital fibroblasts are activated by cellular interactions with immune cells and the soluble factors they secrete. Orbital fibroblasts, especially from GO patients, express the thyrotropin receptor (TSH-receptor; TSHR), and activation of the orbital fibroblast population by stimulatory autoantibodies directed against the TSHR may provide an important link between GD and GO. Furthermore, stimulatory autoantibodies directed against the insulin-like growth factor-1 receptor have been proposed to contribute to orbital fibroblast activation in GO. Activated orbital fibroblasts produce inflammatory mediators thereby contributing to the orbital inflammatory process in GO. Moreover, orbital fibroblasts exhibit robust proliferative activity and extracellular matrix (especially hyaluronan) synthesizing capacity and can differentiate into adipocytes and myofibroblasts with disease progression, thereby contributing to tissue expansion/remodeling and fibrosis in GO. Orbital fibroblasts, especially those from GO patients, exhibit a hyper-responsive phenotype when compared to fibroblasts from other anatomical regions, which may further contribute to GO pathogenesis. Fibrocytes have been identified as additional source of orbital fibroblasts in GO, where they may contribute to orbital tissue inflammation, adipogenesis and remodeling/fibrosis. This review addresses our current view on the role that orbital fibroblasts fulfill in GO pathogenesis and both established as well as less established not fully crystallized concepts that need future studies will be discussed.

  12. The application of the fibroblast activation protein α-targeted immunotherapy strategy.

    Science.gov (United States)

    Jiang, Guan-Min; Xu, Wei; Du, Jun; Zhang, Kun-Shui; Zhang, Qiu-Gui; Wang, Xiao-Wei; Liu, Zhi-Gang; Liu, Shuang-Quan; Xie, Wan-Ying; Liu, Hui-Fang; Liu, Jing-Shi; Wu, Bai-Ping

    2016-05-31

    Cancer immunotherapy has primarily been focused on attacking tumor cells. However, given the close interaction between tumor cells and cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME), CAF-targeted strategies could also contribute to an integrated cancer immunotherapy. Fibroblast activation protein α (FAP α) is not detectible in normal tissues, but is overexpressed by CAFs and is the predominant component of the stroma in most types of cancer. FAP α has both dipeptidyl peptidase and endopeptidase activities, cleaving substrates at a post-proline bond. When all FAP α-expressing cells (stromal and cancerous) are destroyed, tumors rapidly die. Furthermore, a FAP α antibody, FAP α vaccine, and modified vaccine all inhibit tumor growth and prolong survival in mouse models, suggesting FAP α is an adaptive tumor-associated antigen. This review highlights the role of FAP α in tumor development, explores the relationship between FAP α and immune suppression in the TME, and discusses FAP α as a potential immunotherapeutic target.

  13. Lidocaine-induced apoptosis of gingival fibroblasts: participation of cAMP and PKC activity.

    Science.gov (United States)

    Villarruel, Emmanuel Quinteros; Borda, Enri; Sterin-Borda, Leonor; Orman, Betina

    2011-08-01

    Local anaesthetics are drugs that prevent or relieve pain by interrupting nervous conduction and are the most commonly used drugs in dentistry. Their main targets of action are voltage-dependent Na+ channels. The Na+ channel is modulated by phosphorylation of two enzymes: PKA (protein kinase A) and PKC (protein kinase C). We studied the ability of lidocaine to modulate programmed cell death of human gingival fibroblasts and the mechanisms involved in this process. Lidocaine (10-5 to 10-7 M) stimulated apoptosis in primary cultures and the caspase-3 activity in a concentration-dependent manner. The stimulatory effect of lidocaine on apoptosis was attenuated in the presence of HA 1004 (PKA inhibitor) and stimulated by staurosporine and Go 6976 (PKC inhibitors). Lidocaine-induced apoptotic nuclei correlated positively with cAMP accumulation and negatively with PKC activity. These results show that lidocaine promotes apoptosis in human gingival fibroblasts at concentrations used for local anaesthesia. The mechanism involves PKA stimulation and PKC inhibition, which in turn stimulates caspase-3 and leads to programmed cell death.

  14. Sodium arsenite induces chromosome endoreduplication and inhibits protein phosphatase activity in human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Rong-Nan Huang; I-Ching Ho; Ling-Hui Yih [Institute of Biomedical Sciences, Taiwan (China)] [and others

    1995-08-01

    Arsenic, strongly associated with increased risks of human cancers, is a potent clastogen in a variety of mammalian cell systems. The effect of sodium arsenite (a trivalent arsenic compound) on chromatid separation was studied in human skin fibroblasts (HFW). Human fibroblasts were arrested in S phase by the aid of serum starvation and aphidicolin blocking and then these cells were allowed to synchronously progress into G2 phase. Treatment of the G2-enriched HFW cells with sodium arsenite (0-200 {mu}M) resulted in arrest of cells in the G2 phase, interference with mitotic division, inhibition of spindle assembly, and induction of chromosome endoreduplication in their second mitosis. Sodium arsenite treatment also inhibited the activities of serine/threonine protein phosphatases and enhanced phosphorylation levels of a small heat shock protein (HSP27). These results suggest that sodium arsenite may mimic okadaic acid to induce chromosome endoreduplication through its inhibitory effect on protein phosphatase activity. 61 refs., 6 figs., 2 tabs.

  15. Thy-1 attenuates TNF-alpha-activated gene expression in mouse embryonic fibroblasts via Src family kinase.

    Directory of Open Access Journals (Sweden)

    Bin Shan

    Full Text Available Heterogeneous surface expression of Thy-1 in fibroblasts modulates inflammation and may thereby modulate injury and repair. As a paradigm, patients with idiopathic pulmonary fibrosis, a disease with pathologic features of chronic inflammation, demonstrate an absence of Thy-1 immunoreactivity within areas of fibrotic activity (fibroblast foci in contrast to the predominant Thy-1 expressing fibroblasts in the normal lung. Likewise, Thy-1 deficient mice display more severe lung fibrosis in response to an inflammatory injury than wildtype littermates. We investigated the role of Thy-1 in the response of fibroblasts to the pro-inflammatory cytokine TNF-alpha. Our study demonstrates distinct profiles of TNF-alpha-activated gene expression in Thy-1 positive (Thy-1+ and negative (Thy-1- subsets of mouse embryonic fibroblasts (MEF. TNF-alpha induced a robust activation of MMP-9, ICAM-1, and the IL-8 promoter driven reporter in Thy-1- MEFs, in contrast to only a modest increase in Thy-1+ counterparts. Consistently, ectopic expression of Thy-1 in Thy-1- MEFs significantly attenuated TNF-alpha-activated gene expression. Mechanistically, TNF-alpha activated Src family kinase (SFK only in Thy-1- MEFs. Blockade of SFK activation abrogated TNF-alpha-activated gene expression in Thy-1- MEFs, whereas restoration of SFK activation rescued the TNF-alpha response in Thy-1+ MEFs. Our findings suggest that Thy-1 down-regulates TNF-alpha-activated gene expression via interfering with SFK- and NF-kappaB-mediated transactivation. The current study provides a novel mechanistic insight to the distinct roles of fibroblast Thy-1 subsets in inflammation.

  16. Expression and role of fibroblast activation protein-alpha in microinvasive breast carcinoma

    Directory of Open Access Journals (Sweden)

    Hua Xing

    2011-11-01

    Full Text Available Abstract Background Diagnosis of ductal carcinoma in situ (DCIS in breast cancer cases is challenging for pathologist due to a variety of in situ patterns and artefacts, which could be misinterpreted as stromal invasion. Microinvasion is detected by the presence of cytologically malignant cells outside the confines of the basement membrane and myoepithelium. When malignant cells invade the stroma, there is tissue remodeling induced by perturbed stromal-epithelial interactions. Carcinoma-associated fibroblasts (CAFs are main cells in the microenvironment of the remodeled tumor-host interface. They are characterized by the expression of the specific fibroblast activation protein-alpha (FAP-α, and differ from that of normal fibroblasts exhibiting an immunophenotype of CD34. We hypothesized that staining for FAP-α may be helpful in determining whether DCIS has microinvasion. Methods 349 excised breast specimens were immunostained for smooth muscle actin SMA, CD34, FAP-α, and Calponin. Study material was divided into 5 groups: group 1: normal mammary tissues of healthy women after plastic surgery; group 2: usual ductal hyperplasia (UDH; group 3: DCIS without microinvasion on H & E stain; group 4: DCIS with microinvasion on H & E stain (DCIS-MI, and group 5: invasive ductal carcinoma (IDC. A comparative evaluation of the four immunostains was conducted. Results Our results demonstrated that using FAP-α and Calponin adjunctively improved the sensitivity of pathological diagnosis of DCIS-MI by 11.29%, whereas the adjunctive use of FAP-α and Calponin improved the sensitivity of pathological diagnosis of DCIS by 13.6%. Conclusions This study provides the first evidence that immunostaining with FAP-α and Calponin can serve as a novel marker for pathologically diagnosing whether DCIS has microinvasion.

  17. Evaluation of anti-proliferative and anti-inflammatory activities of Pelagia noctiluca venom in Lipopolysaccharide/Interferon-γ stimulated RAW264.7 macrophages.

    Science.gov (United States)

    Ayed, Yosra; Sghaier, Rabiaa Manel; Laouini, Dhafer; Bacha, Hassen

    2016-12-01

    Components of Pelagia noctiluca (P. noctiluca) venom were evaluated for their anticancer and nitric Oxide (NO) inhibition activities. Three fractions, out of four, obtained by gel filtration on Sephadex G75 of P. noctiluca venom revealed an important selective anti-proliferative activity on several cell lines such as human bladder carcinoma (RT112), human glioblastoma (U87), and human myelogenous leukemia (K562) but not on mitogen-stimulated peripheral blood mononuclear cells. Interestingly, P. noctiluca components showed an important dose-dependent anti-inflammatory activity, through inhibition of NO production via transcriptional regulation of Inducible NO Synthase (iNOS), in IFN-γ/LPS stimulated RAW 264.7 macrophages. These data strongly suggest that P. noctiluca venom could be used as a natural inhibitor of cancer cell lines and a potent anti-inflammatory agent for the treatment of anti-inflammatory diseases.

  18. A peroxisome proliferator-activated receptor ligand MCC-555 imparts anti-proliferative response in pancreatic cancer cells by PPARgamma-independent up-regulation of KLF4

    Energy Technology Data Exchange (ETDEWEB)

    Min, Kyung-Won [Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Zhang, Xiaobo [Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi, 712100 (China); Imchen, Temjenmongla [Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Baek, Seung Joon, E-mail: sbaek2@utk.edu [Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States)

    2012-09-01

    MCC-555 is a novel PPARα/γ dual ligand of the thiazolidinedione class and was recently developed as an anti-diabetic drug with unique properties. MCC-555 also has anti-proliferative activity through growth inhibition and apoptosis induction in several cancer cell types. Our group has shown that MCC-555 targets several proteins in colorectal tumorigenesis including nonsteroidal anti-inflammatory drug (NSAID)-activated gene (NAG-1) which plays an important role in chemoprevention responsible for chemopreventive compounds. NAG-1 is a member of the TGF-β superfamily and is involved in tumor progression and development; however, NAG-1's roles in pancreatic cancer have not been studied. In this report, we found that MCC-555 alters not only NAG-1 expression, but also p21 and cyclin D1 expression. NAG-1 and p21 expression was not blocked by PPARγ-specific antagonist GW9662, suggesting that MCC-555-induced NAG-1 and p21 expression is independent of PPARγ activation. However, decreasing cyclin D1 by MCC-555 seems to be affected by PPARγ activation. Further, we found that the GC box located in the NAG-1 promoter play an important role in NAG-1 transactivation by MCC-555. Subsequently, we screened several transcription factors that may bind to the GC box region in the NAG-1 promoter and found that KLF4 potentially binds to this region. Expression of KLF4 precedes NAG-1 and p21 expression in the presence of MCC-555, whereas blocking KLF4 expression using specific KLF4 siRNA showed that both NAG-1 and p21 expression by MCC-555 was blocked. In conclusion, MCC-555's actions on anti-proliferation involve both PPARγ-dependent and -independent pathways, thereby enhancing anti-tumorigenesis in pancreatic cancer cells. -- Highlights: ► PPARα/γ ligand MCC-555 exhibits anti-proliferative activity in pancreatic cancer cells. ► MCC-555 affects KLF4 expression following by NAG-1 and p21 expression in a PPARγ independent manner. ► MCC-555 also affects cyclin D1 down

  19. Screening of plants acting against Heterometrus laoticus scorpion venom activity on fibroblast cell lysis.

    Science.gov (United States)

    Uawonggul, Nunthawun; Chaveerach, Arunrat; Thammasirirak, Sompong; Arkaravichien, Tarinee; Chuachan, Chattong; Daduang, Sakda

    2006-01-16

    The aqueous extracts of 64 plant species, listed as animal- or insect-bite antidotes in old Thai drug recipes were screened for their activity against fibroblast cell lysis after Heterometrus laoticus scorpion venom treatment. The venom was preincubated with plant extract for 30 min and furthered treated to confluent fibroblast cells for 30 min. More than 40% efficiency (test/control) was obtained from cell treatment with venom preincubated with extracts of Andrographis paniculata Nees (Acanthaceae), Barringtonia acutangula (L.) Gaertn. (Lecythidaceae), Calamus sp. (Palmae), Clinacanthus nutans Lindau (Acanthaceae), Euphorbia neriifolia L. (Euphorbiaceae), Ipomoea aquatica Forssk (Convolvulaceae), Mesua ferrea L. (Guttiferae), Passiflora laurifolia L. (Passifloraceae), Plectranthus amboinicus (Lour.) Spreng. (Labiatae), Ricinus communis L. (Euphorbiaceae), Rumex sp. (Polygonaceae) and Sapindus rarak DC. (Sapindaceae), indicating that they had a tendency to be scorpion venom antidotes. However, only Andrographis paniculata and Barringtonia acutangula extracts provided around 50% viable cells from extract treatments without venom preincubation. These two plant extracts are expected to be scorpion venom antidotes with low cytotoxicity.

  20. Recombinant N-Terminal Slit2 Inhibits TGF-β-Induced Fibroblast Activation and Renal Fibrosis.

    Science.gov (United States)

    Yuen, Darren A; Huang, Yi-Wei; Liu, Guang-Ying; Patel, Sajedabanu; Fang, Fei; Zhou, Joyce; Thai, Kerri; Sidiqi, Ahmad; Szeto, Stephen G; Chan, Lauren; Lu, Mingliang; He, Xiaolin; John, Rohan; Gilbert, Richard E; Scholey, James W; Robinson, Lisa A

    2016-09-01

    Fibrosis and inflammation are closely intertwined injury pathways present in nearly all forms of CKD for which few safe and effective therapies exist. Slit glycoproteins signaling through Roundabout (Robo) receptors have been described to have anti-inflammatory effects through regulation of leukocyte cytoskeletal organization. Notably, cytoskeletal reorganization is also required for fibroblast responses to TGF-β Here, we examined whether Slit2 also controls TGF-β-induced renal fibrosis. In cultured renal fibroblasts, which we found to express Slit2 and Robo-1, the bioactive N-terminal fragment of Slit2 inhibited TGF-β-induced collagen synthesis, actin cytoskeletal reorganization, and Smad2/3 transcriptional activity, but the inactive C-terminal fragment of Slit2 did not. In mouse models of postischemic renal fibrosis and obstructive uropathy, treatment with N-terminal Slit2 before or after injury inhibited the development of renal fibrosis and preserved renal function, whereas the C-terminal Slit2 had no effect. Our data suggest that administration of recombinant Slit2 may be a new treatment strategy to arrest chronic injury progression after ischemic and obstructive renal insults by not only attenuating inflammation but also, directly inhibiting renal fibrosis.

  1. Evaluation of cytotoxicity and gelatinases activity in 3T3 fibroblast cell by root repair materials

    Directory of Open Access Journals (Sweden)

    Varol Basak

    2016-09-01

    Full Text Available The aim of this study was to investigate the effects of calcium silicate-based products on cytotoxicity in the 3T3 fibroblast and gelatinolytic activity of matrix metalloproteinases (MMPs. 3T3 fibroblasts were incubated directly with Ortho Mineral trioxide aggregate (MTA, BioAggregate, Biodentine, MTA Plus, MTA Angelus and MTA Cerkamed for 24 hours and seven days. The cytotoxicity was determined using an MTT assay. Supernatants were collected to determine MMP-2 and MMP-9. Data were analysed using IBM SPSS 22. Seventh day extracts of Ortho MTA and Biodentine showed reduced cell viability. Specific characterization of MMPs in cell culture demonstrated that MMP-2 (62 kPa in the cell culture supernatants by gelatin zymography showed induced expression in four out of seven groups by 3T3 cells. No MMP-9 expression was observed. The cytotoxicity of materials revealed a significant difference in cell viability between the groups on the first and seventh days. The results of this study revealed minor cytotoxic effects for Ortho MTA and Biodentine. This study suggests that endodontic sealers induced production of MMP-2. MMP-9 might be expressed in small amounts when compared with MMP-2.

  2. Fibroblast growth factor 9 activates akt and MAPK pathways to stimulate steroidogenesis in mouse leydig cells.

    Science.gov (United States)

    Lai, Meng-Shao; Cheng, Yu-Sheng; Chen, Pei-Rong; Tsai, Shaw-Jenq; Huang, Bu-Miin

    2014-01-01

    Fibroblast growth factor 9 (FGF9) is a multifunctional polypeptide belonging to the FGF family and has functions related to bone formation, lens-fiber differentiation, nerve development, gap-junction formation and sex determination. In a previous study, we demonstrated that FGF9 stimulates the production of testosterone in mouse Leydig cells. In the present study, we used both primary mouse Leydig cells and MA-10 mouse Leydig tumor cells to further investigate the molecular mechanism of FGF9-stimulated steroidogenesis. Results showed that FGF9 significantly activated steroidogenesis in both mouse primary and tumor Leydig cells (psteroidogenesis in mouse Leydig cells. In conclusion, FGF9 specifically activated the Akt and ERK1/2 in normal mouse Leydig cells and the Akt, JNK and ERK1/2 in MA-10 mouse Leydig tumor cells to stimulate steroidogenesis.

  3. Antibacterial activity of extract and fractions from branches of Protium spruceanum and cytotoxicity on fibroblasts.

    Science.gov (United States)

    Amparo, Tatiane Roquete; Rodrigues, Ivanildes Vasconcelos; Seibert, Janaína Brandão; Souza, Rafaella Hilda Zaniti; Oliveira, Amanda Ribeiro de; Cabral, Vivette Appolinário Rodrigues; Vieira, Paula Melo de Abreu; Brandão, Geraldo Célio; Okuma, Adriana Akemi; Filho, Sidney Augusto Vieira; Teixeira, Luiz Fernando Medeiros; Souza, Gustavo Henrique Bianco de

    2017-07-20

    The crude ethanol extract (CEE) and fractions from branches of Protium spruceanum were subjected to antibacterial and cytotoxicity assays. Compounds of the most active fraction were identified by GC-MS and LC-MS. CEE was active against 19 bacteria and the ethyl acetate fraction (EAF) showed the lowest minimum bactericidal concentration (MBC 0.3-80.0 mg/mL). Through time-kill assay was observed that EAF induced rapid bactericidal effect against Staphylococcus saprophyticus. The cytotoxicity tests against L929 fibroblasts showed great potential of EAF on the treatment of infections caused by five bacteria (MBC < IC50). The results provide in vitro scientific support to the possible application of branches of P. spruceanum as antimicrobial agent that may contribute for treatment of infections.

  4. Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol

    Directory of Open Access Journals (Sweden)

    Donejko M

    2017-03-01

    Full Text Available Magdalena Donejko,1 Edyta Rysiak,2 Elżbieta Galicka,1 Robert Terlikowski,3 Edyta Katarzyna Głażewska,1 Andrzej Przylipiak1 1Department of Esthetic Medicine, 2Department of Medicinal Chemistry, Faculty of Pharmacy, 3Department of Health Restoration, Medical University of Białystok, Białystok, Poland Aim: The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK, and the influence of HA on those processes. Materials and methods: Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Results: Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. Conclusion: This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol. Keywords: apoptosis, skin fibroblast, focal adhesion kinase, hyaluronic acid, ethanol

  5. Prostaglandin E2 regulates angiogenesis via activation of fibroblast growth factor receptor-1.

    Science.gov (United States)

    Finetti, Federica; Solito, Raffaella; Morbidelli, Lucia; Giachetti, Antonio; Ziche, Marina; Donnini, Sandra

    2008-01-25

    Prostaglandin E(2) (PGE(2)) behaves as a mitogen in epithelial tumor cells as well as in many other cell types. We investigated the actions of PGE(2) on microvascular endothelial cells (capillary venular endothelial cells) with the purpose of delineating the signaling pathway leading to the acquisition of the angiogenic phenotype and to new vessel formation. PGE(2) (100 nM) produced activation of the fibroblast growth factor receptor 1 (FGFR-1), as measured by its phosphorylation, but not of vascular endothelial growth factor receptor 2. PGE(2) stimulated the EP3 subtype receptor, as deduced by abrogation of EP3 Galpha(i) subunit activity through pertussis toxin. Consistent with this result, in human umbilical venular endothelial cells missing the EP3 receptor, PGE(2) did not phosphorylate FGFR-1. Upon binding to its receptor, PGE(2) initiated an autocrine/paracrine signaling cascade involving the intracellular activation of c-Src, activation of matrix metalloproteinase (predominantly MMP2), which in turn caused the mobilization of membrane-anchored fibroblast growth factor-2 (FGF-2). In fact, in cells unable to release FGF-2 the transfection with both FGFR-1 and EP3 did not result in FGFR-1 phosphorylation in response to PGE(2). Relevance for the FGF2-FGFR-1 system was highlighted by confocal analysis, showing receptor internalization after cell exposure to the prostanoid. ERK1/2 appeared to be the distal signal involved, its phosphorylation being sensitive to either cSrc inhibitor or FGFR-1 blocker. Finally, PGE(2) stimulated cell migration and capillary formation in aortic rings, which were severely reduced by inhibitors of signaling molecules or by receptor antagonist. In conclusion, this study provides evidence for the involvement of FGFR-1 through FGF2 in eliciting PGE(2) angiogenic responses. This signaling pattern is similar to the autocrine-paracrine mechanism which operates in endothelial cells to support neovascular growth.

  6. A critical role of cardiac fibroblast-derived exosomes in activating renin angiotensin system in cardiomyocytes.

    Science.gov (United States)

    Lyu, Linmao; Wang, Hui; Li, Bin; Qin, Qingyun; Qi, Lei; Nagarkatti, Mitzi; Nagarkatti, Prakash; Janicki, Joseph S; Wang, Xing Li; Cui, Taixing

    2015-12-01

    Chronic activation of the myocardial renin angiotensin system (RAS) elevates the local level of angiotensin II (Ang II) thereby inducing pathological cardiac hypertrophy, which contributes to heart failure. However, the precise underlying mechanisms have not been fully delineated. Herein we report a novel paracrine mechanism between cardiac fibroblasts (CF)s and cardiomyocytes whereby Ang II induces pathological cardiac hypertrophy. In cultured CFs, Ang II treatment enhanced exosome release via the activation of Ang II receptor types 1 (AT1R) and 2 (AT2R), whereas lipopolysaccharide, insulin, endothelin (ET)-1, transforming growth factor beta (TGFβ)1 or hydrogen peroxide did not. The CF-derived exosomes upregulated the expression of renin, angiotensinogen, AT1R, and AT2R, downregulated angiotensin-converting enzyme 2, and enhanced Ang II production in cultured cardiomyocytes. In addition, the CF exosome-induced cardiomyocyte hypertrophy was blocked by both AT1R and AT2R antagonists. Exosome inhibitors, GW4869 and dimethyl amiloride (DMA), inhibited CF-induced cardiomyocyte hypertrophy with little effect on Ang II-induced cardiomyocyte hypertrophy. Mechanistically, CF exosomes upregulated RAS in cardiomyocytes via the activation of mitogen-activated protein kinases (MAPKs) and Akt. Finally, Ang II-induced exosome release from cardiac fibroblasts and pathological cardiac hypertrophy were dramatically inhibited by GW4869 and DMA in mice. These findings demonstrate that Ang II stimulates CFs to release exosomes, which in turn increase Ang II production and its receptor expression in cardiomyocytes, thereby intensifying Ang II-induced pathological cardiac hypertrophy. Accordingly, specific targeting of Ang II-induced exosome release from CFs may serve as a novel therapeutic approach to treat cardiac pathological hypertrophy and heart failure.

  7. [Autoradiographic investigations on postnatal proliferative activity of the telencephalic and diencephalic matrix-zones in the axolotl (Ambystoma mexicanum), with special references to the olfactory organ (author's transl)].

    Science.gov (United States)

    Richter, W; Kranz, D

    1981-01-01

    The localization and proliferative activity of the matrix-zones has been investigated in the telencephalon and in the diencephalon of 21 axolotls (Ambystoma mexicanum) by means of autoradiographs, after injection of tritiated thymidine at different stages of the postnatal life. There are no previous detailed autoradiographical reports on postnatal brain development in the axolotl. Matrix-zones (i.e. ventricular and subventricular zone) exist in the dorsal part and in the ventral part of the telencephalon, we have found these also in the diencephalon in the wall of the preoptic recessus and ventrally of the habenula. The quantitative part of this study indicates high values of the labeling-index in the early postnatal stages. Then, the labeling-index decreases, but also in 3 years old specimens labeled cells were observed in the matrix-zones of the telencephalon; therefore a few of proliferative capacity remains in the central nervous system of adult axolotls. Labeled cells were also found in the olfactory organ of early postnatal and adult axolotls; these are neuroblasts which have relevance for the regeneration of the forebrain.

  8. Proliferative and anti-proliferative effects of dietary levels of phytoestrogens in rat pituitary GH3/B6/F10 cells - the involvement of rapidly activated kinases and caspases

    OpenAIRE

    Watson Cheryl S; Jeng Yow-Jiun

    2009-01-01

    Abstract Background Phytoestogens are a group of lipophillic plant compounds that can have estrogenic effects in animals; both tumorigenic and anti-tumorigenic effects have been reported. Prolactin-secreting adenomas are the most prevalent form of pituitary tumors in humans and have been linked to estrogen exposures. We examined the proliferative effects of phytoestrogens on a rat pituitary tumor cell line, GH3/B6/F10, originally subcloned from GH3 cells based on its ability to express high l...

  9. Proliferative retinopathy predicts nephropathy

    DEFF Research Database (Denmark)

    Karlberg, Charlotte; Falk, Christine; Green, Anders;

    2012-01-01

    We wanted to examine proliferative retinopathy as a marker of incident nephropathy in a 25-year follow-up study of a population-based cohort of Danish type 1 diabetic patients and to examine cross-sectional associations between nephropathy and retinopathy in long-term surviving patients of the same...... photographs at follow-up. Single spot urine was used to evaluate nephropathy at both examinations. Proliferative retinopathy was present in 29 patients (15.8%) at baseline. At follow-up, these patients were more likely to macroalbuminuria (20.7% vs. 6.5%) than patients without proliferative retinopathy...... at baseline. In a multivariate logistic regression adjusted for baseline age, sex, duration of diabetes, smoking, HbA(1,) systolic and diastolic blood pressure, odds ratio of nephropathy (micro- and macroalbuminuria combined) was 2.98 (95% confidence interval 1.18-7.51, p = 0.02) for patients...

  10. Idebenone increases mitochondrial complex I activity in fibroblasts from LHON patients while producing contradictory effects on respiration

    DEFF Research Database (Denmark)

    Angebault, Claire; Gueguen, Naig; Desquiret-Dumas, Valerie

    2011-01-01

    ABSTRACT: BACKGROUND: Leber's hereditary optic neuropathy (LHON) is caused by mutations in the complex I subunits of the respiratory chain. Although patients have been treated with idebenone since 1992, the efficacy of the drug is still a matter of debate. Methods: We evaluated the effect...... of idebenone in fibroblasts from LHON patients using enzymatic and polarographic measurements. Results: Complex I activity was 42% greater in treated fibroblasts compared to controls (p = 0.002). Despite this complex I activity improvement, the effects on mitochondrial respiration were contradictory, leading...

  11. IL-36R signalling activates intestinal epithelial cells and fibroblasts and promotes mucosal healing in vivo.

    Science.gov (United States)

    Scheibe, Kristina; Backert, Ingo; Wirtz, Stefan; Hueber, Axel; Schett, Georg; Vieth, Michael; Probst, Hans Christian; Bopp, Tobias; Neurath, Markus F; Neufert, Clemens

    2017-05-01

    Interleukin (IL)-36R signalling plays a proinflammatory role in different organs including the skin, but the expression of IL-36R ligands and their molecular function in intestinal inflammation are largely unknown. We studied the characteristics of IL-36R ligand expression in IBDs and experimental colitis. The functional role of IL-36R signalling in the intestine was addressed in experimental colitis and wound healing models in vivo by using mice with defective IL-36R signalling (IL-36R-/-) or Myd88, neutralising anti-IL-36R antibodies, recombinant IL-36R ligands and RNA-seq genome expression analysis. Expression of IL-36α and IL-36γ was significantly elevated in active human IBD and experimental colitis. While IL-36γ was predominantly detected in nuclei of the intestinal epithelium, IL-36α was mainly found in the cytoplasm of CD14(+) inflammatory macrophages. Functional studies showed that defective IL-36R signalling causes high susceptibility to acute dextran sodium sulfate colitis and impairs wound healing. Mechanistically, IL-36R ligands released upon mucosal damage activated IL-36R(+) colonic fibroblasts via Myd88 thereby inducing expression of chemokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-6. Moreover, they induced proliferation of intestinal epithelial cells (IECs) and expression of the antimicrobial protein lipocalin 2. Finally, treatment of experimental intestinal wounds with IL-36R ligands significantly accelerated mucosal healing in vivo. IL-36R signalling is activated upon intestinal damage, stimulates IECs and fibroblasts and drives mucosal healing. Modulation of the IL-36R pathway emerges as a potential therapeutic strategy for induction of mucosal healing in IBD. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  12. H-ras transformation sensitizes volume-activated anion channels and increases migratory activity of NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Schneider, Linda; Klausen, Thomas K; Stock, Christian;

    2008-01-01

    The expression of the H-ras oncogene increases the migratory activity of many cell types and thereby contributes to the metastatic behavior of tumor cells. Other studies point to an involvement of volume-activated anion channels (VRAC) in (tumor) cell migration. In this paper, we tested whether...... VRACs are required for the stimulation of cell migration upon expression of the H-ras oncogene. We compared VRAC activation and migration of wild-type and H-ras-transformed NIH3T3 fibroblasts by means of patch-clamp techniques and time-lapse video microscopy. Both cell types achieve the same degree...... of VRAC activation upon maximal stimulation, induced by reducing extracellular osmolarity from 300 to 190 mOsm/l. However, upon physiologically relevant reductions in extracellular osmolarity (275 mOsm/l), the level of VRAC activation is almost three times higher in H-ras-transformed compared to wild...

  13. Anti-Proliferative and Pro-Apoptotic Activities of 4-Methyl-2,6-bis(1-phenylethyl)phenol in Cancer Cells

    Science.gov (United States)

    Sung, Nak Yoon; Kim, Seung Cheol; Kim, Yun Hwan; Kim, Gihyeon; Lee, Yunmi; Sung, Gi-Ho; Kim, Ji Hye; Yang, Woo Seok; Kim, Mi Seon; Baek, Kwang-Soo; Kim, Jong-Hoon; Cho, Jae Youl

    2016-01-01

    It has been found that 4-isopropyl-2,6-bis(1-phenylethyl)phenol (KTH-13), a novel compound isolated from Cordyceps bassiana, is able to suppress tumor cell proliferation by inducing apoptosis. To mass-produce this compound, we established a total synthesis method. Using those conditions, we further synthesized various analogs with structural similarity to KTH-13. In this study, we aimed to test their anti-cancer activity by measuring anti-proliferative and pro-apoptotic activities. Of 8 compounds tested, 4-methyl-2,6-bis(1-phenylethyl)phenol (KTH-13-Me) exhibited the strongest anti-proliferative activity toward MDA-MB 231 cells. KTH-13-Me also similarly suppressed the survival of various cancer cell lines, including C6 glioma, HCT-15, and LoVo cells. Treatment of KTH-13-Me induced several apoptotic signs in C6 glioma cells, such as morphological changes, induction of apoptotic bodies, and nuclear fragmentation and chromatin condensation. Concordantly, early-apoptotic cells were also identified by staining with FITC-Annexin V/PI. Moreover, KTH-13-Me highly enhanced the activation of caspase-3 and caspase-9, and decreased the protein level of Bcl-2. In addition, the phosphorylation levels of Src and STAT3 were diminished in KTH-13-Me-treated C6 cells. Therefore, these results suggest that KTH-13-Me can be developed as a novel anti-cancer drug capable of blocking proliferation, inducing apoptosis, and blocking cell survival signaling in cancer cells. PMID:27068261

  14. Expression of the familial Mediterranean fever gene and activity of the C5a inhibitor in human primary fibroblast cultures.

    Science.gov (United States)

    Matzner, Y; Abedat, S; Shapiro, E; Eisenberg, S; Bar-Gil-Shitrit, A; Stepensky, P; Calco, S; Azar, Y; Urieli-Shoval, S

    2000-07-15

    Familial Mediterranean fever (FMF) is an inherited disease whose manifestations are acute but reversible attacks of sterile inflammation affecting synovial and serosal spaces. The FMF gene (MEFV) was recently cloned, and it codes for a protein (pyrin/marenostrin) homologous to known nuclear factors. We previously reported the deficient activity of a C5a/interleukin (IL)-8 inhibitor, a physiologic regulator of inflammatory processes, in FMF serosal and synovial fluids. We now describe the concomitant expression of MEFV and C5a/IL-8-inhibitor activity in primary cultures of human fibroblasts. Fibroblasts grown from synovial and peritoneal tissues displayed C5a/IL-8-inhibitor activity that could be further induced with phorbol myristate acetate (PMA) and IL-1 beta. Very low levels of chemotactic inhibitor were evident in skin fibroblast cultures or in peritoneal and skin fibroblasts obtained from FMF patients. MEFV was expressed in peritoneal and skin fibroblasts at a lower level than in neutrophils and could be further induced by PMA and IL-1 beta. In the FMF cultures, the MEFV transcript carried the M694V mutation, consistent with the genetic defect found in patients with this disease. MEFV was also expressed in other cell lines that do not produce C5a/IL-8 inhibitor. These findings suggest that human primary fibroblast cultures express MEFV and produce C5a/IL-8-inhibitor activity. The interrelationship between pyrin, the MEFV product, and the C5a/IL-8 inhibitor requires further investigation. (Blood. 2000;96:727-731)

  15. Protease-activated receptor 1 and 2 contribute to angiotensin II-induced activation of adventitial fibroblasts from rat aorta

    Energy Technology Data Exchange (ETDEWEB)

    He, Rui-Qing; Tang, Xiao-Feng; Zhang, Bao-Li [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Shanghai Institute of Hypertension, Shanghai (China); Li, Xiao-Dong [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China); Hong, Mo-Na [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Shanghai Institute of Hypertension, Shanghai (China); Chen, Qi-Zhi [Shanghai Institute of Hypertension, Shanghai (China); Han, Wei-Qing, E-mail: whan020@gmail.com [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China); Gao, Ping-Jin, E-mail: gaopingjin@sibs.ac.cn [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China)

    2016-04-29

    Adventitial fibroblasts (AFs) can be activated by angiotensin II (Ang II) and exert pro-fibrotic and pro-inflammatory effects in vascular remodeling. Protease-activated receptor (PAR) 1 and 2 play a significant role in fibrogenic and inflammatory diseases. The present study hypothesized that PAR1 and PAR2 are involved in Ang II-induced AF activation and contribute to adventitial remodeling. We found that direct activation of PAR1 and PAR2 with PAR1-AP and PAR2-AP led to AF activation, including proliferation and differentiation of AFs, extracellular matrix synthesis, as well as production of pro-fibrotic cytokine TGF-β and pro-inflammatory cytokines IL-6 and MCP-1. Furthermore, PAR1 and PAR2 mediated Ang II-induced AF activation, since both PAR1 and PAR2 antagonists inhibited Ang II-induced proliferation, migration, differentiation, extracellular matrix synthesis and production of pro-fibrotic and pro-inflammatory cytokines in AFs. Finally, mechanistic study showed that Ang II, via Ang II type I receptor (AT1R), upregulated both PAR1 and PAR2 expression, and transactivated PAR1 and PAR2, as denoted by internalization of both proteins. In conclusion, our results suggest that PAR1 and PAR2 play a critical role in Ang II-induced AF activation, and this may contribute to adventitia-related pathological changes. - Highlights: • Direct activation of PAR1 and PAR2 led to adventitial fibroblast (AF) activation. • PAR1 and PAR2 antagonists attenuated Ang II-induced AF activation. • Ang II induced the upregulation and transactivation of PAR1/PAR2 in AFs.

  16. Regulation of protease-activated receptor-1 expression in human buccal fibroblasts stimulated with arecoline.

    Science.gov (United States)

    Tsai, Chung-Hung; Lee, Shiuan-Shinn; Huang, Fu-Mei; Chang, Yu-Chao

    2013-09-01

    The purpose of this study was to compare the major thrombin receptor protease-activated receptor-1 (PAR-1) expression in normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanisms that may lead to induce PAR-1 expression. Thirty OSF and 10 normal buccal mucosa specimens were examined by immunohistochemistry. Buccal mucosal fibroblasts (BMFs) were challenged with arecoline by using Western blot analysis. N-acetyl-L-cysteine (NAC), LY294002, herbimycin A, NS-398, and PD98059 were added to find the possible regulatory mechanisms. PAR-1 expression was significantly higher in OSF specimens (p Arecoline was found to elevate PAR-1 expression in a dose-dependent and time-dependent manner (p arecoline-induced PAR-1 expression (p Arecoline-induced PAR-1 expression was downregulated by NAC, LY294002, herbimycin A, NS398, and PD98059. Copyright © 2012 Wiley Periodicals, Inc.

  17. Fibroblast Activation Protein (FAP) Accelerates Collagen Degradation and Clearance from Lungs in Mice

    DEFF Research Database (Denmark)

    Fan, Ming-Hui; Zhu, Qiang; Li, Hui-Hua

    2016-01-01

    Idiopathic pulmonary fibrosis is a disease characterized by progressive, unrelenting lung scarring, with death from respiratory failure within 2-4 years unless lung transplantation is performed. New effective therapies are clearly needed. Fibroblast activation protein (FAP) is a cell surface......, intratracheal bleomycin instillation and thoracic irradiation, we find increased mortality and increased lung fibrosis in FAP-deficient mice compared with wild-type mice. Lung extracellular matrix analysis reveals accumulation of intermediate-sized collagen fragments in FAP-deficient mouse lungs, consistent...... within vitrostudies showing that FAP mediates ordered proteolytic processing of matrix metalloproteinase (MMP)-derived collagen cleavage products. FAP-mediated collagen processing leads to increased collagen internalization without altering expression of the endocytic collagen receptor, Endo180...

  18. Maintenance of telomeres in SV40-transformed pre-immortal and immortal human fibroblasts.

    Science.gov (United States)

    Small, M B; Hubbard, K; Pardinas, J R; Marcus, A M; Dhanaraj, S N; Sethi, K A

    1996-09-01

    Shortening of telomeres has been hypothesized to contribute to cellular senescence and may play a role in carcinogenesis of human cells. Furthermore, activation of telomerase has frequently been demonstrated in tumor-derived and in vitro immortalized cells. In this study, we have assessed these phenomena during the life span of simian virus 40 (SV40)-transformed preimmortal and immortal human fibroblasts. We observed progressive reduction in telomere length in preimmortal transformed cells with extended proliferative capacity, with the most dramatic shortening at late passage. Telomere lengths became stabilized (or increased) in immortal fibroblasts accompanied, in one case, by the activation of telomerase. However, an independent immortal cell line that displayed stable telomeres did not have detectable telomerase activity. Furthermore, we found significant telomerase activity in two preimmortal derivatives. Our results provide further evidence for maintenance of telomeres in immortalized human fibroblasts, but they suggest a lack of causal relationship between telomerase activation and immortalization.

  19. Bacillus Calmette Guerin induces fibroblast activation both directly and through macrophages in a mouse bladder cancer model.

    Directory of Open Access Journals (Sweden)

    Catalina Lodillinsky

    Full Text Available BACKGROUND: Bacillus Calmette-Guerin (BCG is the most effective treatment for non-muscle invasive bladder cancer. However, a failure in the initial response or relapse within the first five years of treatment has been observed in 20% of patients. We have previously observed that in vivo administration of an inhibitor of nitric oxide improved the response to BCG of bladder tumor bearing mice. It was described that this effect was due to a replacement of tumor tissue by collagen depots. The aim of the present work was to clarify the mechanism involved in this process. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that BCG induces NIH-3T3 fibroblast proliferation by activating the MAPK and PI3K signaling pathways and also differentiation determined by alpha-smooth muscle actin (alpha-SMA expression. In vivo, intratumoral inoculation of BCG also increased alpha-SMA and collagen expression. Oral administration of L-NAME enhanced the pro-fibrotic effect of BCG. Peritoneal macrophages obtained from MB49 tumor-bearing mice treated in vivo with combined treatment of BCG with L-NAME also enhanced fibroblast proliferation. We observed that FGF-2 is one of the factors released by BCG-activated macrophages that is able to induce fibroblast proliferation. The involvement of FGF-2 was evidenced using an anti-FGF2 antibody. At the same time, this macrophage population improved wound healing rate in normal mice and FGF-2 expression was also increased in these wounds. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that fibroblasts are targeted by BCG both directly and through activated macrophages in an immunotherapy context of a bladder murine model. We also described, for the first time, that FGF-2 is involved in a dialog between fibroblasts and macrophages induced after BCG treatment. The fact that L-NAME administration improves the BCG effect on fibroblasts, NO inhibition, might represent a new approach to add to the conventional BCG therapy.

  20. Gene Signature of Human Oral Mucosa Fibroblasts: Comparison with Dermal Fibroblasts and Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Keiko Miyoshi

    2015-01-01

    Full Text Available Oral mucosa is a useful material for regeneration therapy with the advantages of its accessibility and versatility regardless of age and gender. However, little is known about the molecular characteristics of oral mucosa. Here we report the first comparative profiles of the gene signatures of human oral mucosa fibroblasts (hOFs, human dermal fibroblasts (hDFs, and hOF-derived induced pluripotent stem cells (hOF-iPSCs, linking these with biological roles by functional annotation and pathway analyses. As a common feature of fibroblasts, both hOFs and hDFs expressed glycolipid metabolism-related genes at higher levels compared with hOF-iPSCs. Distinct characteristics of hOFs compared with hDFs included a high expression of glycoprotein genes, involved in signaling, extracellular matrix, membrane, and receptor proteins, besides a low expression of HOX genes, the hDFs-markers. The results of the pathway analyses indicated that tissue-reconstructive, proliferative, and signaling pathways are active, whereas senescence-related genes in p53 pathway are inactive in hOFs. Furthermore, more than half of hOF-specific genes were similarly expressed to those of hOF-iPSC genes and might be controlled by WNT signaling. Our findings demonstrated that hOFs have unique cellular characteristics in specificity and plasticity. These data may provide useful insight into application of oral fibroblasts for direct reprograming.

  1. Immunohistochemical Estimates of Angiogenesis, Proliferative Activity, p53 Expression, and Multiple Drug Resistance Have No Prognostic Impact in Osteosarcoma: A Comparative Clinicopathological Investigation

    DEFF Research Database (Denmark)

    Sørensen, Flemming Brandt; Jensen, Kenneth; Vaeth, Michael;

    2008-01-01

    regarding angiogenesis (CD34), proliferative activity (MIB-1), and the expression of p53 and MDR (P-glycoprotein (Pgp); clones JSB-1, C494, and MRK16). Quantitative and semiquantitative scores of immunoreactive cells were analyzed statistically along with retrospectively obtained clinicopathologic variables...... (P = .64) and p53 (P > .32), whereas the MIB-1 index was reduced in the post-chemotherapy specimens (P = .02). The overall, disease-specific survival was 47%, increasing to 54% in patients receiving pre-operative chemotherapy. Statistical analyses showed prognostic impact exclusively by patient age...... and type of osteosarcoma. Discussion. The studied series of patients documented already prior to the chemotherapy era, a rather excellent survival and estimates of angiogenesis, proliferation, p53, and Pgp expressions, did not demonstrate sufficient power to serve as predictors of treatment response...

  2. Migration and Proliferative Activity of Mesenchymal Stem Cells in 3D Polylactide Scaffolds Depends on Cell Seeding Technique and Collagen Modification.

    Science.gov (United States)

    Rodina, A V; Tenchurin, T Kh; Saprykin, V P; Shepelev, A D; Mamagulashvili, V G; Grigor'ev, T E; Lukanina, K I; Orekhov, A S; Moskaleva, E Yu; Chvalun, S N

    2016-11-01

    We analyzed viability of mesenchymal stem cells seeded by static and dynamic methods to highly porous fibrous 3D poly-L-lactide scaffolds with similar physical and chemical properties, but different spatial organization modified with collagen. Standard collagen coating promoted protein adsorption on the scaffold surface and improved adhesive properties of 100 μ-thick scaffolds. Modification of 600-μ scaffolds with collagen under pressure increased proliferative activity of mesenchymal stem cells seeded under static and dynamic (delivery of 100,000 cells in 10 ml medium in a perfusion system at a rate of 1 ml/min) conditions by 47 and 648%, respectively (measured after 120-h culturing by MTT test). Dynamic conditions provide more uniform distribution of collagen on scaffold fibers and promote cell penetration into 3D poly-L-lactide scaffolds with thickness >600 μ.

  3. Proliferative activity (MIB-1 index) is an independent prognostic parameter in patients with high-grade soft tissue sarcomas of subtypes other than malignant fibrous histiocytomas

    DEFF Research Database (Denmark)

    Jensen, V; Sørensen, Flemming Brandt; Bentzen, S M

    1998-01-01

    . The proliferative activity was assessed by use of the monoclonal antibody MIB-1 and evaluated in multiple, random systematic sampled fields of vision. The percentage of proliferating cells (the MIB-1 index) ranged between 1% and 85% (median 12%). A significant increase in mean MIB-1 index was seen with increasing....... Univariate analysis identified patient age, tumour size, histological grade of malignancy, MIB-1 index and p53 accumulation as significant prognostic parameters. Multivariate Cox analysis, including tests for interaction terms between histological subtypes and MIB-1 index, showed independent prognostic...... effect of MIB-1 index and tumour size in patients with high-grade tumours of other subtypes than malignant fibrous histiocytoma (MFH). CONCLUSIONS: Histopathological malignancy grading is the most important single prognostic factor for overall survival in STS, but estimation of MIB-1 index is useful...

  4. Neuroligin-1 induces neurite outgrowth through interaction with neurexin-1ß and activation of fibroblast growth factor receptor-1

    DEFF Research Database (Denmark)

    Gjørlund, Michelle D; Nielsen, Janne; Pankratova, Stanislava;

    2012-01-01

    the neuritogenic effect of NLGN1 in cultures of hippocampal neurons. Our results show that NLGN1, both in soluble and membrane-bound forms, induces neurite outgrowth that depends on the interaction with NRXN1ß and on activation of fibroblast growth factor receptor-1. In addition, we demonstrate that a synthetic...

  5. The Influence of Spirulina platensis Filtrates on Caco-2 Proliferative Activity and Expression of Apoptosis-Related microRNAs and mRNA.

    Science.gov (United States)

    Śmieszek, Agnieszka; Giezek, Ewa; Chrapiec, Martyna; Murat, Martyna; Mucha, Aleksandra; Michalak, Izabela; Marycz, Krzysztof

    2017-03-07

    Spirulina platensis (SP) is a blue-green microalga that has recently raised attention not only as a nutritional component, but also as a source of bioactivities that have therapeutic effects and may find application in medicine, including cancer treatment. In the present study we determined the cytotoxic effect of S. platensis filtrates (SPF) on human colon cancer cell line Caco-2. Three concentrations of SPF were tested-1.25%, 2.5%, and 5% (v/v). We have found that the highest concentration of SPF exerts the strongest anti-proliferative and pro-apoptotic effect on Caco-2 cultures. The SPF negatively affected the morphology of Caco-2 causing colony shrinking and significant inhibition of metabolic and proliferative activity of cells. The wound-healing assay showed that the SPF impaired migratory capabilities of Caco-2. This observation was consistent with lowered mRNA levels for metalloproteinases. Furthermore, SPF decreased the transcript level of pro-survival genes (cyclin D1, surviving, and c-Myc) and reduced the autocrine secretion of Wnt-10b. The cytotoxic effect of SPF involved the modulation of the Bax and Bcl-2 ratio and a decrease of mitochondrial activity, and was related with increased levels of intracellular reactive oxygen species (ROS) and nitric oxide (NO). Moreover, the SPF also caused an increased number of cells in the apoptotic sub-G0 phase and up-regulated expression of mir-145, simultaneously decreasing expression of mir-17 and 146. Obtained results indicate that SPF can be considered as an agent with anti-cancer properties that may be used for colon cancer prevention and treatment.

  6. Antioxidant and anti-lipid peroxidation activities of Tamarindus indica seed coat in human fibroblast cells.

    Science.gov (United States)

    Nakchat, Oranuch; Meksuriyen, Duangdeun; Pongsamart, Sunanta

    2014-02-01

    Antioxidant activity and total phenolic content of tamarind seed coat extracts (TSCEs) were compared between the two extracts using boiling-water (TSCE-W) and 70% ethanol (TSCE-E) for extraction. TSCE-W, consisting of the highest phenolic content, possessed 2,2-diphenyl-1 -picrylhydrazyl (DPPH) radical scavenging and anti-lipid peroxidation activities much higher than TSCE-E and Trolox. Additionally, both TSCEs also exhibited superoxide anion and hydrogen peroxide scavenging activities higher than Trolox and BHA. Anti-lipid peroxidation and cytotoxicity of TSCE-W were also studied in human foreskin fibroblast CCD-1064Sk cells. Cytotoxic effect was not observed when exposed to TSCE-W up to 1 mg/mL for 12-48 h. However, TSCE-W significantly attenuated lipid peroxidation in H202-damaged cells. HPLC analysis showed the presence of (+)-catechin, (-)-epicatechin, and procyanidin B2 in TSCE-W, which could be responsible for antioxidant and anti-lipid peroxidation activities. The results suggest that an inexpensive and simple boiling-water extraction of TSCE-W may provide a valuable natural antioxidant source having anti-lipid peroxidation for health food additives, nutraceuticals as well as cosmeceuticals.

  7. Suppression of angiogenic activity of sera from diabetic patients with non-proliferative retinopathy by compounds of herbal origin and sulindac sulfone.

    Science.gov (United States)

    Skopinski, Piotr; Szaflik, Jerzy; Duda-Król, Barbara; Nartowska, Jadwiga; Sommer, Ewa; Chorostowska-Wynimko, Joanna; Demkow, Urszula; Skopinska-Rózewska, Ewa

    2004-10-01

    Angiogenesis, the process of new blood vessel formation, is the key event in the mechanism of several pathological processes including diabetic retinopathy. The physiological control of angiogenesis depends on the balance between stimulatory and inhibitory factors. Therefore, a number of anti-angiogenic approaches has been developed, many of them based on the inhibition of the functional activity of pro-angiogenic factors. The aim of the present study was to compare the anti-angiogenic effectiveness of sulindac sulfone and some herbal compounds in the serum-induced angiogenesis test performed in Balb/c mice. Pooled sera from 35 patients with diabetes type 2 and retinopathy were used as pro-angiogenic stimuli. The strongest inhibitory effect was observed for the sulindac sulfone and ursolic acid in the highest concentration of 200 micro g/ml, as well as for the low-dosage concomitant treatment with 2 micro g/ml of epigallocatechin gallate (EGCG, green tea flavanol), ursolic acid (plant-derived triterpenoid), sulindac sulfone and convalamaroside (steroidal saponin). Combination treatment was significantly more effective than monotherapy with medium (20 micro g/ml) or lowest doses of tested compounds. The present study is the first to demonstrate the potent anti-angiogenic effect of the combination therapy comprising of plant-derived extracts and sulindac sulfone, as tested in the in vivo angiogenesis experimental model with sera of non-proliferative diabetic retinopathy patients used as the pro-angiogenic stimuli. We think that it might be the first step toward application of some of these compounds, in the future, in preventive anti-angiogenic therapy of these patients, as well, as in the treatment of later, proliferative stage of this disease.

  8. Ras Activated ERK and PI3K Pathways Differentially Affect Directional Movement of Cultured Fibroblasts

    Directory of Open Access Journals (Sweden)

    Leandra Sepe

    2013-01-01

    Full Text Available Background: Cell migration is essential in physiological and pathological processes, such as wound healing and metastasis formation. Ras involvement in these processes has been extensively demonstrated. This work attempts to characterize Ras regulation of the phenomena determining directional cell migration by separately analyzing the role of its principal effector pathways, MAPK and PI3K. Methods: NIH3T3 and NIHRasV12 fibroblasts were followed in wound healing assays to study, in time and under a directional stimulus, cell migration both under standard conditions and in presence of MAPK and PI3K inhibitors. Several parameters, descriptive of specific aspects of cell motion, were evaluated by coupling dynamic microscopy with quantitative and statistical methods. Quantitative Western Blots coupled with immunofluorescence stainings, were used to evaluate ERK activation. Results: Constitutive RasV12 activation confers to NIH3T3 the ability to close the wound faster. Neither increased cell proliferation nor higher speed explains the accelerated healing, but the increased directional migration drives the wound closure. Inhibition of ERK activation, which occurs immediately after wound, greatly blocks the directional migration, while inhibition of PI3K pathway reduces cell speed but does not prevent wound closure. Conclusion: Ras is greatly involved in determining and regulating directionality, ERK is its key effector for starting, driving and regulating directional movement.

  9. Anti-inflammatory activity of fisetin in human gingival fibroblasts treated with lipopolysaccharide.

    Science.gov (United States)

    Gutiérrez-Venegas, Gloria; Contreras-Sánchez, Anabel; Ventura-Arroyo, Jairo Agustín

    2014-10-01

    Fisetin is an anti-inflammatory flavonoid; however, its anti-inflammatory mechanism is not yet understood. In this study, we evaluated the anti-inflammatory effect of fisetin and its association with mitogen-activated protein kinase (MAPK) and nuclear factor kappa-beta pathways in human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS) obtained from Porphyromonas gingivalis. The cell signaling, cell viability, and cyclooxygenase-2 (COX-2) expression of HGFs treated with various concentrations (0, 1, 5, 10, and 15 μM) of fisetin were measured by cell viability assay (MTT), Western blotting, and reverse transcriptase polymerase chain reaction analysis on COX-2. We found that fisetin significantly reduced the synthesis and expression of prostaglandin E2 in HGFs treated with LPS. Activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK was suppressed consistently by fisetin in HGFs treated with LPS. The data indicate that fisetin inhibits MAPK activation and COX-2 expression without affecting cell viability. These findings may be valuable for understanding the mechanism of the effect of fisetin on periodontal disease.

  10. MORPHOFUNCTIONAL CHARACTERISTICS OF FIBROBLASTS (MCCOY CELL LINE CULTURED WITH MAGNESIUM PREPARATIONS

    Directory of Open Access Journals (Sweden)

    L. V. Didenko

    2015-01-01

    Full Text Available Aim. To study the effect of magnesium orotate, magnesium/pyridoxine combination and magnesium sulfate on fibroblast morphofunctional characteristics in cell culture of fibroblasts (McCoy line.Material and methods. The study of fibroblasts (McCoy line with the addition of magnesium-containing preparations (magnesium orotate, magnesium/pyridoxine combination, magnesium sulphate to the culture medium was performed using scanning and transmission electron microscopy.Results. When adding into the media magnesium orotate or magnesium/pyridoxine combination, significant changes in morphofunctional state of fibroblasts were noted, which were absent when adding magnesium sulfate. At that fibroblasts significantly promoted synthetic and secretory activity. This was expressed in the formation of amorphous and fibrous components in well-formed cell layers.Conclusion. Stimulation by magnesium ions of the proliferative activity of fibroblasts is shown in the morphological analysis of the effect of magnesium orotate or magnesium in combination with pyridoxine and magnesium sulfate on morphological and functional organization of fibroblasts. Revealed the predominant influence of magnesium orotate, and a less pronounced effect of magnesium in combination with pyridoxine on biosynthetic processes in the cells, including the synthesis of amorphous and fibrous components (protocollagen and elastin of the extracellular matrix founded.

  11. Ultraviolet A Enhances Cathepsin L Expression and Activity via JNK Pathway in Human Dermal Fibroblasts

    Science.gov (United States)

    Xu, Qing-Fang; Zheng, Yue; Chen, Jian; Xu, Xin-Ya; Gong, Zi-Jian; Huang, Yun-Fen; Lu, Chun; Maibach, Howard I; Lai, Wei

    2016-01-01

    Background: Cathepsin L (CatL) is a cysteine protease with strong matrix degradation activity that contributes to photoaging. Mannose phosphate-independent sorting pathways mediate ultraviolet A (UVA)-induced alternate trafficking of CatL. Little is known about signaling pathways involved in the regulation of UVA-induced CatL expression and activity. This study aims to investigate whether a single UVA irradiation affects CatL expression and activity and whether mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) pathway is involved in the regulation of UVA-induced CatL expression and activity in human dermal fibroblasts (HDFs). Methods: Primary HDFs were exposed to UVA. Cell proliferation was determined by a cell counting kit. UVA-induced CatL production and activity were studied with quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and fluorimetric assay in cell lysates collected on three consecutive days after irradiation. Time courses of UVA-activated JNK and p38MAPK signaling were examined by Western blotting. Effects of MAPK inhibitors and knockdown of Jun and Fos on UVA-induced CatL expression and activity were investigated by RT-PCR, Western blotting, and fluorimetric assay. Data were analyzed by one-way analysis of variance. Results: UVA significantly increased CatL gene expression, protein abundance, and enzymatic activity for three consecutive days after irradiation (F = 83.11, 56.14, and 71.19, respectively; all P < 0.05). Further investigation demonstrated phosphorylation of JNK and p38MAPK activated by UVA. Importantly, inactivation of JNK pathway significantly decreased UVA-induced CatL expression and activity, which were not affected by p38MAPK inhibition. Moreover, knockdown of Jun and Fos significantly attenuated basal and UVA-induced CatL expression and activity. Conclusions: UVA enhances CatL production and activity in HDFs, probably by activating JNK and downstreaming AP-1. These

  12. Ultraviolet A Enhances Cathepsin L Expression and Activity via JNK Pathway in Human Dermal Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Qing-Fang Xu; Yue Zheng; Jian Chen; Xin-Ya Xu; Zi-Jian Gong; Yun-Fen Huang; Chun Lu

    2016-01-01

    Background:Cathepsin L (CatL) is a cysteine protease with strong matrix degradation activity that contributes to photoaging.Mannose phosphate-independent sorting pathways mediate ultraviolet A (UVA)-induced alternate trafficking of CatL.Little is known about signaling pathways involved in the regulation of UVA-induced CatL expression and activity.This study aims to investigate whether a single UVA irradiation affects CatL expression and activity and whether mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) pathway is involved in the regulation of UVA-induced CatL expression and activity in human dermal fibroblasts (HDFs).Methods:Primary HDFs were exposed to UVA.Cell proliferation was determined by a cell counting kit.UVA-induced CatL production and activity were studied with quantitative real-time reverse transcription polymerase chain reaction (RT-PCR),Western blotting,and fluorimetric assay in cell lysates collected on three consecutive days after irradiation.Time courses of UVA-activated JNK and p38MAPK signaling were examined by Western blotting.Effects ofMAPK inhibitors and knockdown of Jun and Fos on UVA-induced CatL expression and activity were investigated by RT-PCR,Western blotting,and fluorimetric assay.Data were analyzed by one-way analysis of variance.Results:UVA significantly increased CatL gene expression,protein abundance,and enzymatic activity for three consecutive days after irradiation (F =83.11,56.14,and 71.19,respectively;all P < 0.05).Further investigation demonstrated phosphorylation of JNK and p38MAPK activated by UVA.Importantly,inactivation of JNK pathway significantly decreased UVA-induced CatL expression and activity,which were not affected by p38MAPK inhibition.Moreover,knockdown of Jun and Fos significantly attenuated basal and UVA-induced CatL expression and activity.Conclusions:UVA enhances CatL production and activity in HDFs,probably by activating JNK and downstreaming AP-1.These findings provide a new possible

  13. Change of digestive enzyme activity in porcine proliferative enteritis%猪增生性肠炎消化系统酶活性的变化

    Institute of Scientific and Technical Information of China (English)

    陈彤; 杨小燕; 祁保民

    2011-01-01

    采用酶学反应方法,检测了增生性肠炎阳性猪与阴性猪的胰腺、十二指肠、空肠、回肠淀粉酶、胰蛋白酶、脂肪酶及碱性磷酸酶的活性变化.结果表明:(1)猪增生性肠炎阳性猪胰腺、十二指肠、空肠、回肠胰蛋白酶活性同比阴性猪均下降,其中空肠、回肠的同比差异显著(P<0.05);(2)阳性猪回肠碱性磷酸酶活性显著下降(P<0.05),胰腺、十二指肠、空肠碱性磷酸酶活性与阴性猪相比差异不显著(P>0.05);(3)阳性猪胰腺、十二指肠、空肠、回肠的淀粉酶和脂肪酶活性均下降,但差异不显著(P>0.05).猪增生性肠炎病猪空肠、回肠胰蛋白酶以及回肠碱性磷酸酶活性显著降低,酶活性降低引起了增生性肠炎猪消化机能障碍.%The enzyme activities of amylase, trypsin, lipase and alkaline phosphatase in pancreas, duodenum,jejunum and ileum were detected in the swine of porcine proliferative enteritis by enzymatic reaction method. The results show as follows. (1) Compared with the negative swine, the activity of the trypsin in pancreas, duodenum, jejunum, and ileum of the positive swine all decreased, among them the difference of comparison in jejunum and ileum is significant (P<0.05) ; (2) Compared with the negative swine, the activities of the alkaline enzymes in ileum are significant (P<0.05), and no obvious difference in pancreas, duodenum, and jejunum (P>0.05); (3) The activities of amylase and the lipase in pancreas, duodenum, jejunum and ileum have no obvious difference between the positive and negative swines (P>0.05). These results suggest that the activity of trypsin in jejunum, ileum and alkaline enzymes in ileum decreased significantly. The decrease of enzyme activities could led to disorder of digestion in the swine of porcine proliferative enteritis.

  14. Multiwall carbon nanotubes directly promote fibroblast-myofibroblast and epithelial-mesenchymal transitions through the activation of the TGF-β/Smad signaling pathway.

    Science.gov (United States)

    Wang, Peng; Wang, Yue; Nie, Xin; Braïni, Céline; Bai, Ru; Chen, Chunying

    2015-01-27

    A number of studies have demonstrated that MWCNTs induce granuloma formation and fibrotic responses in vivo, and it has been recently reported that MWCNT-induced macrophage activation and subsequent TGF-β secretion contribute to pulmonary fibrotic responses. However, their direct effects against alveolar type-II epithelial cells and fibroblasts and the corresponding underlying mechanisms remain largely unaddressed. Here, MWCNTs are reported to be able to directly promote fibroblast-to-myofibroblast conversion and the epithelial-mesenchymal transition (EMT) through the activation of the TGF-β/Smad signaling pathway. Both of the cell transitions may play important roles in MWCNT-induced pulmonary fibrosis. Firstly, in-vivo and in-vitro data show that long MWCNTs can directly interact with fibroblasts and epithelial cells, and some of them may be uptaken into fibroblasts and epithelial cells by endocytosis. Secondly, long MWCNTs can directly activate fibroblasts and increase both the basal and TGF-β1-induced expression of the fibroblast-specific protein-1, α-smooth muscle actin, and collagen III. Finally, MWCNTs can induce the EMT through the activation of TGF-β/Smad2 signaling in alveolar type-II epithelial cells, from which some fibroblasts involved in pulmonary fibrosis are thought to originate. These observations suggest that the activation of the TGF-β/Smad2 signaling plays a critical role in the process of the fibroblast-to-myofibroblast transition and the EMT induced by MWCNTs.

  15. Inhibition of aromatase activity by methyl sulfonyl PCB metabolites in primary culture of human mammary fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Berg, M. van den; Heneweer, M.; Geest, M. de; Sanderson, T. [Inst. for Risk Assessment Sciences and Utrecht Univ. (Netherlands); Jong, P. de [St. Antonius Hospital, Nieuwegein (Netherlands); Bergman, A. [Stockholm Univ., Stockholm (Sweden)

    2004-09-15

    Methyl sulfonyl PCB metabolites (MeSO2-PCBs) are persistent contaminants and are ubiquitously present in humans and the environment. Lipophilicity of MeSO2- PCB metabolites is similar to the parent compounds and they have been detected in human milk, adipose, liver and lung tissue. 4- MeSO2-PCB-149 is the most abundant PCB metabolite in human adipose tissue and milk at a level of 1.5 ng/g lipids. Human blood concentration of 4-MeSO2-PCB-149 is approximately 0.03 nM. 3- MeSO2-PCB-101 is the predominant PCB metabolite in muscle and blubber in wildlife, such as otter, mink and grey seal. In the environment, they have been linked to chronic and reproductive toxicity in exposed mink. Additionaly, some MeSO{sub 2}-PCBs have been shown to be glucocorticoid receptor (GR) antagonists. Since approximately 60% of all breast tumors are estrogen responsive, exposure to compounds that are able to alter estrogen synthesis through interference with the aromatase enzyme, can lead to changes in estrogen levels and possibly to accelerated or inhibit breast tumor growth. Therefore, it is important to identify exogenous compounds that can alter aromatase activity in addition to those compounds which have direct interaction with the estrogen receptor (ER). Aromatase (CYP19) comprises the ubiquitous flavoprotein, NADPH-cytochrome P450 reductase, and a unique cytochrome P450 that is exclusively expressed in estrogen producing cells. Previous studies have revealed that expression of the aromatase gene is regulated in a species- and tissue specific manner. In healthy breast tissue, the predominantly active aromatase promoter region I.4 is regulated by glucocorticoids and class I cytokines. Therefore, it is important to investigate possible aromatase inhibiting properties of MeSO{sub 2}-PCBs (as anti glucocorticoids?) in relevant human tissues. We used primary human mammary fibroblasts because of their role in breast cancer development. We compared the results in primary fibroblasts with

  16. Activation of the innate immune response against DENV in normal non-transformed human fibroblasts.

    Directory of Open Access Journals (Sweden)

    José Bustos-Arriaga

    2011-12-01

    Full Text Available BACKGROUND: When mosquitoes infected with DENV are feeding, the proboscis must traverse the epidermis several times ("probing" before reaching a blood vessel in the dermis. During this process, the salivary glands release the virus, which is likely to interact first with cells of the various epidermal and dermal layers, cells which could be physiologically relevant to DENV infection and replication in humans. However, important questions are whether more abundant non-hematopoietic cells such as fibroblasts become infected, and whether they play any role in antiviral innate immunity in the very early stages of infection, or even if they might be used by DENV as primary replication cells. METHODOLOGY/PRINCIPAL FINDINGS: Fibroblasts freshly released from healthy skin and infected 12 hours after their isolation show a positive signal for DENV. In addition, when primary skin fibroblast cultures were established and subsequently infected, we showed DENV-2 antigen-positive intracellular signal at 24 hours and 48 hours post-infection. Moreover, the fibroblasts showed productive infection in a conventional plaque assay. The skin fibroblasts infected with DENV-2 underwent potent signaling through both TLR3 and RIG- 1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5 and β defensin 2 (HβD2. In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN regulatory factor 3 (IRF3, but not interferon regulatory factor 7 (IRF7, when compared with mock-infected fibroblasts. CONCLUSIONS/SIGNIFICANCE: In this work, we demonstrated the high susceptibility to DENV infection by primary fibroblasts from normal human skin, both in situ and in vitro. Our results suggest that these cells may contribute to the pro-inflammatory and anti-viral microenvironment in the early stages of interaction with DENV-2. Furthermore, the data suggest that fibroblast may also be used as a primary site of DENV replication and

  17. Co-culture of 3D tumor spheroids with fibroblasts as a model for epithelial–mesenchymal transition in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun-Ah, E-mail: j.sarah.k@gmail.com [Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Lee, Eun Kyung, E-mail: leeek@catholic.ac.kr [Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Cancer Evolution Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Kuh, Hyo-Jeong, E-mail: hkuh@catholic.ac.kr [Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Cancer Evolution Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of)

    2015-07-15

    Epithelial–mesenchymal transition (EMT) acts as a facilitator of metastatic dissemination in the invasive margin of malignant tumors where active tumor–stromal crosstalks take place. Co-cultures of cancer cells with cancer-associated fibroblasts (CAFs) are often used as in vitro models of EMT. We established a tumor–fibroblast proximity co-culture using HT-29 tumor spheroids (TSs) with CCD-18co fibroblasts. When co-cultured with TSs, CCD-18co appeared activated, and proliferative activity as well as cell migration increased. Expression of fibronectin increased whereas laminin and type I collagen decreased in TSs co-cultured with fibroblasts compared to TSs alone, closely resembling the margin of in vivo xenograft tissue. Active TGFβ1 in culture media significantly increased in TS co-cultures but not in 2D co-cultures of cancer cells–fibroblasts, indicating that 3D context-associated factors from TSs may be crucial to crosstalks between cancer cells and fibroblasts. We also observed in TSs co-cultured with fibroblasts increased expression of α-SMA, EGFR and CTGF; reduced expression of membranous β-catenin and E-cadherin, together suggesting an EMT-like changes similar to a marginal region of xenograft tissue in vivo. Overall, our in vitro TS–fibroblast proximity co-culture mimics the EMT-state of the invasive margin of in vivo tumors in early metastasis. - Highlights: • An adjacent co-culture of tumor spheroids and fibroblasts is presented as EMT model. • Activation of fibroblasts and increased cell migration were shown in co-culture. • Expression of EMT-related factors in co-culture was similar to that in tumor tissue. • Crosstalk between spheroids and fibroblasts was demonstrated by secretome analysis.

  18. Fucoidan is the active component of fucus vesiculosus that promotes contraction of fibroblast-populated collagen gels.

    Science.gov (United States)

    Fujimura, T; Shibuya, Y; Moriwaki, S; Tsukahara, K; Kitahara, T; Sano, T; Nishizawa, Y; Takema, Y

    2000-10-01

    The fibroblast-populated collagen gel culture method has been evaluated as a dermal model of wound contraction and granulation in tissues during the wound healing process and as an in vitro model of dermal tissue. We previously reported that an extract of Fucus vesiculosus promoted fibroblast-populated collagen gel contraction and that the promotion of the gel contraction was due to the increased expression of integrin alpha2beta1 on the surface of the fibroblasts. In this study, we investigated the active component of the extract of this alga using extraction and fractionation techniques. Water extraction of the alga was followed by precipitation with excess ethanol and then gel filtration with the boundary molecular weight of 30,000. The high molecular weight fraction obtained from gel filtration was fractionated by ion exchange chromatography on diethylaminoethyl cellulose column to give active fractions that have more polar properties. These polar, high molecular weight fractions which contained molecules with fucose and sulfate groups showed significant gel contraction-promoting activity and integrin expression-enhancing activity, and were estimated to be the sulfated-polysaccharide fucoidan. Commercially available fucoidan showed similar activities to the above-described fraction of this alga. Although it remains necessary to precisely identify the specific active component, the above results indicate that fucoidan is the active component which promotes collagen gel contraction, and also indicate the possibility that it dose so by enhancing the integrin alpha2beta1 expression.

  19. Identification of Sp1 as a Transcription Activator to Regulate Fibroblast Growth Factor 21 Gene Expression

    Directory of Open Access Journals (Sweden)

    Shuqin Chen

    2017-01-01

    Full Text Available Fibroblast growth factor 21 (FGF21 is a metabolic hormone with multiple beneficial effects on lipid and glucose homeostasis. Previous study demonstrated that FGF21 might be one of the Sp1 target genes. However, the transcriptional role of Sp1 on FGF21 in adipose tissue and liver has not been reported. In this study, we found that the proximal promoter of mouse FGF21 is located between −63 and −20 containing two putative Sp1-binding sites. Sp1 is a mammalian transcription factor involved in the regulation of many genes during physiological and pathological processes. Our study showed that overexpression of Sp1 or suppressing Sp1 expression resulted in increased or reduced FGF21 promoter activity, respectively. Mutation analysis demonstrated that the Sp1-binding site located between −46 and −38 plays a primary role in transcription of FGF21. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis indicated that Sp1 specifically bound to this region. Furthermore, the binding activity of Sp1 was significantly increased in adipose tissues of HFD-induced obese mouse and liver of DEN-treated mouse. Thus, our results demonstrate that Sp1 positively regulates the basal transcription of FGF21 in the liver and adipose tissue and contributes to the obesity-induced FGF21 upregulation in mouse adipose tissue and hepatic FGF21 upregulation in hepatocarcinogenesis.

  20. Cucurbitacins-type triterpene with potent activity on mouse embryonic fibroblast from Cucumis prophetarum, cucurbitaceae

    Directory of Open Access Journals (Sweden)

    Seif-Eldin N Ayyad

    2011-01-01

    Full Text Available Background: Higher plants are considered as a well-known source of the potent anticancer metabolites with diversity of chemical structures. For instance, taxol is an amazing diterpene alkaloid had been lunched since 1990. Objective: To isolate the major compounds from the fruit extract of Cucumis prophetarum, Cucurbitaceae, which are mainly responsible for the bioactivities as anticancer. Materials and Methods: Plant material was shady air dried, extracted with equal volume of chloroform/methanol, and fractionated with different adsorbents. The structures of obtained pure compounds were elucidated with different spectroscopic techniques employing 1D ( 1 H and 13 C and 2D (COSY, HMQC and HMBC NMR (Nuclear Magnetic Resonance Spectrometry and ESI-MS (Eelectrospray Ionization Mass Spectrometry spectroscopy. The pure isolates were tested towards human cancer cell lines, mouse embryonic fibroblast (NIH3T3 and virally transformed form (KA3IT. Results: Two cucurbitacins derivatives, dihydocucurbitacin B (1 and cucurbitacin B (2, had been obtained. Compounds 1 and 2 showed potent inhibitory activities toward NIH3T3 and KA31T with IC 50 0.2, 0.15, 2.5 and 2.0 μg/ml, respectively. Conclusion: The naturally cucurbitacin derivatives (dihydocucurbitacin B and cucurbitacin B showed potent activities towards NIH3T3 and KA31T, could be considered as a lead of discovering a new anticancer natural drug.

  1. AP-2{alpha} suppresses skeletal myoblast proliferation and represses fibroblast growth factor receptor 1 promoter activity

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, Darrion L. [Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064 (United States); DiMario, Joseph X., E-mail: joseph.dimario@rosalindfranklin.edu [Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064 (United States)

    2010-01-15

    Skeletal muscle development is partly characterized by myoblast proliferation and subsequent differentiation into postmitotic muscle fibers. Developmental regulation of expression of the fibroblast growth factor receptor 1 (FGFR1) gene is required for normal myoblast proliferation and muscle formation. As a result, FGFR1 promoter activity is controlled by multiple transcriptional regulatory proteins during both proliferation and differentiation of myogenic cells. The transcription factor AP-2{alpha} is present in nuclei of skeletal muscle cells and suppresses myoblast proliferation in vitro. Since FGFR1 gene expression is tightly linked to myoblast proliferation versus differentiation, the FGFR1 promoter was examined for candidate AP-2{alpha} binding sites. Mutagenesis studies indicated that a candidate binding site located at - 1035 bp functioned as a repressor cis-regulatory element. Furthermore, mutation of this site alleviated AP-2{alpha}-mediated repression of FGFR1 promoter activity. Chromatin immunoprecipitation studies demonstrated that AP-2{alpha} interacted with the FGFR1 promoter in both proliferating myoblasts and differentiated myotubes. In total, these results indicate that AP-2{alpha} is a transcriptional repressor of FGFR1 gene expression during skeletal myogenesis.

  2. Intracellular Oxidant Activity, Antioxidant Enzyme Defense System, and Cell Senescence in Fibroblasts with Trisomy 21

    Directory of Open Access Journals (Sweden)

    Víctor Rodríguez-Sureda

    2015-01-01

    Full Text Available Down’s syndrome (DS is characterized by a complex phenotype associated with chronic oxidative stress and mitochondrial dysfunction. Overexpression of genes on chromosome-21 is thought to underlie the pathogenesis of the major phenotypic features of DS, such as premature aging. Using cultured fibroblasts with trisomy 21 (T21F, this study aimed to ascertain whether an imbalance exists in activities, mRNA, and protein expression of the antioxidant enzymes SOD1, SOD2, glutathione-peroxidase, and catalase during the cell replication process in vitro. T21F had high SOD1 expression and activity which led to an interenzymatic imbalance in the antioxidant defense system, accentuated with replicative senescence. Intracellular ROS production and oxidized protein levels were significantly higher in T21F compared with control cells; furthermore, a significant decline in intracellular ATP content was detected in T21F. Cell senescence was found to appear prematurely in DS cells as shown by SA-β-Gal assay and p21 assessment, though not apoptosis, as neither p53 nor the proapoptotic proteins cytochrome c and caspase 9 were altered in T21F. These novel findings would point to a deleterious role of oxidatively modified molecules in early cell senescence of T21F, thereby linking replicative and stress-induced senescence in cultured cells to premature aging in DS.

  3. Fine mapping and functional activity of the adenosine deaminase origin in murine embryonic fibroblasts.

    Science.gov (United States)

    Sibani, Sahar; Rampakakis, Emmanouil; Di Paola, Domenic; Zannis-Hadjopoulos, Maria

    2008-06-01

    DNA replication initiates at origins within the genome. The late-firing murine adenosine deaminase (mAdA) origin is located within a 2 kb fragment of DNA, making it difficult to examine by realtime technology. In this study, fine mapping of the mAdA region by measuring the abundance of nascent strand DNA identified two origins, mAdA-1 and mAdA-C, located 397 bp apart from each other. Both origins conferred autonomous replication to plasmids transfected in murine embryonic fibroblasts (MEFs), and exhibited similar activities in vivo and in vitro. Furthermore, both were able to recruit the DNA replication initiator proteins Cdc6 and Ku in vitro, similar to other bona fide replication origins. When tested in a murine Ku80(-/-) cell line, both origins exhibited replication activities comparable to those observed in wildtype cells, as did the hypoxanthine-guanine phosphoribosyltransferase (HPRT) and c-myc origins. This contrasts with previously published studies using Ku80-deficient human cells lines and suggests differences in the mechanism of initiation of DNA replication between the murine and human systems.

  4. SPIN90 Depletion and Microtubule Acetylation Mediate Stromal Fibroblast Activation in Breast Cancer Progression.

    Science.gov (United States)

    You, Eunae; Huh, Yun Hyun; Kwon, Ahreum; Kim, So Hee; Chae, In Hee; Lee, Ok-Jun; Ryu, Je-Hwang; Park, Min Ho; Kim, Ga-Eon; Lee, Ji Shin; Lee, Kun Ho; Lee, Yong-Seok; Kim, Jung-Woong; Rhee, Sangmyung; Song, Woo Keun

    2017-09-01

    Biomechanical remodeling of stroma by cancer-associated fibroblasts (CAF) in early stages of cancer is critical for cancer progression, and mechanical cues such as extracellular matrix stiffness control cell differentiation and malignant progression. However, the mechanism by which CAF activation occurs in low stiffness stroma in early stages of cancer is unclear. Here, we investigated the molecular mechanism underlying CAF regulation by SPIN90 and microtubule acetylation under conditions of mechanically soft matrices corresponding to normal stromal rigidity. SPIN90 was downregulated in breast cancer stroma but not tumor, and this low stromal expression correlated with decreased survival in breast cancer patients. Spin90 deficiency facilitated recruitment of mDia2 and APC complex to microtubules, resulting in increased microtubule acetylation. This increased acetylation promoted nuclear localization of YAP, which upregulated expression of myofibroblast marker genes on soft matrices. Spin90 depletion enhanced tumor progression, and blockade of microtubule acetylation in CAF significantly inhibited tumor growth in mice. Together, our data demonstrate that loss of SPIN90-mediated microtubule acetylation is a key step in CAF activation in low stiffness stroma. Moreover, correlation among these factors in human breast cancer tissue supports the clinical relevance of SPIN90 and microtubule acetylation in tumor development. Cancer Res; 77(17); 4710-22. ©2017 AACR. ©2017 American Association for Cancer Research.

  5. Hormonal regulation of dipeptidyl-aminopeptidase I activity in cultured human fibroblasts.

    Science.gov (United States)

    Davis, M H

    1987-05-01

    Human male fibroblasts, cell line GM2987, were grown in 10% Nu-Serum or fetal bovine serum. Dipeptidyl-aminopeptidase I (DAP-I) activity was higher in cells grown with Nu-Serum and cells grown in 10% fetal bovine serum purchased from Grand Island Biological Company (GIBCO) and lower in cells grown in 10% fetal bovine serum obtained from Sterile Systems, Inc. (Hyclone). The addition of 0.3 microM cortisol to all three types of sera resulted in cells that had similar levels of DAP-I activity (maximum of 800-900 nmol of beta-naphthylamine released from glycyl-L-phenylanine-beta-naphthylamine per hour per milligram of cellular protein). The addition of cortisol to Hyclone fetal bovine serum increased the DAP-I levels by up to threefold with a half-maximal response occurring at 30 nM cortisol. Triiodothyronine also could increase DAP-I levels, but only between 1.5- and 2.0-fold. Testosterone propionate increased DAP-I levels by 1.4-fold. These changes in growth media and hormones had little effect on other lysosomal enzymes or the growth characteristics of the cells.

  6. Reverse transcriptase activity in chicken embryo fibroblast culture supernatants is associated with particles containing endogenous avian retrovirus EAV-0 RNA.

    OpenAIRE

    Weissmahr, R N; Schüpbach, J; Böni, J

    1997-01-01

    We have recently shown that live attenuated virus vaccines produced on chicken-derived cells contain low levels of particle-associated reverse transcriptase (RT). In both virus and corresponding control harvests produced on chicken embryo fibroblasts, these activities were present at significantly higher concentrations than in the vaccines. In order to identify the putative retrovirus sequence responsible for this activity, a novel method for the selective PCR amplification of particle-associ...

  7. Quantitation of fibroblast activation protein (FAP-specific protease activity in mouse, baboon and human fluids and organs

    Directory of Open Access Journals (Sweden)

    Fiona M. Keane

    2014-01-01

    Full Text Available The protease fibroblast activation protein (FAP is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.

  8. A Blockade of IGF Signaling Sensitizes Human Ovarian Cancer Cells to the Anthelmintic Niclosamide-Induced Anti-Proliferative and Anticancer Activities

    Directory of Open Access Journals (Sweden)

    Youlin Deng

    2016-08-01

    Full Text Available Background/Aims: Ovarian cancer is the most lethal gynecologic malignancy, and there is an unmet clinical need to develop new therapies. Although showing promising anticancer activity, Niclosamide may not be used as a monotherapy. We seek to investigate whether inhibiting IGF signaling potentiates Niclosamide's anticancer efficacy in human ovarian cancer cells. Methods: Cell proliferation and migration are assessed. Cell cycle progression and apoptosis are analyzed by flow cytometry. Inhibition of IGF signaling is accomplished by adenovirus-mediated expression of siRNAs targeting IGF-1R. Cancer-associated pathways are assessed using pathway-specific reporters. Subcutaneous xenograft model is used to determine anticancer activity. Results: We find that Niclosamide is highly effective on inhibiting cell proliferation, cell migration, and cell cycle progression, and inducing apoptosis in human ovarian cancer cells, possibly by targeting multiple signaling pathways involved in ELK1/SRF, AP-1, MYC/MAX and NFkB. Silencing IGF-1R exert a similar but weaker effect than that of Niclosamide's. However, silencing IGF-1R significantly sensitizes ovarian cancer cells to Niclosamide-induced anti-proliferative and anticancer activities both in vitro and in vivo. Conclusion: Niclosamide as a repurposed anticancer agent may be more efficacious when combined with agents that target other signaling pathways such as IGF signaling in the treatment of human cancers including ovarian cancer.

  9. Erucin, a new promising cancer chemopreventive agent from rocket salads, shows anti-proliferative activity on human lung carcinoma A549 cells.

    Science.gov (United States)

    Melchini, A; Costa, C; Traka, M; Miceli, N; Mithen, R; De Pasquale, R; Trovato, A

    2009-07-01

    Erucin (ER) is a dietary isothiocyanate present in cruciferous vegetables, such as rocket salads (Erucasativa Mill., Diplotaxis sp.), that has been recently considered a promising cancer chemopreventive phytochemical. Biological activity of ER was investigated on human lung adenocarcinoma A549 cells, analyzing its effects on molecular pathways involved in apoptosis and cell cycle arrest, such as PARP-1 cleavage, p53 and p21 protein expression. Our results show that ER affects the A549 cell proliferation, enhancing significantly p53 and p21 protein expression in a dose-dependent manner (pinduction of p53, p21 and PARP-1 cleavage may participate in the anti-proliferative activity of ER in human lung adenocarcinoma A549 cells. Comparison of data with those obtained with the isothiocyanate sulforaphane (SF), structurally related to ER, underlines the strong relationship between structural analogy of ITCs and their biological activity. The ability of dietary compounds to modulate molecular mechanisms that affect cancer cell proliferation is certainly a key point of the cancer prevention potential by functional foods.

  10. Conformational study reveals amino acid residues essential for hemagglutinating and anti-proliferative activities of Clematis montana lectin

    National Research Council Canada - National Science Library

    Bangmin Lu Bin Zhang Wei Qi Yanan Zhu Yan Zhao Nan Zhou Rong Sun Jinku Bao Chuanfang Wu

    2014-01-01

    Clematis montana lectin (CML), a novel mannose-binding lectin purified from C. montana Buch.-Ham stem (Ranunculaceae), has been proved to have hemagglutinat- ing activity in rabbit erythrocytes and apoptosis-inducing activity in tumor cells...

  11. Effect of pirfenidone on proliferation, TGF-β-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts.

    Science.gov (United States)

    Conte, Enrico; Gili, Elisa; Fagone, Evelina; Fruciano, Mary; Iemmolo, Maria; Vancheri, Carlo

    2014-07-16

    Pirfenidone is an orally active small molecule that has been shown to inhibit the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis. Although pirfenidone exhibits well documented antifibrotic and antiinflammatory activities, in vitro and in vivo, its molecular targets and mechanisms of action have not been elucidated. In this study, we investigated the effects of pirfenidone on proliferation, TGF-β-induced differentiation and fibrogenic activity of primary human lung fibroblasts (HLFs). Pirfenidone reduced fibroblast proliferation and attenuated TGF-β-induced α-smooth muscle actin (SMA) and pro-collagen (Col)-I mRNA and protein levels. Importantly, pirfenidone inhibited TGF-β-induced phosphorylation of Smad3, p38, and Akt, key factors in the TGF-β pathway. Together, these results demonstrate that pirfenidone modulates HLF proliferation and TGF-β-mediated differentiation into myofibroblasts by attenuating key TGF-β-induced signaling pathways.

  12. The stromal cell-surface protease fibroblast activation protein-α localizes to lipid rafts and is recruited to invadopodia.

    Science.gov (United States)

    Knopf, Julia D; Tholen, Stefan; Koczorowska, Maria M; De Wever, Olivier; Biniossek, Martin L; Schilling, Oliver

    2015-10-01

    Fibroblast activation protein alpha (FAPα) is a cell surface protease expressed by cancer-associated fibroblasts in the microenvironment of most solid tumors. As there is increasing evidence for proteases having non-catalytic functions, we determined the FAPα interactome in cancer-associated fibroblasts using the quantitative immunoprecipitation combined with knockdown (QUICK) method. Complex formation with adenosin deaminase, erlin-2, stomatin, prohibitin, Thy-1 membrane glycoprotein, and caveolin-1 was further validated by immunoblotting. Co-immunoprecipitation (co-IP) of the known stoichiometric FAPα binding partner dipeptidyl-peptidase IV (DPPIV) corroborated the proteomic strategy. Reverse co-IPs validated the FAPα interaction with caveolin-1, erlin-2, and stomatin while co-IP upon RNA-interference mediated knock-down of DPPIV excluded adenosin deaminase as a direct FAPα interaction partner. Many newly identified FAPα interaction partners localize to lipid rafts, including caveolin-1, a widely-used marker for lipid raft localization. We hypothesized that this indicates a recruitment of FAPα to lipid raft structures. In density gradient centrifugation, FAPα co-fractionates with caveolin-1. Immunofluorescence optical sectioning microscopy of FAPα and lipid raft markers further corroborates recruitment of FAPα to lipid rafts and invadopodia. FAPα is therefore an integral component of stromal lipid rafts in solid tumors. In essence, we provide one of the first interactome analyses of a cell surface protease and translate these results into novel biological aspects of a marker protein for cancer-associated fibroblasts.

  13. A Murine Fibroblast Growth Factor (FGF) Receptor Expressed in CHO Cells is Activated by Basic FGF and Kaposi FGF

    Science.gov (United States)

    Mansukhani, Alka; Moscatelli, David; Talarico, Daniela; Levytska, Vera; Basilico, Claudio

    1990-06-01

    We have cloned a murine cDNA encoding a tyrosine kinase receptor with about 90% similarity to the chicken fibroblast growth factor (FGF) receptor and the human fms-like gene (FLG) tyrosine kinase. This mouse receptor lacks 88 amino acids in the extracellular portion, leaving only two immunoglobulin-like domains compared to three in the chicken FGF receptor. The cDNA was cloned into an expression vector and transfected into receptor-negative CHO cells. We show that cells expressing the receptor can bind both basic FGF and Kaposi FGF. Although the receptor binds basic FGF with a 15- to 20-fold higher affinity, Kaposi FGF is able to induce down-regulation of the receptor to the same extent as basic FGF. The receptor is phosphorylated upon stimulation with both FGFs, DNA synthesis is stimulated, and a proliferative response is produced in cells expressing the receptor, whereas cells expressing the cDNA in the antisense orientation show none of these responses to basic FGF or Kaposi FGF. Thus this receptor can functionally interact with two growth factors of the FGF family.

  14. The Cancer Cell Oxygen Sensor PHD2 Promotes Metastasis via Activation of Cancer-Associated Fibroblasts

    Directory of Open Access Journals (Sweden)

    Anna Kuchnio

    2015-08-01

    Full Text Available Several questions about the role of the oxygen sensor prolyl-hydroxylase 2 (PHD2 in cancer have not been addressed. First, the role of PHD2 in metastasis has not been studied in a spontaneous tumor model. Here, we show that global PHD2 haplodeficiency reduced metastasis without affecting tumor growth. Second, it is unknown whether PHD2 regulates cancer by affecting cancer-associated fibroblasts (CAFs. We show that PHD2 haplodeficiency reduced metastasis via two mechanisms: (1 by decreasing CAF activation, matrix production, and contraction by CAFs, an effect that surprisingly relied on PHD2 deletion in cancer cells, but not in CAFs; and (2 by improving tumor vessel normalization. Third, the effect of concomitant PHD2 inhibition in malignant and stromal cells (mimicking PHD2 inhibitor treatment is unknown. We show that global PHD2 haplodeficiency, induced not only before but also after tumor onset, impaired metastasis. These findings warrant investigation of PHD2’s therapeutic potential.

  15. Activation of Sirt1 by resveratrol inhibits TNF-α induced inflammation in fibroblasts.

    Directory of Open Access Journals (Sweden)

    Xiaoxia Zhu

    Full Text Available Inflammation is one of main mechanisms of autoimmune disorders and a common feature of most diseases. Appropriate suppression of inflammation is a key resolution to treat the diseases. Sirtuin1 (Sirt1 has been shown to play a role in regulation of inflammation. Resveratrol, a potent Sirt1 activator, has anti-inflammation property. However, the detailed mechanism is not fully understood. In this study, we investigated the anti-inflammation role of Sirt1 in NIH/3T3 fibroblast cell line. Upregulation of matrix metalloproteinases 9 (MMP-9, interleukin-1beta (IL-1β, IL-6 and inducible nitric oxide synthase (iNOS were induced by tumor necrosis factor alpha (TNF-α in 3T3 cells and resveratrol suppressed overexpression of these pro-inflammatory molecules in a dose-dependent manner. Knockdown of Sirt1 by RNA interference caused 3T3 cells susceptible to TNF-α stimulation and diminished anti-inflammatory effect of resveratrol. We also explored potential anti-inflammatory mechanisms of resveratrol. Resveratrol reduced NF-κB subunit RelA/p65 acetylation, which is notably Sirt1 dependent. Resveratrol also attenuated phosphorylation of mammalian target of rapamycin (mTOR and S6 ribosomal protein (S6RP while ameliorating inflammation. Our data demonstrate that resveratrol inhibits TNF-α-induced inflammation via Sirt1. It suggests that Sirt1 is an efficient target for regulation of inflammation. This study provides insight on treatment of inflammation-related diseases.

  16. Alterations in ROS activity and lysosomal pH account for distinct patterns of macroautophagy in LINCL and JNCL fibroblasts.

    Directory of Open Access Journals (Sweden)

    José Manuel Vidal-Donet

    Full Text Available Neuronal ceroid lipofuscinoses (NCL are lysosomal storage disorders characterized by the accumulation of lipofuscin within lysosomes. Late infantile (LINCL and juvenile (JNCL are their most common forms and are caused by loss-of-function mutations in tripeptidyl peptidase 1 (TPP1, a lysosomal endopeptidase, and CLN3 protein (CLN3p, whose location and function is still controversial. LINCL patients suffer more severely from NCL consequences than JNCL patients, in spite of having in common an abnormal accumulation of material with a similar composition in the lysosomes. To identify distinctive characteristics that could explain the differences in the severity of LINCL and JNCL pathologies, we compared the protein degradation mechanisms in patientś fibroblasts. Pulse-chase experiments show a significant decrease in protein degradation by macroautophagy in fibroblasts bearing TPP1 (CLN2 and CLN3p (CLN3 mutations. In CLN2 fibroblasts, LC3-II levels and other procedures indicate an impaired formation of autophagosomes, which confirms the pulse-chase experiments. This defect is linked to an accumulation of reactive oxygen species (ROS, an upregulation of the Akt-mTOR signalling pathway and increased activities of the p38α and ERK1/2 MAPKs. In CLN3 fibroblasts, LC3-II analysis indicates impairment in autophagosome maturation and there is also a defect in fluid phase endocytosis, two alterations that can be related to an observed increase of 0.5 units in lysosomal pH. CLN3 fibroblasts also accumulate ROS but to a lower extent than CLN2. TPP1 activity is completely abrogated in CLN2 and partially diminished in CLN3 fibroblasts. TPP1 cleaves small hydrophobic proteins like subunit c of mitochondrial ATP synthase and the lack or a lower activity of this enzyme can contribute to lipofuscin accumulation. These alterations in TPP1 activity lead to an increased ROS production, especially in CLN2 in which it is aggravated by a decrease in catalase activity

  17. Estrogenic potency of benzophenone UV filters in breast cancer cells: proliferative and transcriptional activity substantiated by docking analysis.

    Directory of Open Access Journals (Sweden)

    Gwenneg Kerdivel

    Full Text Available The results from recent studies show that some benzophenones (BPs and their hydroxylated metabolites can function as weak estrogens (E2 in the environment. However, little is known about the structure-activity relationship of these molecules. We have examined the effects of exposure to ten different BPs on the proliferation of estrogen receptor (ER-positive breast cancer cells and on the transcriptional activity of E2-target genes. We analyzed two genes that are tightly linked with estrogen-mediated proliferation, the CXCL12 and amphiregulin genes and two classical estrogen-responsive genes, the pS2 and progesterone receptor. Significant differences in the BPs efficiency to induce cell proliferation and endogenous E2-target gene expressions were observed. Using ERE-, Sp1-, AP1- and C3-reporter genes that contain different ER-binding sites in their promoter, we also showed significant differences in the BPs efficiency in activation of the ER transactivation. Together, our analyzes showed that the most active molecule is 4-hydroxy-BP. Docking analysis of the interaction of BPs in the ligand-binding pocket of ERα suggests that the minimum structural requirement for the estrogenic activity of BPs is a hydroxyl (OH group in the phenyl A-ring that allows interaction with Glu-353, Arg-394 or Phe-404, which enhances the stability between BPs and ERα. Our modeling also indicates a loss of interaction between the OH groups of the phenyl B-ring and His-524. In addition, the presence of some OH groups in the phenyl B-ring can create repulsion forces, which may constrain helix 12 in an unfavorable position, explaining the differential estrogenic effects of BPs. These results, together with our analysis of BPs for their potency in activation of cell proliferation and ER-mediated transcription, report an improved understanding of the mechanism and structure-activity relationship of BPs.

  18. DDR2 plays a role in fibroblast migration independent of adhesion ligand and collagen activated DDR2 tyrosine kinase.

    Science.gov (United States)

    Herrera-Herrera, Mireya Liliana; Quezada-Calvillo, Roberto

    2012-12-07

    Discoidin domain receptor-2 (DDR2) is a cell surface tyrosine kinase receptor that can be activated by soluble collagen and has been implicated in diverse physiological functions including organism growth and wound repair. In the current studies, we used fibronectin and collagen-coated 2D surfaces and collagen matrices in combination with siRNA technology to investigate the role of DDR2 in a range of fibroblast motile activities. Silencing DDR2 with siRNA inhibited cell spreading and migration, and similar inhibition occurred regardless whether cells were interacting with fibronectin or collagen surfaces. Under the assay conditions used, DDR2 tyrosine kinase activation was not observed unless soluble collagen was added to the incubation medium. Finally silencing DDR2 also inhibited human fibroblast migration in 3D collagen matrices but had no effect on 3D collagen matrix remodeling and contraction. Taken together, our findings suggest that DDR2 is required for normal fibroblast spreading and migration independent of adhesion ligand and collagen activation of DDR2 tyrosine kinase.

  19. Idebenone increases mitochondrial complex I activity in fibroblasts from LHON patients while producing contradictory effects on respiration

    Directory of Open Access Journals (Sweden)

    Angebault Claire

    2011-12-01

    Full Text Available Abstract Background Leber's hereditary optic neuropathy (LHON is caused by mutations in the complex I subunits of the respiratory chain. Although patients have been treated with idebenone since 1992, the efficacy of the drug is still a matter of debate. Methods We evaluated the effect of idebenone in fibroblasts from LHON patients using enzymatic and polarographic measurements. Results Complex I activity was 42% greater in treated fibroblasts compared to controls (p = 0.002. Despite this complex I activity improvement, the effects on mitochondrial respiration were contradictory, leading to impairment in some cases and stimulation in others. Conclusion These results indicate that idebenone is able to compensate the complex I deficiency in LHON patient cells with variable effects on respiration, indicating that the patients might not be equally likely to benefit from the treatment.

  20. Idebenone increases mitochondrial complex I activity in fibroblasts from LHON patients while producing contradictory effects on respiration

    DEFF Research Database (Denmark)

    Angebault, Claire; Gueguen, Naig; Desquiret-Dumas, Valerie;

    2011-01-01

    to impairment in some cases and stimulation in others. Conclusion: These results indicate that idebenone is able to compensate the complex I deficiency in LHON patient cells with variable effects on respiration, indicating that the patients might not be equally likely to benefit from the treatment.......ABSTRACT: BACKGROUND: Leber's hereditary optic neuropathy (LHON) is caused by mutations in the complex I subunits of the respiratory chain. Although patients have been treated with idebenone since 1992, the efficacy of the drug is still a matter of debate. Methods: We evaluated the effect...... of idebenone in fibroblasts from LHON patients using enzymatic and polarographic measurements. Results: Complex I activity was 42% greater in treated fibroblasts compared to controls (p = 0.002). Despite this complex I activity improvement, the effects on mitochondrial respiration were contradictory, leading...

  1. The KDM5 family is required for activation of pro-proliferative cell cycle genes during adipocyte differentiation

    DEFF Research Database (Denmark)

    Brier, Ann-Sofie B; Loft, Anne; Madsen, Jesper G S

    2017-01-01

    The KDM5 family of histone demethylases removes the H3K4 tri-methylation (H3K4me3) mark frequently found at promoter regions of actively transcribed genes and is therefore generally considered to contribute to corepression. In this study, we show that knockdown (KD) of all expressed members of th...

  2. Upregulation of the N-formyl Peptide receptors in scleroderma fibroblasts fosters the switch to myofibroblasts.

    Science.gov (United States)

    Rossi, Francesca Wanda; Napolitano, Filomena; Pesapane, Ada; Mascolo, Massimo; Staibano, Stefania; Matucci-Cerinic, Marco; Guiducci, Serena; Ragno, Pia; di Spigna, Gaetano; Postiglione, Loredana; Marone, Gianni; Montuori, Nunzia; de Paulis, Amato

    2015-06-01

    Systemic sclerosis (SSc) is characterized by chronic inflammation and fibrosis. N-Formyl peptide (fMLF) receptors (FPRs) are chemotactic receptors involved in inflammation. Three FPRs have been identified: FPR1, FPR2, and FPR3. We have examined, by RT-PCR, Western blot and immunohistochemistry, FPRs expression in skin fibroblasts from 10 normal subjects and 10 SSc patients, showing increased expression in SSc fibroblasts. Several functions of FPRs occur through the interaction with a region of the urokinase-type plasminogen activator receptor (uPAR88-92), able to interact with FPRs and to mediate urokinase (uPA) or fMLF-dependent cell migration. Soluble uPAR84-95 peptide can act as a direct ligand of FPRs. Furthermore, uPA or its aminoterminal fragment (ATF) can promote the exposure of the uPAR88-92 region. The WKYMVm peptide is a FPRs pan-agonist. We investigated the functional effects of these agonists on normal and SSc fibroblasts. ATF, uPAR84-95, and WKYMVm regulated adhesion, migration, and proliferation of normal fibroblasts. Despite FPR overexpression, the response of SSc fibroblasts to the same agonists was greatly reduced, except for the proliferative response to ATF. SSc fibroblasts showed increased α-smooth muscle actin expression and improved capability to induce wound closure. Indeed, they overexpressed a cleaved uPAR form, exposing the uPAR88-92 region, and vitronectin, both involved in fibrosis and in the fibroblast-to-myofibroblast transition. FPR stimulation promoted α-smooth muscle actin expression in normal fibroblasts as well as motility, matrix deposition, αvβ5 integrin expression, and radical oxygen species generation in normal and SSc fibroblasts. This study provides evidence that FPRs may play a role in fibrosis and in the fibroblast-to-myofibroblast transition. Copyright © 2015 by The American Association of Immunologists, Inc.

  3. Cardiac fibroblasts contribute to myocardial dysfunction in mice with sepsis: the role of NLRP3 inflammasome activation.

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    Wenbo Zhang

    Full Text Available Myocardial contractile dysfunction in sepsis is associated with the increased morbidity and mortality. Although the underlying mechanisms of the cardiac depression have not been fully elucidated, an exaggerated inflammatory response is believed to be responsible. Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3 inflammasome is an intracellular platform that is involved in the maturation and release of interleukin (IL-1β. The aim of the present study is to evaluate whether sepsis activates NLRP3 inflammasome/caspase-1/IL-1β pathway in cardiac fibroblasts (CFs and whether this cytokine can subsequently impact the function of cardiomyocytes (cardiac fibroblast-myocyte cross-talk. We show that treatment of CFs with lipopolysaccharide (LPS induces upregulation of NLRP3, activation of caspase-1, as well as the maturation (activation and release of IL-1β. In addition, the genetic (small interfering ribonucleic acid [siRNA] and pharmacological (glyburide inhibition of the NLRP3 inflammasome in CFs can block this signaling pathway. Furthermore, the inhibition of the NLRP3 inflammasome in cardiac fibroblasts ameliorated the ability of LPS-challenged CFs to impact cardiomyocyte function as assessed by intracellular cyclic adenosine monophosphate (cAMP responses in cardiomyocytes. Salient features of this the NLP3 inflammasome/ caspase-1 pathway were confirmed in in vivo models of endotoxemia/sepsis. We found that inhibition of the NLRP3 inflammasome attenuated myocardial dysfunction in mice with LPS and increased the survival rate in mice with feces-induced peritonitis. Our results indicate that the activation of the NLRP3 inflammasome in cardiac fibroblasts is pivotal in the induction of myocardial dysfunction in sepsis.

  4. Fibroblast growth factor 2 dimer with superagonist in vitro activity improves granulation tissue formation during wound healing.

    Science.gov (United States)

    Decker, Caitlin G; Wang, Yu; Paluck, Samantha J; Shen, Lu; Loo, Joseph A; Levine, Alex J; Miller, Lloyd S; Maynard, Heather D

    2016-03-01

    Site-specific chemical dimerization of fibroblast growth factor 2 (FGF2) with the optimal linker length resulted in a FGF2 homodimer with improved granulation tissue formation and blood vessel formation at exceptionally low concentrations. Homodimers of FGF2 were synthesized through site-specific linkages to both ends of different molecular weight poly(ethylene glycols) (PEGs). The optimal linker length was determined by screening dimer-induced metabolic activity of human dermal fibroblasts and found to be that closest to the inter-cysteine distance, 70 Å, corresponding to 2 kDa PEG. A straightforward analysis of the kinetics of second ligand binding as a function of tether length showed that, as the polymerization index (the number of monomer repeat units in the polymer, N) of the tether decreases, the mean time for second ligand capture decreases as ∼N(3/2), leading to an enhancement of the number of doubly bound ligands in steady-state for a given (tethered) ligand concentration. FGF2-PEG2k-FGF2 induced greater fibroblast metabolic activity than FGF2 alone, all other dimers, and all monoconjugates, at each concentration tested, with the greatest difference observed at low (0.1 ng/mL) concentration. FGF2-PEG2k-FGF2 further exhibited superior activity compared to FGF2 for both metabolic activity and migration in human umbilical vein endothelial cells, as well as improved angiogenesis in a coculture model in vitro. Efficacy in an in vivo wound healing model was assessed in diabetic mice. FGF2-PEG2k-FGF2 increased granulation tissue and blood vessel density in the wound bed compared to FGF2. The results suggest that this rationally designed construct may be useful for improving the fibroblast matrix formation and angiogenesis in chronic wound healing.

  5. Potential Wound Healing Activities of Galla Rhois in Human Fibroblasts and Keratinocytes.

    Science.gov (United States)

    Park, Hyo-Hyun; Park, Na-Young; Kim, Sun-Gun; Jeong, Kyu-Tae; Lee, Eu-Jin; Lee, Eunkyung

    2015-01-01

    Wound healing is a complex process orchestrated by the regeneration of the epithelium and the remodeling of the extracellular matrix through processes like collagen deposition. Galla Rhois has been widely used in traditional Korean medicine for its various pharmacological effects, including an anticoccidial effect, however, little is known about its healing activity. The purpose of this study was to determine the effects of Galla Rhois ethanol extract (GRE) on wound healing activities, including H2O2-induced oxidative stress, cell migration, and lactate dehydrogenase (LDH) release assays using human keratinocyte (HaCaT) and dermal fibroblasts (CCD-986SK). In addition, total soluble collagen deposition and collagen gene expression for Type I and III collagen were evaluated in CCD-986SK. Total tannin and flavonoid contents for GRE were measured. GRE induced a significant increase in the number and migration of cells, along with a decrease in cell death and LDH release. In addition, it also induced the over-expression of collagen Type I and III mRNA and caused increased synthesis of total soluble collagen. The contents of total tannin and flavonoid for GRE were 55.7% ([Formula: see text][Formula: see text]mg/g) and 62.9% ([Formula: see text][Formula: see text]mg/g), respectively. The results suggest that GRE can cause accelerated wound healing by increasing cell survival, proliferation, migration, and collagen synthesis along with a potential anti-oxidant property. This evidence provides novel insight into natural therapy for tissue injury.

  6. Novel pyrazole and indazole derivatives: synthesis and evaluation of their anti-proliferative and anti-angiogenic activities

    OpenAIRE

    Tzanetou, Evangelia; Liekens, Sandra; Kasiotis, Konstantinos M.; Fokialakis, Nikolas; Haroutounian, Serkos A

    2012-01-01

    The synthesis of several new pyrazole and indazole derivatives from acetophenone and tetralone substrates is reported. The bioactivities of the new compounds were evaluated through in vitro assays for endothelial cell proliferation and tube formation. Results herein indicate that the easily prepared compounds containing the indazole structural framework exhibit potent cytostatic properties against all cell lines tested, with compounds 13 and 14 being the most active displaying IC(50) values o...

  7. Proliferative Activity of Liver Growth Factor is Associated with an Improvement of Cigarette Smoke-Induced Emphysema in Mice

    Science.gov (United States)

    Terrón-Expósito, Raúl; Díaz-Gil, Juan José; González-Mangado, Nicolás; Peces-Barba, Germán

    2014-01-01

    Cigarette smoke (CS)-induced emphysema is a major component of chronic obstructive pulmonary disease (COPD). COPD treatment is based on the administration of bronchodilators and corticosteroids to control symptoms and exacerbations, however, to date, there are no effective therapies to reverse disease progression. Liver growth factor (LGF) is an albumin-bilirubin complex with mitogenic properties, whose therapeutic effects have previously been reported in a model of emphysema and several rodent models of human disease. To approach the therapeutic effect of LGF in a model of previously established emphysema, morphometric and lung function parameters, matrix metalloproteinase (MMP) activity and the expression of several markers, such as VEGF, PCNA, 3NT and Nrf2, were assessed in air-exposed and CS-exposed C57BL/6J male mice with and without intraperitoneal (i.p.) injection of LGF. CS-exposed mice presented a significant enlargement of alveolar spaces, higher alveolar internal area and loss of lung function that correlated with higher MMP activity, higher expression of 3NT and lower expression of VEGF. CS-exposed mice injected with LGF, showed an amelioration of emphysema and improved lung function, which correlated with lower MMP activity and 3NT expression and higher levels of VEGF, PCNA and Nrf2. Taken together, this study suggests that LGF administration ameliorates CS-induced emphysema, highlights the ability of LGF to promote alveolar cell proliferation and may be a promising strategy to revert COPD progression. PMID:25401951

  8. Mesenchymal stem cells suppress fibroblast proliferation and reduce skin fibrosis through a TGF-β3-dependent activation.

    Science.gov (United States)

    Wu, Yan; Peng, Yan; Gao, Dongyun; Feng, Changjiang; Yuan, Xiaohuan; Li, Houzhong; Wang, Ying; Yang, Lan; Huang, Sha; Fu, Xiaobing

    2015-03-01

    Recent studies showed that transplantation of mesenchymal stem cells (MSCs) significantly decreased tissue fibrosis; however, little attention has been paid to its efficacy on attenuating skin fibrosis, and the mechanism involved in its effect is poorly understood. In this work, we investigated the effects of MSCs on keloid fibroblasts and extracellular matrix deposition through paracrine actions and whether the antifibrotic properties of MSCs involved transforming growth factor-β (TGF-β)-dependent activation. In vitro experiments showed that conditioned media (CM) from MSCs decreased viability, a-smooth muscle actin expression, and collagen secretion of human keloid fibroblasts. In addition, TGF-β3 secreted by MSCs was expressed at high level under inflammatory environment, and blocking the activity of TGF-β3 apparently antagonized the suppressive activity of MSC CM, which demonstrated that TGF-β3 played a preponderant role in preventing collagen accumulation. In vivo studies showed that MSC CM infusion in a mouse dermal fibrosis model induced a significant decrease in skin fibrosis. Histological examination of tissue sections and immunohistochemical analysis for α-smooth muscle actin revealed that TGF-β3 of CM-mediated therapeutic effects could obviously attenuate matrix production and myofibroblast proliferation and differentiation. These findings suggest that TGF-β3 mediates the attenuating effect of MSCs on both the proliferation and extracellular matrix production of human keloid fibroblasts and decreases skin fibrosis of mouse model, thus providing new understanding and MSC-based therapeutic strategy for cutaneous scar treatment.

  9. Suppression of Tumor Growth in Mice by Rationally Designed Pseudopeptide Inhibitors of Fibroblast Activation Protein and Prolyl Oligopeptidase

    Directory of Open Access Journals (Sweden)

    Kenneth W. Jackson

    2015-01-01

    Full Text Available Tumor microenvironments (TMEs are composed of cancer cells, fibroblasts, extracellular matrix, microvessels, and endothelial cells. Two prolyl endopeptidases, fibroblast activation protein (FAP and prolyl oligopeptidase (POP, are commonly overexpressed by epithelial-derived malignancies, with the specificity of FAP expression by cancer stromal fibroblasts suggesting FAP as a possible therapeutic target. Despite overexpression in most cancers and having a role in angiogenesis, inhibition of POP activity has received little attention as an approach to quench tumor growth. We developed two specific and highly effective pseudopeptide inhibitors, M83, which inhibits FAP and POP proteinase activities, and J94, which inhibits only POP. Both suppressed human colon cancer xenograft growth >90% in mice. By immunohistochemical stains, M83- and J94-treated tumors had fewer microvessels, and apoptotic areas were apparent in both. In response to M83, but not J94, disordered collagen accumulations were observed. Neither M83- nor J94-treated mice manifested changes in behavior, weight, or gastrointestinal function. Tumor growth suppression was more extensive than noted with recently reported efforts by others to inhibit FAP proteinase function or reduce FAP expression. Diminished angiogenesis and the accompanying profound reduction in tumor growth suggest that inhibition of either FAP or POP may offer new therapeutic approaches that directly target TMEs.

  10. Endothelin-1 activation of ETB receptors leads to a reduced cellular proliferative rate and an increased cellular footprint

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Jamie L.; Taylor, Linda; Polgar, Peter, E-mail: peterp@bu.edu

    2012-06-10

    Endothelin-1 (ET-1) is a vasoactive peptide which signals through two G-protein coupled receptors, endothelin receptor A (ETA) and B (ETB). We determined that ET-1 activation of its ETB receptor in stably cDNA transfected CHO cells leads to a 55% reduction in cell number by end-point cell counting and a 35% decrease in cell growth by a real-time cell-substrate impedance-based assay after 24 h of cell growth. When CHO ETB cells were synchronized in the late G1 cell cycle phase, ET-1 delayed their S phase progression compared to control by 30% as determined by [{sup 3}H]-thymidine incorporation. On the other hand, no such delay was observed during late G2/M to G1 transit when cells were treated with ET-1 after release from mitotic arrest. Using the cell-substrate impedance-based assay, we observed that ET-1 induces opposing morphological changes in CHO ETA and CHO ETB cells with ETB causing an increase in the cell footprint and ETA a decrease. Likewise, in pulmonary artery smooth muscle cells, which express both ETA and ETB receptors, ET-1 induces an ETA-dependent contraction and an ETB dependent dilation. These results are shedding light on a possible beneficial role for ETB in diseases involving ET-1 dysfunction such as pulmonary hypertension. -- Highlights: Black-Right-Pointing-Pointer ET- hinders cell proliferation in CHO cells transfected with ETB. Black-Right-Pointing-Pointer ET-1 also decreases the rate of DNA synthesis in CHO ETB cells. Black-Right-Pointing-Pointer JNK and PI3K appear to be involved in this reduction of DNA synthesis. Black-Right-Pointing-Pointer ETB activation in CHO ETB cells and hSMCs leads to dilatory morphological changes. Black-Right-Pointing-Pointer In CHO ETA and hSMCs, ETA activation leads to constrictive morphological changes.

  11. SMI of Bcl-2 TW-37 is active across a spectrum of B-cell tumors irrespective of their proliferative and differentiation status.

    Science.gov (United States)

    Al-Katib, Ayad M; Sun, Yuan; Goustin, Anton Scott; Azmi, Asfar Sohail; Chen, Ben; Aboukameel, Amro; Mohammad, Ramzi M

    2009-02-16

    The Bcl-2 family of proteins is critical to the life and death of malignant B-lymphocytes. Interfering with their activity using small-molecule inhibitors (SMI) is being explored as a new therapeutic strategy for treating B-cell tumors. We evaluated the efficacy of TW-37, a non-peptidic SMI of Bcl-2 against a range spectrum of human B-cell lines, fresh patient samples and animal xenograft models. Multiple cytochemical and molecular approaches such as acridine orange/ethidium bromide assay for apoptosis, co-immunoprecipitation of complexes and western blot analysis, caspase luminescent activity assay and apoptotic DNA fragmentation assay were used to demonstrate the effect of TW-37 on different B-cell lines, patient derived samples, as well as in animal xenograft models. Nanomolar concentrations of TW-37 were able to induce apoptosis in both fresh samples and established cell lines with IC50 in most cases of 165-320 nM. Apoptosis was independent of proliferative status or pathological classification of B-cell tumor. TW-37 was able to block Bim-Bcl-XL and Bim-Mcl-1 heterodimerization and induced apoptosis via activation of caspases -9, -3, PARP and DNA fragmentation. TW-37 administered to tumor-bearing SCID mice led to significant tumor growth inhibition (T/C), tumor growth delay (T-C) and Log10kill, when used at its maximum tolerated dose (40 mg/kg x 3 days) via tail vein. TW-37 failed to induce changes in the Bcl-2 proteins levels suggesting that assessment of baseline Bcl-2 family proteins can be used to predict response to the drug. These findings indicate activity of TW-37 across the spectrum of human B-cell tumors and support the concept of targeting the Bcl-2 system as a therapeutic strategy regardless of the stage of B-cell differentiation.

  12. Synthesis, characterization, DNA interactions, DNA cleavage, radical scavenging activity, antibacterial, anti-proliferative and docking studies of new transition metal complexes.

    Science.gov (United States)

    Chennam, Kishan Prasad; Ravi, Mudavath; Ushaiah, B; Srinu, V; Eslavath, Ravi Kumar; Devi, Ch Sarala

    2016-01-01

    The compound N-(2-hydroxybenzylidene)-1-ethyl-1, 4-dihydro-7-methyl-4-oxo-1, 8 naphthyridine-3-carbohydrazide (LH) and its Cu (II), Co (II) and Zn (II) complexes were synthesized and characterized. The absorption spectral titrations and competitive DNA binding studies depicted those complexes of title compound bind to CT-DNA through intercalation. Interestingly [Cu (II)-(L2)] showed relatively high binding constant value (6.61 x 10(5) M(-1)) compared to [Co (II)-(L2)] (4.378× 10(5) M(-1)) and [Zn (II)-(L2)] (3.1x10(5) M(-1)). Ligand and its complexes were also examined for DNA nuclease activity against pBR-322 plasmid DNA, which showed that [Cu (II)-(L2)] had the best hydrolytic cleavage property displaying prominent double-strand DNA cleavage. In addition, antioxidant activities of the ligand and its metal complexes investigated through scavenging effects for DPPH radical in- vitro, indicated their potentiality as good antioxidants. The in vitro anti-bacterial study inferred the better anti-bacterial activity of [Cu (II)-(L2)] and this was also correlated theoretically by employing docking studies wherein [Cu (II)-(L2)] displayed good Gold score and Chem score. Finally the in vitro anti- proliferative activity of studied compounds was tested against HeLa and MCF-7 cell lines. Interestingly [Cu (II)-(L2)] displayed lower IC50 value and lower percentage of viability in both HeLa and MCF-7 cell lines.

  13. Resveratrol prevents oxidative stress-induced senescence and proliferative dysfunction by activating the AMPK-FOXO3 cascade in cultured primary human keratinocytes.

    Science.gov (United States)

    Ido, Yasuo; Duranton, Albert; Lan, Fan; Weikel, Karen A; Breton, Lionel; Ruderman, Neil B

    2015-01-01

    The aging process is perceived as resulting from a combination of intrinsic factors such as changes in intracellular signaling and extrinsic factors, most notably environmental stressors. In skin, the relationship between intrinsic changes and keratinocyte function is not clearly understood. Previously, we found that increasing the activity of AMP-activated protein kinase (AMPK) suppressed senescence in hydrogen peroxide (H2O2)-treated human primary keratinocytes, a model of oxidative stress-induced cellular aging. Using this model in the present study, we observed that resveratrol, an agent that increases the activities of both AMPK and sirtuins, ameliorated two age-associated phenotypes: cellular senescence and proliferative dysfunction. In addition, we found that treatment of keratinocytes with Ex527, a specific inhibitor of sirtuin 1 (SIRT1), attenuated the ability of resveratrol to suppress senescence. In keeping with the latter observation, we noted that compared to non-senescent keratinocytes, senescent cells lacked SIRT1. In addition to these effects on H2O2-induced senescence, resveratrol also prevented the H2O2-induced decrease in proliferation (as indicated by 3H-thymidine incorporation) in the presence of insulin. This effect was abrogated by inhibition of AMPK but not SIRT1. Compared to endothelium, we found that human keratinocytes expressed relatively high levels of Forkhead box O3 (FOXO3), a downstream target of both AMPK and SIRT1. Treatment of keratinocytes with resveratrol transactivated FOXO3 and increased the expression of its target genes including catalase. Resveratrol's effects on both senescence and proliferation disappeared when FOXO3 was knocked down. Finally, we performed an exploratory study which showed that skin from humans over 50 years old had lower AMPK activity than skin from individuals under age 20. Collectively, these findings suggest that the effects of resveratrol on keratinocyte senescence and proliferation are regulated by

  14. Transcriptional Regulation of Δ6-Desaturase by Peroxisome Proliferative-Activated Receptor δ Agonist in Human Pancreatic Cancer Cells: Role of MEK/ERK1/2 Pathway

    Directory of Open Access Journals (Sweden)

    Maryam Darabi

    2013-01-01

    Full Text Available The Δ6-desaturase (Δ6D, also known as fatty acid desaturase 2, is a regulatory enzyme in de novo fatty acid synthesis, which has been linked to obesity and diabetes. The aim of the present study was to investigate the effect of peroxisome proliferative-activated receptor δ (PPARδ agonist and MEK/ERK1/2-dependent pathway on the expression of Δ6D in human pancreatic carcinoma cell line PANC-1. PANC-1 cells cultured in RPMI-1640 were exposed to the commonly used ERK1/2 pathway inhibitor PD98059 and PPARδ agonist GW0742. Changes in mRNA and protein expression of Δ6D were then determined using real-time RT-PCR and Western blot, respectively. The expression of Δ6D (P40%, P25%, P<0.05 pretreatment. PPARδ and MEK/ERK1/2 signaling pathways affect differentially the expression of Δ6D in pancreatic cancer cells. Furthermore, there may be an inhibitory crosstalk between these two regulatory pathways on the mRNA expression of Δ6D and subsequently on Δ6D protein expression.

  15. Fetal intestinal fibroblasts respond to insulin-like growth factor (IGF-II better than adult intestinal fibroblasts

    Directory of Open Access Journals (Sweden)

    Fillenwarth Michael J

    2006-01-01

    Full Text Available Abstract Background We compared IGF responses of fetal and adult intestinal fibroblasts to identify a developmental difference in the IGF-axis. Intestinal fibroblasts were isolated from maternal and fetal jejunum. Media was conditioned at confluence and one week afterwards. The proliferative response at confluence to 5 nM IGF-I or -II was compared. Results There were no significant differences in IGFBP expression at confluence. Post-confluence, fetal fibroblasts had no significant changes in IGFBP-2 and IGFBP-3 expression. Post-confluent maternal fibroblasts had increased IGFBP-3 levels that were significant compared to the fetal fibroblasts. IGF-I increased in post-confluent fetal fibroblasts, while in maternal fibroblasts it decreased (p Conclusion Fetal intestinal fibroblasts respond to IGF-II with greater proliferation and do not have the increased IGFBPs seen post-confluence in adult intestinal fibroblasts.

  16. Anti-Proliferative Activity of λ-Carrageenan Through the Induction of Apoptosis in Human Breast Cancer Cells

    Science.gov (United States)

    Jazzara, Marie; Ghannam, Ahmed; Soukkarieh, Chadi; Murad, Hossam

    2016-01-01

    Background Sulfated Polysaccharides (SPs) possess spectrum of pharmacological and therapeutic properties that could attributed to their origins variation, chemical structures and biological activities. Various studies have shown the impact of SPs on proliferation in different cancer cell lines. Objectives In this study, we have evaluated the biological effects of λ-carrageenan, a highly SP, extracted from the red seaweed Laurencia papillosa, on MDA-MB-231 cancer cell line. Materials and Methods MDA-MB-231 cells have treated with λ-carrageenan, the viability and apoptosis have assessed by the appropriate florescent probes on flow cytometer. The expression levels of mRNA of apoptotic genes have detected by real-time PCR analysis. Results Our results have indicated that the signaling pathway of λ-carrageenan inhibited the proliferation of MDA-MB-231 cells by up-regulating the pro-apoptotic genes caspase-8, caspase-9, caspase-3 which have been resulting the increased levels of active caspase-3 protein. Furthermore, This SP had that capacity to disrupt the mitochondrial function by altering the bax/bcl-2 ratio of expression which has considered an important element in apoptosis induction. Conclusions The presented results have signposted that λ-carrageenan was a promising bioactive polymer which could be a potential candidate in preventing or treating breast cancer. PMID:27761203

  17. Paclitaxel attenuates renal interstitial fibroblast activation and interstitial fibrosis by inhibiting STAT3 signaling

    Directory of Open Access Journals (Sweden)

    Zhang L

    2015-04-01

    results suggest that paclitaxel may block the STAT3 activity by disrupting the association of STAT3 with tubulin and inhibiting STAT3 nucleus translocation, consequently leading to the suppression of renal interstitial fibroblast activation and the development of renal fibrosis, and inhibition of proinflammatory cytokine production.Keywords: UUO, tubulointerstitial fibrosis, tubulin, paclitaxel, STAT3

  18. Galphaq signaling is required for Rho-dependent transcriptional activation of the cyclooxygenase-2 promoter in fibroblasts.

    Science.gov (United States)

    Slice, Lee W; Han, Sang-Kyou; Simon, Melvin I

    2003-02-01

    Previously, we demonstrated that the gastrin releasing peptide (GRP) induces cyclooxygenase-2 (COX-2) expression through a Rho-dependent, protein kinase C (PKC)-independent signaling pathway in fibroblasts (Slice et al., 1999, J Biol Chem 274:27562-27566). However, the specific role of heterotrimeric guanine nucleotide binding regulatory proteins (G-proteins) that are coupled to the GRP receptor in Rho-dependent COX-2 expression has not been elucidated. In this report, we utilize embryonic fibroblasts from transgenic mice containing double gene knock-outs (DKO) for Galpha(q/11) and Galpha(12/13) to demonstrate that COX-2 promoter activation by GRP requires Galpha(q). Furthermore, we show that GRP-dependent COX-2 gene expression, as assessed by a COX-2 reporter luciferase assay, was induced in cells lacking Galpha(12/13) but was blocked in cells that did not express Galpha(q/11). GRP-dependent COX-2 promoter induction in Galpha(q/11) deficient cells was rescued by expression of wild type Galpha(q) but blocked by inhibition of calcium signaling in calcium-free media or in cells treated with 2-aminoethoxydiphenylborate (2-APB). Co-stimulation of transfected Galpha(q/11) deficient cells with GRP and thapsigargin (TG) induced the COX-2 promoter. Activation of endogenous Rho by expression of Onco-lbc or expression of Rho A Q63L resulted in COX-2 promoter activation in Galpha(q/11) deficient cells. Inhibition of Rho by Clostridium botulinum C3 toxin blocked COX-2 promoter induction. Expression of Galpha(q) Q209L in the well-characterized fibroblast cell line, NIH3T3, induced the COX-2 promoter which was blocked by expression of C3 toxin. These results demonstrate that calcium signaling mediated by Galpha(q) and Rho play critical roles in GRP-dependent COX-2 expression in fibroblasts.

  19. Novel pyrazole and indazole derivatives: synthesis and evaluation of their anti-proliferative and anti-angiogenic activities.

    Science.gov (United States)

    Tzanetou, Evangelia; Liekens, Sandra; Kasiotis, Konstantinos M; Fokialakis, Nikolas; Haroutounian, Serkos A

    2012-10-01

    The synthesis of several new pyrazole and indazole derivatives from acetophenone and tetralone substrates is reported. The bioactivities of the new compounds were evaluated through in vitro assays for endothelial cell proliferation and tube formation. Results herein indicate that the easily prepared compounds containing the indazole structural framework exhibit potent cytostatic properties against all cell lines tested, with compounds 13 and 14 being the most active displaying IC(50) values of 1.5 ± 0.4 µM and 5.6 ± 2.5 µM, respectively, against MCF-7 cells. In addition, the indazole derivative 16 was assessed as a competent inhibitor of endothelial tube formation at 30 µM.

  20. Cytotoxic Compounds from Juglans sinensis Dode Display Anti-Proliferative Activity by Inducing Apoptosis in Human Cancer Cells.

    Science.gov (United States)

    Lee, Yoo Jin; Cui, Jun; Lee, Jun; Han, Ah-Reum; Lee, Eun Byul; Jang, Ho Hee; Seo, Eun Kyoung

    2016-01-01

    Phytochemical investigation of the bark of Juglans sinensis Dode (Juglandaceae) led to the isolation of two active compounds, 8-hydroxy-2-methoxy-1,4-naphthoquinone (1) and 5-hydroxy-2-methoxy-1,4-naphthoquinone (2), together with 15 known compounds 3-17. All compounds were isolated from this plant for the first time. The structures of 1 and 2 were elucidated by spectroscopic data analysis, including 1D and 2D NMR experiments. Compounds 1-17 were tested for their cytotoxicity against the A549 human lung cancer cell line; compounds 1 and 2 exhibited significant cytotoxicity and additionally had potent cytotoxicity against six human cancer cell lines, MCF7 (breast cancer), SNU423 (liver cancer), SH-SY5Y (neuroblastoma), HeLa (cervical cancer), HCT116 (colorectal cancer), and A549 (lung cancer). In particular, breast, colon, and lung cancer cells were more sensitive to the treatment using compound 1. In addition, compounds 1 and 2 showed strong cytotoxic activity towards human breast cancer cells MCF7, HS578T, and T47D, but not towards MCF10A normal-like breast cells. They also inhibited the colony formation of MCF7, A549, and HCT116 cells in a dose-dependent manner. Flow cytometry analysis revealed that the percentage of apoptotic cells significantly increased in MCF7 cells upon the treatment with compounds 1 and 2. The mechanism of cell death caused by compounds 1 and 2 may be attributed to the upregulation of Bax and downregulation of Bcl2. These findings suggest that compounds 1 and 2 may be regarded as potential therapeutic agents against cancer.

  1. Fibroblast activation protein (FAP is essential for the migration of bone marrow mesenchymal stem cells through RhoA activation.

    Directory of Open Access Journals (Sweden)

    Kuei-Min Chung

    Full Text Available BACKGROUND: The ability of human bone marrow mesenchymal stem cells (BM-MSCs to migrate and localize specifically to injured tissues is central in developing therapeutic strategies for tissue repair and regeneration. Fibroblast activation protein (FAP is a cell surface serine protease expressed at sites of tissue remodeling during embryonic development. It is also expressed in BM-MSCs, but not in normal tissues or cells. The function of FAP in BM-MSCs is not known. PRINCIPAL FINDINGS: We found that depletion of FAP proteins significantly inhibited the migration of BM-MSCs in a transwell chemotaxis assay. Such impaired migration ability of BM-MSCs could be rescued by re-expressing FAP in these cells. We then demonstrated that depletion of FAP activated intracellular RhoA GTPase. Consistently, inhibition of RhoA activity using a RhoA inhibitor rescued its migration ability. Inhibition of FAP activity with an FAP-specific inhibitor did not affect the activation of RhoA or the migration of BM-MSCs. Furthermore, the inflammatory cytokines interleukin-1beta (IL-1β and transforming growth factor-beta (TGF-β upregulated FAP expression, which coincided with better BM-MSC migration. CONCLUSIONS: Our results indicate FAP plays an important role in the migration of BM-MSCs through modulation of RhoA GTPase activity. The peptidase activity of FAP is not essential for such migration. Cytokines IL-1β and TGF-β upregulate the expression level of FAP and thus enhance BM-MSC migration.

  2. The Apoptotic Effects of the P300 Activator on Breast Cancer and Lung Fibroblast Cell Lines

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Salahshoor

    2013-10-01

    Full Text Available Background: P300 is an enzyme that acetylates histones during stress. It alsoacetylates several non-histone proteins, including P53 which is the most important tumorsuppressor gene. P53 plays an important role in the apoptosis of tumor cells. Hereby,this study describes the potency of cholera toxin B subunit as a P300 activator to induceapoptosis in a breast cancer cell line (MCF-7 and a lung fibroblast cell line (MRC-5as a non-tumorigenic control sample. Methods: MCF-7 and MRC-5 were cultured in RPMI-1640 and treated with orwithout cholera toxin B subunit at the concentration of 85.43 μmol/L, based on the half-maximal inhibitory concentration index at different times (24, 48 and 72 h. Thepercentage of apoptotic cells was measured by flow cytometry. Real-time quantitativeRT-PCR was performed to estimate the mRNA expression of P300 in MCF-7 and MRC-5 with cholera toxin B subunit at different times. We used the ELISA and Bradford proteintechniques to detect levels of total and acetylated P53 protein generated in MCF-7 andMRC-5. Results: Our findings indicated that the cholera toxin B subunit effectively andsignificantly induced more apoptosis in MCF-7 compared to MRC-5. We showed thatexpression of P300 up-regulated by increasing the time of the cholera toxin B subunittreatment in MCF-7 but not in MRC-5. In addition, the acetylated and total P53protein levels increased more in MCF-7 cells than in MRC-5 cells.Conclusion: Cholera toxin B subunit induced significant cell death in MCF-7, butit could be well tolerated in MRC-5. Therefore, cholera toxin B subunit can besuggested as an anti-cancer agent.

  3. Two New Oleanane-Type Saponins with Anti-Proliferative Activity from Camellia oleifera Abel. Seed Cake

    Directory of Open Access Journals (Sweden)

    Jian-Fa Zong

    2016-02-01

    Full Text Available Two new oleanane-type saponins, named oleiferasaponins C4 (1 and C5 (2, were isolated from Camellia oleifera Abel. seed cake residue. Their respective structures were identified as 16α-hydroxy-22α-O-angeloyl-23α-aldehyde-28-dihydroxymethylene-olean-12-ene-3β-O-[β-d-galacto-pyranosyl-(1→2]-[β-d-glucopyranosyl-(1→2-β-d-galactopyranosy-(1→3]-β-d-glucopyranosid-uronic acid methyl ester (1 and 16α-hydroxy-22α-O-angeloyl-23α-aldehyde-28-dihydroxy-methylene-olean-12-ene-3β-O-[β-d-galactopyranosyl-(1→2]-[β-d-galactopyranosyl-(1→3]-β-d-glucopyranosiduronic acid methyl ester (2 through 1D- and 2D-NMR, HR-ESI-MS, and GC-MS spectroscopic methods. The two compounds exhibited potent cytotoxic activities against five human tumor cell lines (BEL-7402, BGC-823, MCF-7, HL-60 and KB.

  4. SGR J1550-5418 BURSTS DETECTED WITH THE FERMI GAMMA-RAY BURST MONITOR DURING ITS MOST PROLIFIC ACTIVITY

    Energy Technology Data Exchange (ETDEWEB)

    Van der Horst, A. J.; Finger, M. H. [Universities Space Research Association, NSSTC, Huntsville, AL 35805 (United States); Kouveliotou, C. [Space Science Office, VP62, NASA/Marshall Space Flight Center, Huntsville, AL 35812 (United States); Gorgone, N. M. [Connecticut College, New London, CT 06320 (United States); Kaneko, Y.; Goegues, E.; Lin, L. [Sabanc Latin-Small-Letter-Dotless-I University, Orhanl Latin-Small-Letter-Dotless-I -Tuzla, Istanbul 34956 (Turkey); Baring, M. G. [Department of Physics and Astronomy, Rice University, MS-108, P.O. Box 1892, Houston, TX 77251 (United States); Guiriec, S.; Bhat, P. N.; Chaplin, V. L.; Goldstein, A. [University of Alabama, Huntsville, CSPAR, Huntsville, AL 35805 (United States); Granot, J. [Racah Institute of Physics, Hebrew University, Jerusalem 91904 (Israel); Watts, A. L. [Astronomical Institute ' Anton Pannekoek' , University of Amsterdam, Postbus 94249, 1090 GE Amsterdam (Netherlands); Bissaldi, E.; Gruber, D. [Max Planck Institute for Extraterrestrial Physics, Giessenbachstrasse, Postfach 1312, 85748 Garching (Germany); Gehrels, N.; Harding, A. K. [NASA Goddard Space Flight Center, Greenbelt, MD 20771 (United States); Gibby, M. H.; Giles, M. M., E-mail: A.J.VanDerHorst@uva.nl [Jacobs Technology, Inc., Huntsville, AL (United States); and others

    2012-04-20

    We have performed detailed temporal and time-integrated spectral analysis of 286 bursts from SGR J1550-5418 detected with the Fermi Gamma-ray Burst Monitor (GBM) in 2009 January, resulting in the largest uniform sample of temporal and spectral properties of SGR J1550-5418 bursts. We have used the combination of broadband and high time-resolution data provided with GBM to perform statistical studies for the source properties. We determine the durations, emission times, duty cycles, and rise times for all bursts, and find that they are typical of SGR bursts. We explore various models in our spectral analysis, and conclude that the spectra of SGR J1550-5418 bursts in the 8-200 keV band are equally well described by optically thin thermal bremsstrahlung (OTTB), a power law (PL) with an exponential cutoff (Comptonized model), and two blackbody (BB) functions (BB+BB). In the spectral fits with the Comptonized model, we find a mean PL index of -0.92, close to the OTTB index of -1. We show that there is an anti-correlation between the Comptonized E{sub peak} and the burst fluence and average flux. For the BB+BB fits, we find that the fluences and emission areas of the two BB functions are correlated. The low-temperature BB has an emission area comparable to the neutron star surface area, independent of the temperature, while the high-temperature BB has a much smaller area and shows an anti-correlation between emission area and temperature. We compare the properties of these bursts with bursts observed from other SGR sources during extreme activations, and discuss the implications of our results in the context of magnetar burst models.

  5. Mitotic defects lead to pervasive aneuploidy and accompany loss of RB1 activity in mouse LmnaDhe dermal fibroblasts.

    Directory of Open Access Journals (Sweden)

    C Herbert Pratt

    Full Text Available BACKGROUND: Lamin A (LMNA is a component of the nuclear lamina and is mutated in several human diseases, including Emery-Dreifuss muscular dystrophy (EDMD; OMIM ID# 181350 and the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS; OMIM ID# 176670. Cells from progeria patients exhibit cell cycle defects in both interphase and mitosis. Mouse models with loss of LMNA function have reduced Retinoblastoma protein (RB1 activity, leading to aberrant cell cycle control in interphase, but how mitosis is affected by LMNA is not well understood. RESULTS: We examined the cell cycle and structural phenotypes of cells from mice with the Lmna allele, Disheveled hair and ears (Lmna(Dhe. We found that dermal fibroblasts from heterozygous Lmna(Dhe (Lmna(Dhe/+ mice exhibit many phenotypes of human laminopathy cells. These include severe perturbations to the nuclear shape and lamina, increased DNA damage, and slow growth rates due to mitotic delay. Interestingly, Lmna(Dhe/+ fibroblasts also had reduced levels of hypophosphorylated RB1 and the non-SMC condensin II-subunit D3 (NCAP-D3, a mitosis specific centromere condensin subunit that depends on RB1 activity. Mitotic check point control by mitotic arrest deficient-like 1 (MAD2L1 also was perturbed in Lmna(Dhe/+ cells. Lmna(Dhe/+ fibroblasts were consistently aneuploid and had higher levels of micronuclei and anaphase bridges than normal fibroblasts, consistent with chromosome segregation defects. CONCLUSIONS: These data indicate that RB1 may be a key regulator of cellular phenotype in laminopathy-related cells, and suggest that the effects of LMNA on RB1 include both interphase and mitotic cell cycle control.

  6. [The influence of low-frequency pulsed electric and magnetic signals or their combination on the normal and modified fibroblasts (an experimental study)].

    Science.gov (United States)

    Ulitko, M V; Medvedeva, S Yu; Malakhov, V V

    2016-01-01

    The results of clinical studies give evidence of the beneficial preventive and therapeutic effects of the «Tiline-EM» physiotherapeutic device designed for the combined specific treatment of the skin regions onto which both discomfort and pain sensations are directly projected, reflectively active sites and zones, as well as trigger zones with the use of low-frequency pulsed electric current and magnetic field. The efficient application of the device requires the understanding of the general mechanisms underlying such action on the living systems including those operating at the cellular and subcellular levels. The objective of the present study was the investigation of the specific and complex effects produced by the low-frequency pulses of electric current and magnetic field generated in the physiotherapeutic device «Tiline-EM» on the viability, proliferative activity, and morphofunctional characteristics of normal skin fibroblasts and the transformed fibroblast line K-22. It has been demonstrated that the biological effects of the electric and magnetic signals vary depending on the type of the cell culture and the mode of impact. The transformed fibroblasts proved to be more sensitive to the specific and complex effects of electric and magnetic pulses than the normal skin fibroblasts. The combined action of the electric and magnetic signals was shown to have the greatest influence on both varieties of fibroblasts. It manifests itself in the form of enhanced viability, elevated proliferative and synthetic activity in the cultures of transformed fibroblasts and as the acceleration of cell differentiation in the cultures of normal fibroblasts. The effect of stimulation of dermal fibroblast differentiation in response to the combined treatment by the electric and magnetic signals is of interest from the standpoint of the physiotherapeutic use of the «Tiline-EM» device for the purpose of obtaining fibroblasts cultures to be employed in regenerative therapy and

  7. Antioxidant and potential anti-inflammatory activity of extracts and formulations of white tea, rose, and witch hazel on primary human dermal fibroblast cells

    OpenAIRE

    Hili Pauline; Thring Tamsyn SA; Naughton Declan P

    2011-01-01

    Abstract Background Numerous reports have identified therapeutic roles for plants and their extracts and constituents. The aim of this study was to assess the efficacies of three plant extracts for their potential antioxidant and anti-inflammatory activity in primary human skin fibroblasts. Methods Aqueous extracts and formulations of white tea, witch hazel and rose were subjected to assays to measure anti-collagenase, anti-elastase, trolox equivalent and catalase activities. Skin fibroblast ...

  8. S100A4 amplifies TGF-β-induced fibroblast activation in systemic sclerosis

    DEFF Research Database (Denmark)

    Tomcik, Michal; Palumbo-Zerr, Katrin; Zerr, Pawel

    2015-01-01

    the expression of S100A4 to stimulate the release of collagen in SSc fibroblasts and induce fibrosis. Since S100A4 is essentially required for the pro-fibrotic effects of TGF-β and neutralising antibodies against S100A4 are currently evaluated, S100A4 might be a candidate for novel antifibrotic therapies....

  9. RAGE activation induces invasiveness of RA fibroblast-like synoviocytes in vitro

    NARCIS (Netherlands)

    Steenvoorden, M.M.C.; Toes, R.E.M.; Ronday, H.K.; Huizinga, T.W.J.; Groot, J. de

    2007-01-01

    Ligands for the receptor for advanced glycation endproducts (RAGE) are increased in RA synovial fluid (SF), serum and synovium. Since RAGE is present on fibroblast-like synoviocytes (FLS), the present study investigates whether the RAGE ligands HMGB-1 and AGEs are able to stimulate the characteristi

  10. RAGE activation induces invasiveness of RA fibroblast-like synoviocytes in vitro

    NARCIS (Netherlands)

    Steenvoorden, M.M.C.; Toes, R.E.M.; Ronday, H.K.; Huizinga, T.W.J.; Groot, J. de

    2007-01-01

    Ligands for the receptor for advanced glycation endproducts (RAGE) are increased in RA synovial fluid (SF), serum and synovium. Since RAGE is present on fibroblast-like synoviocytes (FLS), the present study investigates whether the RAGE ligands HMGB-1 and AGEs are able to stimulate the

  11. RAGE activation induces invasiveness of RA fibroblast-like synoviocytes in vitro

    NARCIS (Netherlands)

    Steenvoorden, M.M.C.; Toes, R.E.M.; Ronday, H.K.; Huizinga, T.W.J.; Groot, J. de

    2007-01-01

    Ligands for the receptor for advanced glycation endproducts (RAGE) are increased in RA synovial fluid (SF), serum and synovium. Since RAGE is present on fibroblast-like synoviocytes (FLS), the present study investigates whether the RAGE ligands HMGB-1 and AGEs are able to stimulate the characteristi

  12. Immune gene expression profiling of Proliferative Kidney Disease in rainbow trout Oncorhynchus mykiss reveals a dominance of anti-inflammatory, antibody and T helper cell-like activities.

    Science.gov (United States)

    Gorgoglione, Bartolomeo; Wang, Tiehui; Secombes, Christopher J; Holland, Jason W

    2013-07-16

    The myxozoan Tetracapsuloides bryosalmonae is the causative agent of Proliferative Kidney Disease (PKD) targeting primarily the kidney of infected fish where it causes a chronic lymphoid immunopathology. Although known to be associated with suppression of some cellular aspects of innate immunity and a prominent lymphocytic hyperplasia, there remains a considerable knowledge gap in our understanding of the underlying immune mechanisms driving PKD pathogenesis. To provide further insights, the expression profiles of a panel of innate/inflammatory and adaptive immune molecules were examined in rainbow trout Oncorhynchus mykiss following a natural exposure to the parasite. Relative to controls, fish with early to advanced stages of kidney pathology exhibited up-regulation of the inflammatory cytokines interleukin (IL)-6 and IL-11, although remaining refractory towards genes indicative of macrophage activity. Antimicrobial peptides (AMPs) and anti-inflammatory markers, including cathelicidin (CATH) and IL-10 were markedly up-regulated during clinical disease. Up-regulation of adaptive immune molecules, including cell markers and antibody genes reflect the lymphocytic dominance of this disease and the likely importance of lymphocyte subsets in PKD pathogenesis. Up-regulation of T helper (TH) cell-like response genes and transcription factors implies that T. bryosalmonae may elicit a complex interplay between TH cell subsets. This work, for the first time in the study of fish-myxozoan interactions, suggests that PKD pathogenesis is shaped by an anti-inflammatory phenotype, a profound B cell/antibody response and dysregulated TH cell-like activities. A better understanding of the functional roles of fish immune cells and molecules in PKD pathogenesis may facilitate future development of control measures against this disease.

  13. Comparative Study of Green Sub- and Supercritical Processes to Obtain Carnosic Acid and Carnosol-Enriched Rosemary Extracts with in Vitro Anti-Proliferative Activity on Colon Cancer Cells

    Directory of Open Access Journals (Sweden)

    Andrea del Pilar Sánchez-Camargo

    2016-12-01

    Full Text Available In the present work, four green processes have been compared to evaluate their potential to obtain rosemary extracts with in vitro anti-proliferative activity against two colon cancer cell lines (HT-29 and HCT116. The processes, carried out under optimal conditions, were: (1 pressurized liquid extraction (PLE, using an hydroalcoholic mixture as solvent at lab-scale; (2 Single-step supercritical fluid extraction (SFE at pilot scale; (3 Intensified two-step sequential SFE at pilot scale; (4 Integrated PLE plus supercritical antisolvent fractionation (SAF at pilot scale. Although higher extraction yields were achieved by using PLE (38.46% dry weight, this extract provided the lowest anti-proliferative activity with no observed cytotoxic effects at the assayed concentrations. On the other hand, extracts obtained using the PLE + SAF process provided the most active rosemary extracts against both colon cancer cell lines, with LC50 ranging from 11.2 to 12.4 µg/mL and from 21.8 to 31.9 µg/mL for HCT116 and HT-29, respectively. In general, active rosemary extracts were characterized by containing carnosic acid (CA and carnosol (CS at concentrations above 263.7 and 33.9 mg/g extract, respectively. Some distinct compounds have been identified in the SAF extracts (rosmaridiphenol and safficinolide, suggesting their possible role as additional contributors to the observed strong anti-proliferative activity of CA and CS in SAF extracts.

  14. Comparative Study of Green Sub- and Supercritical Processes to Obtain Carnosic Acid and Carnosol-Enriched Rosemary Extracts with in Vitro Anti-Proliferative Activity on Colon Cancer Cells.

    Science.gov (United States)

    Sánchez-Camargo, Andrea Del Pilar; García-Cañas, Virginia; Herrero, Miguel; Cifuentes, Alejandro; Ibáñez, Elena

    2016-12-07

    In the present work, four green processes have been compared to evaluate their potential to obtain rosemary extracts with in vitro anti-proliferative activity against two colon cancer cell lines (HT-29 and HCT116). The processes, carried out under optimal conditions, were: (1) pressurized liquid extraction (PLE, using an hydroalcoholic mixture as solvent) at lab-scale; (2) Single-step supercritical fluid extraction (SFE) at pilot scale; (3) Intensified two-step sequential SFE at pilot scale; (4) Integrated PLE plus supercritical antisolvent fractionation (SAF) at pilot scale. Although higher extraction yields were achieved by using PLE (38.46% dry weight), this extract provided the lowest anti-proliferative activity with no observed cytotoxic effects at the assayed concentrations. On the other hand, extracts obtained using the PLE + SAF process provided the most active rosemary extracts against both colon cancer cell lines, with LC50 ranging from 11.2 to 12.4 µg/mL and from 21.8 to 31.9 µg/mL for HCT116 and HT-29, respectively. In general, active rosemary extracts were characterized by containing carnosic acid (CA) and carnosol (CS) at concentrations above 263.7 and 33.9 mg/g extract, respectively. Some distinct compounds have been identified in the SAF extracts (rosmaridiphenol and safficinolide), suggesting their possible role as additional contributors to the observed strong anti-proliferative activity of CA and CS in SAF extracts.

  15. Resveratrol Increases Anti-Proliferative Activity of Bestatin Through Downregulating P-Glycoprotein Expression Via Inhibiting PI3K/Akt/mTOR Pathway in K562/ADR Cells.

    Science.gov (United States)

    Wang, Li; Wang, Changyuan; Jia, Yongming; Liu, Zhihao; Shu, Xiaohong; Liu, Kexin

    2016-05-01

    Multidrug resistance (MDR) is a major obstacle in the clinical therapy of hematological malignancies. P-glycoprotein (P-gp) overexpression results in reduction of intracellular drug concentration with a consequence that the cytotoxicity of anti-tumor drugs is decreased, which leads to MDR in K562/ADR cells. In this study, we found that resveratrol enhanced the anti-proliferative activity of bestatin in K562/ADR cells. Co-treatment with resveratrol, IC50 values of bestatin in K562/ADR cells significantly decreased and activation of caspase-3 and caspase-8 increased, which indicated that resveratrol potentiated bestatin-induced apoptosis. Resveratrol increased the intracellular concentration of bestatin through inhibiting P-gp function and downregulating P-gp expression at mRNA and protein levels, which increased anti-proliferative activity of bestatin in K562/ADR cells. Resveratrol decreased the phosphorylation of Akt and mTOR but did not affect the phosphorylations of JNK or ERK1/2. These results demonstrated that resveratrol could increase the anti-proliferative activity of bestatin through downregulating P-gp expression via suppressing the PI3K/Akt/mTOR signaling pathway.

  16. Clinical efficacy and mechanistic evaluation of aflibercept for proliferative diabetic retinopathy (acronym CLARITY): a multicentre phase IIb randomised active-controlled clinical trial.

    Science.gov (United States)

    Sivaprasad, Sobha; Prevost, A Toby; Bainbridge, James; Edwards, Rhiannon Tudor; Hopkins, David; Kelly, Joanna; Luthert, Phil; Murphy, Caroline; Ramu, Jayashree; Sarafraz-Shekary, Negin; Vasconcelos, Joana; White-Alao, Beverley; Hykin, Philip

    2015-09-14

    Proliferative diabetic retinopathy (PDR) is the main cause of severe visual loss in people with diabetes mellitus. The standard treatment for this condition is panretinal photocoagulation (PRP). This laser treatment is inherently destructive, with predictable adverse effects on visual function, and a safer alternative is required. Intravitreal injection of vascular endothelial growth factor (VEGF) inhibitors can induce short-term regression of retinal neovascularisation. The aim of this randomised controlled trial is to determine the efficacy, safety and cost-effectiveness of intravitreal aflibercept, an inhibitor of VEGF-A, VEGF-B and placental growth factor (PLGF), in PDR, and to investigate the impact on local oxygenation. This is a phase IIb randomised controlled single-masked multicentre clinical trial to determine the impact of repeated intravitreal aflibercept injections in the treatment and prevention of PDR. 220 participants with treatment-naïve or treated but active retinal neovascularisation in at least one eye will be randomly allocated 1:1 to intravitreal aflibercept injections or PRP for a period of 52 weeks. The primary outcome is the change in best-corrected visual acuity in the study eye at 52 weeks. Secondary outcomes include changes from baseline in other visual functions, anatomical changes and cost-effectiveness. Ocular and non-ocular adverse events will also be reported over 52 weeks. The study has been approved by the National Research Ethics Service (NRES) committee with respect to scientific content and compliance with applicable research and human subjects' regulations. Findings will be reported through scientific publications and research conferences. The results of this study will provide clinical evidence for the feasibility, efficacy safety and cost-effectiveness of intravitreal aflibercept for PDR. ISRCTN 32207582. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence

  17. A novel dual-functioning ruthenium(II)-arene complex of an anti-microbial ciprofloxacin derivative - Anti-proliferative and anti-microbial activity.

    Science.gov (United States)

    Ude, Ziga; Romero-Canelón, Isolda; Twamley, Brendan; Fitzgerald Hughes, Deirdre; Sadler, Peter J; Marmion, Celine J

    2016-07-01

    7-(4-(Decanoyl)piperazin-1-yl)-ciprofloxacin, CipA, (1) which is an analogue of the antibiotic ciprofloxacin, and its ruthenium(II) complex [Ru(η(6)-p-cymene)(CipA-H)Cl], (2) have been synthesised and the x-ray crystal structures of 1·1.3H2O·0.6CH3OH and 2·CH3OH·0.5H2O determined. The complex adopts a typical pseudo-octahedral 'piano-stool' geometry, with Ru(II) π-bonded to the p-cymene ring and σ-bonded to a chloride and two oxygen atoms of the chelated fluoroquinolone ligand. The complex is highly cytotoxic in the low μM range and is as potent as the clinical drug cisplatin against the human cancer cell lines A2780, A549, HCT116, and PC3. It is also highly cytotoxic against cisplatin- and oxaliplatin-resistant cell lines suggesting a different mechanism of action. The complex also retained low μM cytotoxicity against the human colon cancer cell line HCT116p53 in which the tumour suppressor p53 had been knocked out, suggesting that the potent anti-proliferative properties associated with this complex are independent of the status of p53 (in contrast to cisplatin). The complex also retained moderate anti-bacterial activity in two Escherichia coli, a laboratory strain and a clinical isolate resistant to first, second and third generation β-lactam antibiotics.

  18. Neutrophil elastase cleavage of the gC1q domain impairs the EMILIN1-α4β1 integrin interaction, cell adhesion and anti-proliferative activity

    Science.gov (United States)

    Maiorani, Orlando; Pivetta, Eliana; Capuano, Alessandra; Modica, Teresa Maria Elisa; Wassermann, Bruna; Bucciotti, Francesco; Colombatti, Alfonso; Doliana, Roberto; Spessotto, Paola

    2017-01-01

    The extracellular matrix glycoprotein EMILIN1 exerts a wide range of functions mainly associated with its gC1q domain. Besides providing functional significance for adhesion and migration, the direct interaction between α4β1 integrin and EMILIN1-gC1q regulates cell proliferation, transducing net anti-proliferative effects. We have previously demonstrated that EMILIN1 degradation by neutrophil elastase (NE) is a specific mechanism leading to the loss of functions disabling its regulatory properties. In this study we further analysed the proteolytic activity of NE, MMP-3, MMP-9, and MT1-MMP on EMILIN1 and found that MMP-3 and MT1-MMP partially cleaved EMILIN1 but without affecting the functional properties associated with the gC1q domain, whereas NE was able to fully impair the interaction of gC1q with the α4β1 integrin by cleaving this domain outside of the E933 integrin binding site. By a site direct mutagenesis approach we mapped the bond between S913 and R914 residues and selected the NE-resistant R914W mutant still able to interact with the α4β1 integrin after NE treatment. Functional studies showed that NE impaired the EMILIN1-α4β1 integrin interaction by cleaving the gC1q domain in a region crucial for its proper structural conformation, paving the way to better understand NE effects on EMILIN1-cell interaction in pathological context. PMID:28074935

  19. Inhibition of cyclooxygenase-2 prevents intra-abdominal adhesions by decreasing activity of peritoneal fibroblasts

    Directory of Open Access Journals (Sweden)

    Wei G

    2015-06-01

    Full Text Available Guangbing Wei,1 Xin Chen,2 Guanghui Wang,1 Pengbo Jia,1,3 Qinhong Xu,2 Gaofeng Ping,1 Kang Wang,1 Xuqi Li1 1Department of General Surgery, 2Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi’an Jiaotong University College of Medicine, Xi’an, 3Department of General Surgery, First People’s Hospital of Xianyang City, Xianyang, People’s Republic of China Background: Postoperative intra-abdominal adhesions are common complications after abdominal surgery. The exact molecular mechanisms that are responsible for these complications remain unclear, and there are no effective methods for preventing adhesion formation or reformation. The aim of the study reported here was to investigate the preventive effects and underlying potential molecular mechanisms of selective cyclooxygenase-2 (COX-2 inhibitors in a rodent model of postoperative intra-abdominal adhesions.Materials and methods: The expression of COX-2 in postoperative intra-abdominal adhesions and normal peritoneal tissue was examined by immunohistochemistry and Western blot analysis. Assays were performed to elucidate the effect of COX-2 inhibition on hypoxia-induced fibroblast activity in vitro and on intra-abdominal adhesion formation in vivo.Results: Hypoxia-induced COX-2 expression in peritoneal fibroblasts was increased in postoperative intra-abdominal adhesions. Inhibition of COX-2 attenuated the activating effect of hypoxia on normal peritoneal fibroblasts in vitro. Data indicate that selective COX-2 inhibitor prevents in vivo intra-abdominal adhesion by inhibition of basic fibroblast growth factor and transforming growth factor-beta expression, but not through an antiangiogenic mechanism. Furthermore, using selective COX-2 inhibitors to prevent intra-abdominal adhesions did not adversely affect the weight, bowel motility, or healing of intestinal anastomoses in a rat model.Conclusion: These results show that hypoxia-induced COX-2 expression in peritoneal

  20. Discoidin Domain Receptor 2 Mediates Collagen-Induced Activation of Membrane-Type 1 Matrix Metalloproteinase in Human Fibroblasts.

    Science.gov (United States)

    Majkowska, Iwona; Shitomi, Yasuyuki; Ito, Noriko; Gray, Nathanael S; Itoh, Yoshifumi

    2017-03-07

    Membrane-Type 1 Matrix Metalloproteinase (MT1-MMP) is a membrane-bound MMP that is highly expressed in cells with invading capacity including fibroblasts and invasive cancer cell. A potential physiological stimulus for MT1-MMP expression is fibrillar collagen, and it has been shown that it upregulates both MT1-MMP gene and functions in various cell types. However, the mechanisms of collagen-mediated MT1-MMP activation is not clearly understood. In this study we identified discoidin domain receptor 2 (DDR2) as a crucial receptor that mediates this process in human fibroblasts. Knocking down DDR2, but not β1 integrin subunit, a common subunit for all collagen-binding integrins, inhibited collagen-induced activation of proMMP-2 and upregulation of MT1-MMP at the gene and protein level. Interestingly DDR2 knockdown or pharmacological inhibition of DDR2 also inhibited MT1-MMP-dependent cellular degradation of collagen film, suggesting that cell surface collagen degradation by MT1-MMP involves DDR2-mediated collagen signalling. This DDR2-mediated mechanism is only present in non-transformed mesenchymal cells, as collagen-induced MT1-MMP activation in HT1080 fibrosarcoma cells and MT1-MMP function in MDA-MB231 breast cancer cells were not affected by DDR kinase inhibition. DDR2 activation was found to be noticeably more effective when cells were stimulated by collagen without non-helical telopeptides region compared to intact collagen fibrils. Those data suggest that DDR2 is a microenvironmental sensor that regulates fibroblasts migration in collagen-rich environment.

  1. Stepwise proteolytic activation of type I procollagen to collagen within the secretory pathway of tendon fibroblasts in situ.

    Science.gov (United States)

    Canty-Laird, Elizabeth G; Lu, Yinhui; Kadler, Karl E

    2012-01-15

    Proteolytic cleavage of procollagen I to collagen I is essential for the formation of collagen fibrils in the extracellular matrix of vertebrate tissues. Procollagen is cleaved by the procollagen N- and C-proteinases, which remove the respective N- and C-propeptides from procollagen. Procollagen processing is initiated within the secretory pathway in tendon fibroblasts, which are adept in assembling an ordered extracellular matrix of collagen fibrils in vivo. It was thought that intracellular processing was restricted to the TGN (trans-Golgi network). In the present study, brefeldin A treatment of tendon explant cultures showed that N-proteinase activity is present in the resulting fused ER (endoplasmic reticulum)-Golgi compartment, but that C-proteinase activity is restricted to the TGN in embryonic chick tendon fibroblasts. In late embryonic and postnatal rat tail and postnatal mouse tail tendon, C-proteinase activity was detected in TGN and pre-TGN compartments. Preventing activation of the procollagen N- and C-proteinases with the furin inhibitor Dec-RVKR-CMK (decanoyl-Arg-Val-Lys-Arg-chloromethylketone) indicated that only a fraction of intracellular procollagen cleavage was mediated by newly activated proteinases. In conclusion, the N-propeptides are removed earlier in the secretory pathway than the C-propeptides. The removal of the C-propeptides in post-Golgi compartments most probably indicates preparation of collagen molecules for fibril formation at the cell-matrix interface.

  2. Analysis of Activated Platelet-Derived Growth Factor β Receptor and Ras-MAP Kinase Pathway in Equine Sarcoid Fibroblasts

    Directory of Open Access Journals (Sweden)

    Gennaro Altamura

    2013-01-01

    Full Text Available Equine sarcoids are skin tumours of fibroblastic origin affecting equids worldwide. Bovine papillomavirus type-1 (BPV-1 and, less commonly, type-2 are recognized as etiological factors of sarcoids. The transforming activity of BPV is related to the functions of its major oncoprotein E5 which binds to the platelet-derived growth factor β receptor (PDGFβR causing its phosphorylation and activation. In this study, we demonstrate, by coimmunoprecipitation and immunoblotting, that in equine sarcoid derived cell lines PDGFβR is phosphorylated and binds downstream molecules related to Ras-mitogen-activated protein kinase-ERK pathway thus resulting in Ras activation. Imatinib mesylate is a tyrosine kinase receptors inhibitor which selectively inhibits the activation of PDGFβR in the treatment of several human and animal cancers. Here we show that imatinib inhibits receptor phosphorylation, and cell viability assays demonstrate that this drug decreases sarcoid fibroblasts viability in a dose-dependent manner. This study contributes to a better understanding of the molecular mechanisms involved in the pathology of sarcoids and paves the way to a new therapeutic approach for the treatment of this common equine skin neoplasm.

  3. Hypoxia induces NO-dependent release of heparan sulfate in fibroblasts from the Alzheimer mouse Tg2576 by activation of nitrite reduction.

    Science.gov (United States)

    Cheng, Fang; Bourseau-Guilmain, Erika; Belting, Mattias; Fransson, Lars-Åke; Mani, Katrin

    2016-06-01

    There is a functional relationship between the heparan sulfate proteoglycan glypican-1 and the amyloid precursor protein (APP) of Alzheimer disease. In wild-type mouse embryonic fibroblasts, expression and processing of the APP is required for endosome-to-nucleus translocation of anhydromannose-containing heparan sulfate released from S-nitrosylated glypican-1 by ascorbate-induced, nitrosothiol-catalyzed deaminative cleavage. In fibroblasts from the transgenic Alzheimer mouse Tg2576, there is increased processing of the APP to amyloid-β peptides. Simultaneously, there is spontaneous formation of anhydromannose-containing heparan sulfate by an unknown mechanism. We have explored the effect of hypoxia on anhydromannose-containing heparan sulfate formation in wild-type and Tg2576 fibroblasts by deconvolution immunofluorescence microscopy and flow cytometry using an anhydromannose-specific monoclonal antibody and by (35)SO4-labeling experiments. Hypoxia prevented ascorbate-induced heparan sulfate release in wild-type fibroblasts, but induced an increased formation of anhydromannose-positive and (35)S-labeled heparan sulfate in Tg2576 fibroblasts. This appeared to be independent of glypican-1 S-nitrosylation as demonstrated by using a monoclonal antibody specific for S-nitrosylated glypican-1. In hypoxic wild-type fibroblasts, addition of nitrite to the medium restored anhydromannose-containing heparan sulfate formation. The increased release of anhydromannose-containing heparan sulfate in hypoxic Tg2576 fibroblasts did not require addition of nitrite. However, it was suppressed by inhibition of the nitrite reductase activity of xanthine oxidoreductase/aldehyde oxidase or by inhibition of p38 mitogen-activated protein kinase or by chelation of iron. We propose that normoxic Tg2576 fibroblasts maintain a high level of anhydromannose-containing heparan sulfate production by a stress-activated generation of nitric oxide from endogenous nitrite. This activation is enhanced

  4. Hypoxia-Induced Collagen Synthesis of Human Lung Fibroblasts by Activating the Angiotensin System

    OpenAIRE

    Shan-Shan Liu; Hao-Yan Wang; Jun-Ming Tang; Xiu-Mei Zhou

    2013-01-01

    The exact molecular mechanism that mediates hypoxia-induced pulmonary fibrosis needs to be further clarified. The aim of this study was to explore the effect and underlying mechanism of angiotensin II (Ang II) on collagen synthesis in hypoxic human lung fibroblast (HLF) cells. The HLF-1 cell line was used for in vitro studies. Angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) expression levels in human ...

  5. Pro-life role for c-Jun N-terminal kinase and p38 mitogen-activated protein kinase at rostral ventrolateral medulla in experimental brain stem death

    Directory of Open Access Journals (Sweden)

    Chang Alice YW

    2012-11-01

    Full Text Available Abstract Background Based on an experimental brain stem death model, we demonstrated previously that activation of the mitogen-activated protein kinase kinase 1/2 (MEK1/2/extracellular signal-regulated kinase 1/2 (ERK1/2/mitogen-activated protein kinase signal-interacting kinase 1/2 (MNK1/2 cascade plays a pro-life role in the rostral ventrolateral medulla (RVLM, the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life and decreases (pro-death to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients. The present study assessed the hypothesis that, in addition to ERK1/2, c-Jun NH2-terminal kinase (JNK and p38 mitogen-activated protein kinase (p38MAPK, the other two mammalian members of MAPKs that are originally identified as stress-activated protein kinases, are activated specifically by MAPK kinase 4 (MAP2K4 or MAP2K6 and play a pro-life role in RVLM during experimental brain stem death. We further delineated the participation of phosphorylating activating transcriptional factor-2 (ATF-2 and c-Jun, the classical transcription factor activated by JNK or p38MAPK, in this process. Results An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol bilaterally into RVLM of Sprague–Dawley rats was used, alongside cardiovascular, pharmacological and biochemical evaluations. Results from ELISA showed that whereas the total JNK, p38MAPK, MAP2K4 and MAP2K6 were not affected, augmented phosphorylation of JNK at Thr183 and Tyr185 and p38MAPK at Thr180 and Tyr182, accompanied by phosphorylation of their upstream activators MAP2K4 at Ser257 and Thr261 and MAP2K6 at Ser207 and Thr211 in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Moreover, the activity of transcription factors ATF-2 at Thr71 and

  6. ShcA regulates neurite outgrowth stimulated by neural cell adhesion molecule but not by fibroblast growth factor 2: evidence for a distinct fibroblast growth factor receptor response to neural cell adhesion molecule activation

    DEFF Research Database (Denmark)

    Hinsby, Anders M; Lundfald, Line; Ditlevsen, Dorte K;

    2004-01-01

    by two principal routes of signaling: NCAM/Fyn and NCAM/fibroblast growth factor receptor (FGFR), respectively. Previous studies have shown that activation of mitogen-activated protein kinases is a pivotal point of convergence in NCAM signaling, but the mechanisms behind this activation are not clear....... Here, we investigated the involvement of adaptor proteins in NCAM and fibroblast growth factor 2 (FGF2)-mediated neurite outgrowth in the PC12-E2 cell line. We found that both FGFR substrate-2 and Grb2 play important roles in NCAM as well as in FGF2-stimulated events. In contrast, the docking protein...... ShcA was pivotal to neurite outgrowth induced by NCAM, but not by FGF2, in PC12 cells. Moreover, in rat cerebellar granule neurons, phosphorylation of ShcA was stimulated by an NCAM mimicking peptide, but not by FGF2. This activation was blocked by inhibitors of both FGFR and Fyn, indicating that NCAM...

  7. TGF-β1 activates the canonical NF-κB signaling to promote cell survival and proliferation in dystrophic muscle fibroblasts in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Zhen-Yu [Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, No. 58 Zhongshan 2nd Road, Guangzhou 510080, Guangdong Province (China); Department of Neurology, The Second Affiliated Hospital, Guangzhou Medical University, No.250 Changgang East Road, Guangzhou 510260, Guangdong Province (China); Zhong, Zhi-Gang; Qiu, Meng-Yao; Zhong, Yu-Hua [Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, No. 58 Zhongshan 2nd Road, Guangzhou 510080, Guangdong Province (China); Zhang, Wei-Xi, E-mail: weixizhang@qq.com [Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, No. 58 Zhongshan 2nd Road, Guangzhou 510080, Guangdong Province (China)

    2016-03-18

    Activated fibroblasts continue to proliferate at injury sites, leading to progressive muscular fibrosis in Duchenne muscular dystrophy (DMD). TGF-β1 is a dominant profibrotic mediator thought to play a critical role in muscle fibrosis; however, the implicated mechanisms are not fully understood. Here we showed that TGF-β1 increased the resistance to apoptosis and stimulated cell cycle progression in dystrophic muscle fibroblasts under serum deprivation conditions in vitro. TGF-β1 treatment activated the canonical NF-κB pathway; and we found that pharmacological inhibition of IKKβ with IMD-0354 and RelA gene knockdown with siRNA attenuated these effects of TGF-β1 on dystrophic muscle fibroblasts. Collectively, our data suggest that TGF-β1 prevents apoptosis and cell cycle arrest in dystrophic muscle fibroblasts through the canonical NF-κB signaling pathway. - Highlights: • TGF-β1 promotes survival and proliferation in dystrophic muscle fibroblasts. • TGF-β1 activated the canonical NF-κB pathway in dystrophic muscle fibroblasts. • Canonical NF-κB pathway mediates these effects of TGF-β1.

  8. Separating mitogenic and metabolic activities of fibroblast growth factor 19 (FGF19).

    Science.gov (United States)

    Wu, Xinle; Ge, Hongfei; Lemon, Bryan; Vonderfecht, Steven; Baribault, Helene; Weiszmann, Jennifer; Gupte, Jamila; Gardner, Jonitha; Lindberg, Richard; Wang, Zhulun; Li, Yang

    2010-08-10

    FGF19 and FGF21 are distinctive members of the FGF family that function as endocrine hormones. Their potent effects on normalizing glucose, lipid, and energy homeostasis in disease models have made them an interesting focus of research for combating the growing epidemics of diabetes and obesity. Despite overlapping functions, FGF19 and FGF21 have many discrete effects, the most important being that FGF19 has both metabolic and proliferative effects, whereas FGF21 has only metabolic effects. Here we identify the structural determinants dictating differential receptor interactions that explain and distinguish these two physiological functions. We also have generated FGF19 variants that have lost the ability to induce hepatocyte proliferation but that still are effective in lowering plasma glucose levels and improving insulin sensitivity in mice. Our results add valuable insight into the structure-function relationship of FGF19/FGF21 and identify the structural basis underpinning the distinct proliferative feature of FGF19 compared with FGF21. In addition, these studies provide a road map for engineering FGF19 as a potential therapeutic candidate for treating diabetes and obesity.

  9. SIRT1 overexpression antagonizes cellular senescence with activated ERK/S6k1 signaling in human diploid fibroblasts.

    Directory of Open Access Journals (Sweden)

    Jing Huang

    Full Text Available Sir2, a NAD-dependent deacetylase, modulates lifespan in yeasts, worms and flies. The SIRT1, mammalian homologue of Sir2, regulates signaling for favoring survival in stress. But whether SIRT1 has the function to influence cell viability and senescence under non-stressed conditions in human diploid fibroblasts is far from unknown. Our data showed that enforced SIRT1 expression promoted cell proliferation and antagonized cellular senescence with the characteristic features of delayed Senescence-Associated beta-galactosidase (SA-beta-gal staining, reduced Senescence-Associated Heterochromatic Foci (SAHF formation and G1 phase arrest, increased cell growth rate and extended cellular lifespan in human fibroblasts, while dominant-negative SIRT1 allele (H363Y did not significantly affect cell growth and senescence but displayed a bit decreased lifespan. Western blot results showed that SIRT1 reduced the expression of p16(INK4A and promoted phosphorylation of Rb. Our data also exposed that overexpression of SIRT1 was accompanied by enhanced activation of ERK and S6K1 signaling. These effects were mimicked in both WI38 cells and 2BS cells by concentration-dependent resveratrol, a SIRT1 activator. It was noted that treatment of SIRT1-.transfected cells with Rapamycin, a mTOR inhibitor, reduced the phosphorylation of S6K1 and the expression of Id1, implying that SIRT1-induced phosphorylation of S6K1 may be partly for the decreased expression of p16(INK4A and promoted phosphorylation of Rb in 2BS. It was also observed that the expression of SIRT1 and phosphorylation of ERK and S6K1 was declined in senescent 2BS. These findings suggested that SIRT1-promoted cell proliferation and antagonized cellular senescence in human diploid fibroblasts may be, in part, via the activation of ERK/ S6K1 signaling.

  10. Fibroblast growth factors activate mitogen-activated protein kinase pathways to promote migration in ovine trophoblast cells.

    Science.gov (United States)

    Yang, Qi En; Giassetti, Mariana I; Ealy, Alan D

    2011-05-01

    Fibroblast growth factors (FGFs) 2 and FGF10 are uterine- and conceptus-derived factors that mediate trophoblast activities in cattle and sheep. To extend our understanding of how FGFs may control peri-implantation development in ruminants, we determined whether FGF2 and FGF10 impact trophoblast cell migration. Transwell inserts containing 8 μm pores were used to examine whether FGF2 or FGF10 supplementation increased oTr1 cell migration. Supplementation with 0.5 ng/ml FGF2 or FGF10 did not affect oTr1 cell migration number, but exposure to 5 or 50 ng/ml FGF2 or FGF10 increased (P<0.05) oTr1 cell migration when compared with controls. The involvement of specific MAP kinase (MAPK) cascades in mediating this FGF response was examined by using pharmacological inhibitors of specific MAPKs. Western blot analysis indicated that FGF2 and FGF10 increased phosphorylation status of MAPKs 1, 3, 8, 9, and 14. Exposure to specific inhibitors blocked FGF induction of each MAPK. Exposure to inhibitors before supplementation with FGF2 or FGF10 prevented FGF induction of cell migration, indicating that each of these signaling molecules was required for FGF effects. A final series of studies examined whether FGF2 and FGF10 also mediated the migration of a bovine trophoblast line (CT1 cell). Increases in migration were detected in each cell line by supplementing 5 or 50 ng/ml FGF2 or FGF10 (P<0.05). In summary, FGF2 and FGF10 regulate migratory activity of ovine trophoblast cells through MAPK-dependent pathways. These outcomes provide further evidence that FGFs function as mediators of peri-implantation conceptus development in cattle and sheep.

  11. Proliferative multifocal leukoplakia better name that proliferative verrucous leukoplakia

    OpenAIRE

    Aguirre-Urizar Jose M

    2011-01-01

    Abstract In this letter I propose the name "Proliferative Multifocal Leukoplakia" with the goal of reducing under-diagnosis of this disease, improve the early diagnosis, try to make an early therapy and control, and prevent its malignant transformation.

  12. Cyclic adenosine 3'-5'-monophosphate (cAMP) exerts proliferative and anti-proliferative effects in pituitary cells of different types by activating both cAMP-dependent protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac).

    Science.gov (United States)

    Vitali, E; Peverelli, E; Giardino, E; Locatelli, M; Lasio, G B; Beck-Peccoz, P; Spada, A; Lania, A G; Mantovani, G

    2014-03-05

    In the pituitary the activation of cyclic adenosine 3'-5'-monophosphate (cAMP) dependent pathways generates proliferative signals in somatotrophs, whereas in pituitary cells of other lineages its effect remains uncertain. Moreover, the specific role of the two main cAMP effectors, protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac), has not been defined. Aim of this study was to investigate the effect of cAMP on pituitary adenomatous cells proliferation and to identify PKA and Epac differential involvement. We found that cAMP increased DNA synthesis and cyclin D1 expression in somatotropinomas, whereas it reduced both parameters in prolactinomas and nonfunctioning adenomas, these effects being replicated in corresponding cell lines. Moreover, the divergent cAMP effects were mimicked by Epac and PKA analogs, which activated Rap1 and CREB, respectively. In conclusion, we demonstrated that cAMP exerted opposite effects on different pituitary cell types proliferation, these effects being mediated by both Epac and PKA.

  13. Exposure to parabens at the concentration of maximal proliferative response increases migratory and invasive activity of human breast cancer cells in vitro.

    Science.gov (United States)

    Khanna, Sugandha; Dash, Philip R; Darbre, Philippa D

    2014-09-01

    Alkyl esters of p-hydroxybenzoic acid (parabens) are widely used as preservatives in personal care products, foods and pharmaceuticals. Their oestrogenic activity, their measurement in human breast tissue and their ability to drive proliferation of oestrogen-responsive human breast cancer cells has opened a debate on their potential to influence breast cancer development. As proliferation is not the only hallmark of cancer cells, we have investigated the effects of exposure to parabens at concentrations of maximal proliferative response on migratory and invasive properties using three oestrogen-responsive human breast cancer cell lines (MCF-7, T-47-D, ZR-75-1). Cells were maintained short-term (1 week) or long-term (20 ± 2 weeks) in phenol-red-free medium containing 5% charcoal-stripped serum with no addition, 10(-8)  M 17β-oestradiol, 1-5 × 10(-4)  M methylparaben, 10(-5)  M n-propylparaben or 10(-5)  M n-butylparaben. Long-term exposure (20 ± 2 weeks) of MCF-7 cells to methylparaben, n-propylparaben or n-butylparaben increased migration as measured using a scratch assay, time-lapse microscopy and xCELLigence technology: invasive properties were found to increase in matrix degradation assays and migration through matrigel on xCELLigence. Western immunoblotting showed an associated downregulation of E-cadherin and β-catenin in the long-term paraben-exposed cells which could be consistent with a mechanism involving epithelial to mesenchymal transition. Increased migratory activity was demonstrated also in long-term paraben-exposed T-47-D and ZR-75-1 cells using a scratch assay and time-lapse microscopy. This is the first report that in vitro, parabens can influence not only proliferation but also migratory and invasive properties of human breast cancer cells.

  14. The effect of activated alveolar macrophages on experimental lung emphysema development. II. The study of fibroblast and alveolar macrophage co-culture.

    Science.gov (United States)

    Sulkowska, M; Wołczyński, S; Sulkowski, S; Sobaniec-Lotowska, M; Chyczewski, L; Sulik, M; Kulikowski, M; Dziecioł, J; Berger, W

    1995-01-01

    The cell-cell interaction between fibroblasts and alveolar macrophages was examined using a co-culture system. Alveolar macrophages (AM) were harvested from the bronchoalveolar lavages (BAL) of rats with papain induced lung emphysema. The BCG-vaccine was applied as a macrophage mobilizing and activating agent. The morphological examinations carried out in scanning electron microscope (SEM) as well as the evaluation of the uptake of 3H-thymidine did not show any significant differences between respective co-cultures of fibroblasts and AM isolated both from the lungs of control and experimental animals (treated with BCG or papain, and BCG+papain). However, significant growth were noted in 3H-thymidine uptake between fibroblast cultures done with or without cells isolated from the lungs. The results obtained suggest that AM can promote fibroblast proliferation during the progression of experimental lung emphysema.

  15. Effect of storage media on the proliferation of periodontal ligament fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Lauer, H.C.; Mueller, J.G.; Gross, J.; Horster, M.F.

    1987-07-01

    The effect of storage media, which are routinely used in replantation, upon the proliferative capacity of periodontal ligament fibroblasts, was compared with the effect of a tissue culture medium. The periodontal tissue was obtained from mandibular central incisors of White New Zealand rabbits. The experiments were performed in fibroblasts derived during second subculture. The storage media were physiologic salt solution, Ringer's solution and Rivanol; the tissue culture medium was alpha-minimum essential medium without nucleosides. The incubation period was 1 hour. (/sup 3/H)-thymidine incorporation and cell counts were taken to indicate changes in the proliferative capacity of the fibroblasts. The tissue culture experiments showed that the proliferative ability of the periodontal ligament fibroblasts was dependent upon the composition of the storage medium. Physiologic salt solution, Ringer's solution and Rivanol were unable to maintain the metabolism of the fibroblasts. alpha-MEM medium, however, was capable of stimulating proliferation of the periodontal ligament fibroblasts.

  16. Curcumin Triggers p16-Dependent Senescence in Active Breast Cancer-Associated Fibroblasts and Suppresses Their Paracrine Procarcinogenic Effects

    Directory of Open Access Journals (Sweden)

    Siti-Fauziah Hendrayani

    2013-06-01

    Full Text Available Activated cancer-associated fibroblasts (CAFs or myofibroblasts not only facilitate tumor growth and spread but also affect tumor response to therapeutic agents. Therefore, it became clear that efficient therapeutic regimens should also take into account the presence of these supportive cells and inhibit their paracrine effects. To this end, we tested the effect of low concentrations of curcumin, a pharmacologically safe natural product, on patient-derived primary breast CAF cells. We have shown that curcumin treatment upregulates p16INK4A and other tumor suppressor proteins while inactivates the JAK2/STAT3 pathway. This reduced the level of alpha-smooth muscle actin (α-SMA and the migration/invasion abilities of these cells. Furthermore, curcumin suppressed the expression/secretion of stromal cell-derived factor-1 (SDF-1, interleukin-6 (IL-6, matrix metalloproteinase-2 (MMP-2, MMP-9, and transforming growth factor-β, which impeded their paracrine procarcinogenic potential. Intriguingly, these effects were sustained even after curcumin withdrawal and cell splitting. Therefore, using different markers of senescence [senescence-associated β-galactosidase (SA-β-gal activity, Ki-67 and Lamin B1 levels, and bromodeoxyuridine incorporation], we have shown that curcumin markedly suppresses Lamin B1 and triggers DNA damage-independent senescence in proliferating but not quiescent breast stromal fibroblasts. Importantly, this curcumin-related senescence was p16INK4A-dependent and occurred with no associated inflammatory secretory phenotype. These results indicate the possible inactivation of cancer-associated myofibroblasts and present the first indication that curcumin can trigger DNA damage-independent and safe senescence in stromal fibroblasts.

  17. Thimerosal induces DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured human neurons and fibroblasts.

    Science.gov (United States)

    Baskin, David S; Ngo, Hop; Didenko, Vladimir V

    2003-08-01

    Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. Little is known about the reactions of human neuronal and skin cells to its micro- and nanomolar concentrations, which can occur after using thimerosal-containing products. A useful combination of fluorescent techniques for the assessment of thimerosal toxicity is introduced. Short-term thimerosal toxicity was investigated in cultured human cerebral cortical neurons and in normal human fibroblasts. Cells were incubated with 125-nM to 250-microM concentrations of thimerosal for 45 min to 24 h. A 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) dye exclusion test was used to identify nonviable cells and terminal transferase-based nick-end labeling (TUNEL) to label DNA damage. Detection of active caspase-3 was performed in live cell cultures using a cell-permeable fluorescent caspase inhibitor. The morphology of fluorescently labeled nuclei was analyzed. After 6 h of incubation, the thimerosal toxicity was observed at 2 microM based on the manual detection of the fluorescent attached cells and at a 1-microM level with the more sensitive GENios Plus Multi-Detection Microplate Reader with Enhanced Fluorescence. The lower limit did not change after 24 h of incubation. Cortical neurons demonstrated higher sensitivity to thimerosal compared to fibroblasts. The first sign of toxicity was an increase in membrane permeability to DAPI after 2 h of incubation with 250 microM thimerosal. A 6-h incubation resulted in failure to exclude DAPI, generation of DNA breaks, caspase-3 activation, and development of morphological signs of apoptosis. We demonstrate that thimerosal in micromolar concentrations rapidly induce membrane and DNA damage and initiate caspase-3-dependent apoptosis in human neurons and fibroblasts. We conclude that a proposed combination of fluorescent techniques can be useful in analyzing the toxicity of thimerosal.

  18. Hmgb1 promotes wound healing of 3T3 mouse fibroblasts via RAGE-dependent ERK1/2 activation.

    Science.gov (United States)

    Ranzato, Elia; Patrone, Mauro; Pedrazzi, Marco; Burlando, Bruno

    2010-05-01

    HMGb1 is a nuclear protein playing a role in DNA architecture and transcription. This protein has also been shown to function as a cytokine and to stimulate keratinocyte scratch wound healing. Due to the importance of finding new wound healing molecules, we have studied the effects of HMGb1 on fibroblasts, another major skin cell type, using the NIH 3T3 line. HMGb1 expression in these cells was assessed by Western blot, while its nuclear localization was pointed out by confocal immunofluorescence. HMGb1-induced cell proliferation with a maximum at a concentration of 10 nM, and such a dose also stimulated cell migration and scratch wound healing. Western blot analysis showed that HMGb1 activates ERK1/2, while the use of an anti-RAGE receptor-blocking antibody and of the selective MEK1/2 inhibitor PD98059 blocked ERK1/2 activation and wound healing responses to HMGb1. Taken together data show that HMGb1 promotes 3T3 fibroblast wound healing by inducing cell proliferation and migration, and that this occurs through the activation of the RAGE/MEK/ERK pathway. In conclusion, HMGb1 seems a good candidate for the development of medical treatments to be used on chronic or severe wounds.

  19. Human ovarian neoplasm cell CD147 stimulates production and activation of matrix metalloproteinases in co-cultures with mouse fibroblasts

    Institute of Scientific and Technical Information of China (English)

    YANG Hong; ZOU Wei; XIN Xiao-yan

    2005-01-01

    Objective: To investigate the expression of CD147 on human ovarian neoplasm cell lines and its influence on production and activation of matrix metallproteinases(MMPs). Methods: The expression of CD147 on different human ovarian neoplasm cell lines was studied by western blotting. Co-culture was carried out to investigate the stimulative effect of the positive expression CD147 cell HO-8910 on the production of MMPs of fibroblast cell in vitro. Zymography and immune blotting were used to study the production and activity of positive MMPs, at the time, to explore the relation between CD147 and MMPs. Results: CD147 was positively presented in 2 ovarian neoplasm cell lines(HO-8910,3-AO), but in SKOV3, TC-1,NIN3T3 cell was negative. MMP-2 and MMP-9 were detected by HO-8910 cell line, mouse fibroblast cell and co-culture cells; but the expression in co-culture cell is obviously higher than individual cultures of each type alone.CD147 stimulated MMPs in dose-dependent manner. Conclusion: CD147 causes increased production and activation of MMP-2, MMP-9.CD147 is probably a indirect marker of some ovarian cancer cells with invasion and metastasis.

  20. Quercetin Inhibits Fibroblast Activation and Kidney Fibrosis Involving the Suppression of Mammalian Target of Rapamycin and β-catenin Signaling.

    Science.gov (United States)

    Ren, Jiafa; Li, Jianzhong; Liu, Xin; Feng, Ye; Gui, Yuan; Yang, Junwei; He, Weichun; Dai, Chunsun

    2016-04-07

    Quercetin, a flavonoid found in a wide variety of plants and presented in human diet, displays promising potential in preventing kidney fibroblast activation. However, whether quercetin can ameliorate kidney fibrosis in mice with obstructive nephropathy and the underlying mechanisms remain to be further elucidated. In this study, we found that administration of quercetin could largely ameliorate kidney interstitial fibrosis and macrophage accumulation in the kidneys with obstructive nephropathy. MTORC1, mTORC2, β-catenin as well as Smad signaling were activated in the obstructive kidneys, whereas quercetin could markedly reduce their abundance except Smad3 phosphorylation. In cultured NRK-49F cells, quercetin could inhibit α-SMA and fibronectin (FN) expression induced by TGFβ1 treatment. MTORC1, mTORC2, β-catenin and Smad signaling pathways were stimulated by TGFβ1 at a time dependent manner. Similar to those findings in the obstructive kidneys, mTORC1, mTORC2 and β-catenin, but not Smad signaling pathways were remarkably blocked by quercetin treatment. Together, these results suggest that quercetin inhibits fibroblast activation and kidney fibrosis involving a combined inhibition of mTOR and β-catenin signaling transduction, which may act as a therapeutic candidate for patients with chronic kidney diseases.

  1. Basement membrane reconstruction in human skin equivalents is regulated by fibroblasts and/or exogenously activated keratinocytes

    NARCIS (Netherlands)

    El Ghalbzouri, A; Jonkman, MF; Dijkman, R; Ponec, M

    2005-01-01

    This study was undertaken to examine the role fibroblasts play in the formation of the basement membrane (BM) in human skin equivalents. For this purpose, keratinocytes were seeded on top of fibroblast-free or fibroblast-populated collagen matrix or de-epidermized dermis and cultured in the absence

  2. Secretion by stimulated murine macrophages of a heparin-binding fibroblast growth activity, distinct from basic FGE and IL-1

    Energy Technology Data Exchange (ETDEWEB)

    Rappolee, D.A.; Banda, M.J.; Werb, Z.

    1986-03-01

    Wound healing requires granulation and formation of neovascularization tissue. These two events require increases in fibroblasts, vascular endothelial, and smooth muscle cells. Macrophages may produce several growth factors which participate in these would healing events. To test this hypothesis they have partially purified a fibroblast growth promoting activity from a murine macrophage cell line (P388 Dl). The activity causes growth in Balb/c and Swiss 3T3 cells as measured by thymidine uptake, nuclear labeling and increase in cell number. PDGF, Basic FGF, and EGF are also mitogenic by thymidine uptake, but purified human IL-1 and recombinant murine IL-1 are not. The activity is pH 2.5-, freeze/thaw-, and dialysis/lyphilyzation-stable. The activity elutes from heparin-Sepharose at 2.0M, but not 0.15m, 0.5M, or 3.0M NaCl. Basic FGF elutes from the same heparin-Sepharose batch at 3.0M, but not at the other three NaCl concentrations. The growth activity is secreted by viable murine macrophage line cells (P388D1, WEHI-3, RAW 264.7) at a 48 hour peak after activating (LPS) or phagocytic stimuli. Unstimulated P388D1 caused growth 1.7 times control whereas stimulation increases the growth 5.1 to 7.1 times control. The optimal activity concentration fails to complement insulin in an assay in which optimal basic FGF concentration complements insulin. These data suggest that the activity may contain a progression factor.

  3. Antioxidant Activity of Ixora parviflora in a Cell/Cell-Free System and in UV-Exposed Human Fibroblasts

    Directory of Open Access Journals (Sweden)

    Hsiu-Mei Chiang

    2011-07-01

    Full Text Available Polyphenols and flavonoids possess a variety of biological activities including antioxidant and anti-tumor activities. Ixora parviflora is a member of the flavonoid-rich Rubiaceae family of flowering plants and used as folk medicine in India. The aim of this study was to investigate the antioxidant activity of Ixora parviflora extract (IPE in a cell-free system and erythrocytes, and the ability of IPE to inhibit reactive oxygen species (ROS generation in human fibroblasts (Hs68 after ultraviolet (UV exposure. Various in vitro antioxidant assays were employed in this study. The extraction yield of IPE was 17.4 ± 3.9%, the total phenolic content of IPE was 26.2 μg gallic acid equivalent (GAE/mg leaves dry weight and the total flavonoids content was 54.2 ± 4.4 μg quercetin equvalent (QE/mg extract. The content of chlorogenic acid was 9.7 ± 1.2 mg/g extract. IPE at 1000 μg/mL exhibited a reducing capacity of 90.5 ± 0.6%, a 1,1-diphenyl-2-picrylhydrazy (DPPH radical scavenging activity of 96.0 ± 0.4%, a ferrous chelating activity of 72.2 ± 3.5%, a hydroxyl radical scavenging activity of 96.8 ± 1.4%, and a hydrogen peroxide scavenging activity of 99.5 ± 3.3%. IPE at 500 μg/mL also possessed inhibitory activity against 2,2'-azobis (2-methylpropionamidine dihydrochloride (AAPH-induced hemolysis of erythrocytes (89.4 ± 1.8% and resulted in a 52.9% reduction in ROS generation in UV-exposed fibroblasts. According to our findings, IPE is a potent antioxidant and a potential anti-photoaging agent.

  4. Anti-biofilm activity of chitosan gels formulated with silver nanoparticles and their cytotoxic effect on human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Pérez-Díaz, M.; Alvarado-Gomez, E. [Facultad de Ciencias Quimicas, Universidad Autonoma de San Luis Potosi (Mexico); Magaña-Aquino, M. [Servicio de Epidemiologia del Hospital Central “Dr. Ignacio Morones Prieto”, San Luis Potosi (Mexico); Sánchez-Sánchez, R.; Velasquillo, C. [Laboratorio de Biotecnologia, Instituto Nacional de Rehabilitacion, Mexico, D.F. (Mexico); Gonzalez, C. [Facultad de Ciencias Quimicas, Universidad Autonoma de San Luis Potosi (Mexico); Ganem-Rondero, A. [Division de Estudios de Posgrado (Tecnologia Farmaceutica), Facultad de Estudios Superiores Cuautitlan, Universidad Nacional Autonoma de Mexico, Cuautitlan Izcalli, Estado de Mexico (Mexico); Martínez-Castañon, G.; Zavala-Alonso, N. [Doctorado en Ciencias Odontológicas Facultad de Estomatologia, UASLP (Mexico); Martinez-Gutierrez, F. [Facultad de Ciencias Quimicas, Universidad Autonoma de San Luis Potosi (Mexico)

    2016-03-01

    The development of multi-species biofilms in chronic wounds is a serious health problem that primarily generates strong resistance mechanisms to antimicrobial therapy. The use of silver nanoparticles (AgNPs) as a broad-spectrum antimicrobial agent has been studied previously. However, their cytotoxic effects limit its use within the medical area. The purpose of this study was to evaluate the anti-biofilm capacity of chitosan gel formulations loaded with AgNPs, using silver sulfadiazine (SSD) as a standard treatment, on strains of clinical isolates, as well as their cytotoxic effect on human primary fibroblasts. Multi-species biofilm of Staphylococcus aureus oxacillin resistant (MRSA) and Pseudomonas aeruginosa obtained from a patient with chronic wound infection were carried out using a standard Drip Flow Reactor (DFR) under conditions that mimic the flow of nutrients in the human skin. Anti-biofilm activity of chitosan gels and SSD showed a log-reduction of 6.0 for MRSA when chitosan gel with AgNPs at a concentration of 100 ppm was used, however it was necessary to increase the concentration of the chitosan gel with AgNPs to 1000 ppm to get a log-reduction of 3.3, while the SSD showed a total reduction of both bacteria in comparison with the negative control. The biocompatibility evaluation on primary fibroblasts showed better results when the chitosan gels with AgNPs were tested even in the high concentration, in contrast with SSD, which killed all the primary fibroblasts. In conclusion, chitosan gel formulations loaded with AgNPs effectively prevent the formation of biofilm and kill bacteria in established biofilm, which suggest that chitosan gels with AgNPs could be used for prevention and treatment of infections in chronic wounds. The statistic significance of the biocompatibility of chitosan gel formulations loaded with AgNPs represents an advance; however further research and development are necessary to translate this technology into therapeutic and

  5. Protein kinase Cζ regulates phospholipase D activity in rat-1 fibroblasts expressing the α1A adrenergic receptor

    Directory of Open Access Journals (Sweden)

    Bourgoin Sylvain G

    2004-01-01

    Full Text Available Abstract Background Phenylephrine (PHE, an α1 adrenergic receptor agonist, increases phospholipase D (PLD activity, independent of classical and novel protein kinase C (PKC isoforms, in rat-1 fibroblasts expressing α1A adrenergic receptors. The aim of this study was to determine the contribution of atypical PKCζ to PLD activation in response to PHE in these cells. Results PHE stimulated a PLD activity as demonstrated by phosphatidylethanol production. PHE increased PKCζ translocation to the particulate cell fraction in parallel with a time-dependent decrease in its activity. PKCζ activity was reduced at 2 and 5 min and returned to a sub-basal level within 10–15 min. Ectopic expression of kinase-dead PKCζ, but not constitutively active PKCζ, potentiated PLD activation elicited by PHE. A cell-permeable pseudosubstrate inhibitor of PKCζ reduced basal PKCζ activity and abolished PHE-induced PLD activation. Conclusion α1A adrenergic receptor stimulation promotes the activation of a PLD activity by a mechanism dependent on PKCζ; Our data also suggest that catalytic activation of PKCζ is not required for PLD stimulation.

  6. In normal human fibroblasts variation in DSB repair capacity cannot be ascribed to radiation-induced changes in the localisation, expression or activity of major NHEJ proteins

    DEFF Research Database (Denmark)

    Kasten-Pisula, Ulla; Vronskaja, Svetlana; Overgaard, Jens

    2008-01-01

    BACKGROUND AND PURPOSE: The aim of the present study was to test whether for normal human fibroblasts the variation in double-strand break (DSB) repair capacity results from radiation-induced differences in localisation, expression or activity of major non-homologous end-joining (NHEJ) proteins....... MATERIALS AND METHODS: Experiments were performed with 11 normal human fibroblast strains AF01-11. NHEJ proteins were determined by Western blot and DNA-PK activity by pulldown-assay. RESULTS: The four NHEJ proteins tested (Ku70, Ku80, XRCC4 and DNA-PKcs) were found to be localised almost exclusively...... in the activity of the DNA-PK complex induced upon irradiation. CONCLUSIONS: For normal human fibroblasts, the level or activity of NHEJ proteins measured prior to or after irradiation cannot be used to predict the DSB repair capacity or cellular radiosensitivity. Udgivelsesdato: 2008-Mar...

  7. Mutant p53 attenuates the anti-tumorigenic activity of fibroblasts-secreted interferon beta.

    Directory of Open Access Journals (Sweden)

    Shalom Madar

    Full Text Available Mutations in the p53 tumor suppressor protein are highly frequent in tumors and often endow cells with tumorigenic capacities. We sought to examine a possible role for mutant p53 in the cross-talk between cancer cells and their surrounding stroma, which is a crucial factor affecting tumor outcome. Here we present a novel model which enables individual monitoring of the response of cancer cells and stromal cells (fibroblasts to co-culturing. We found that fibroblasts elicit the interferon beta (IFNβ pathway when in contact with cancer cells, thereby inhibiting their migration. Mutant p53 in the tumor was able to alleviate this response via SOCS1 mediated inhibition of STAT1 phosphorylation. IFNβ on the other hand, reduced mutant p53 RNA levels by restricting its RNA stabilizer, WIG1. These data underscore mutant p53 oncogenic properties in the context of the tumor microenvironment and suggest that mutant p53 positive cancer patients might benefit from IFNβ treatment.

  8. Scopoletin has a potential activity for anti-aging via autophagy in human lung fibroblasts.

    Science.gov (United States)

    Nam, Hyang; Kim, Moon-Moo

    2015-03-15

    Autophagy was known to be associated with aging in addition to cancer and neurodegeneration. The effects of scopoletin on autophagy and anti-aging were investigated in human lung fibroblast cell line, IMR 90. Here we show that scopoletin induces autophagy. It is also identified that the modulation of p53 by scopoletin are related to the induction of autophagy. Moreover, the level of SA-β-Gal staining, an aging marker, is reduced by scopoletin. In addition, while the expression levels of histone deacetylases such as HDAC1, SIRT1 and SIRT6 are increased in IMR 90 cells in the presence of scopoletin, the expression levels of histone acetyltransferases are decreased. Furthermore, scopoletin enhances the level of transcription factors such as Nrf-2and p-FoxO1 related to anti-aging. In addition, scopoletin modulates the reprogramming proteins. Therefore, these findings suggest that scopoletin could exert a positive effect on anti-aging related to autophagy through modulation of p53 in human lung fibroblasts. Copyright © 2015 Elsevier GmbH. All rights reserved.

  9. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts.

    Science.gov (United States)

    Hasaneen, Nadia A; Cao, Jian; Pulkoski-Gross, Ashleigh; Zucker, Stanley; Foda, Hussein D

    2016-02-17

    EMMPRIN blocking antibody markedly inhibited TGF-β1 induced proliferation, migration, and differentiation of fibroblasts to myofibroblasts. EMMPRIN overexpression in lung fibroblasts was found to induce an increase in TOPFLASH luciferase reporter activity when compared with control fibroblasts. These findings indicate that TGF-β1 induces the release of EMMPRIN that activates β-catenin/canonical Wnt signaling pathway. EMMPRIN overexpression induces an anti-apoptotic and pro-fibrotic phenotype in lung fibroblasts that may contribute to the persistent fibro-proliferative state seen in IPF.

  10. PGE2 induces IL-6 in orbital fibroblasts through EP2 receptors and increased gene promoter activity: implications to thyroid-associated ophthalmopathy.

    Directory of Open Access Journals (Sweden)

    Nupur Raychaudhuri

    Full Text Available BACKGROUND: IL-6 plays an important role in the pathogenesis of Graves' disease and its orbital component, thyroid-associated ophthalmopathy (TAO. Orbital tissues become inflamed in TAO, a process in which prostanoids have been implicated. Orbital fibroblasts both generate and respond to PGE(2, underlying the inflammatory phenotype of these cells. METHODOLOGY/PRINCIPAL FINDINGS: Using cultured orbital and dermal fibroblasts, we characterized the effects of PGE(2 on IL-6 expression. We found that the prostanoid provokes substantially greater cytokine synthesis in orbital fibroblasts, effects that are mediated through cell-surface EP(2 receptors and increased steady-state IL-6 mRNA levels. The pre-translational up-regulation of IL-6 results from increased gene promoter activity and can be reproduced with the PKA agonist, Sp-cAMP and blocked by interrupting the PKA pathway. PGE(2-induced production of cAMP in orbital fibroblasts was far greater than that in dermal fibroblasts, resulting from higher levels of adenylate cyclase. PGE(2 provokes CREB phosphorylation, increases the pCREB/CREB ratio, and initiates nuclear localization of the pCREB/CREB binding protein/p300 complex (CBP preferentially in orbital fibroblasts. Transfection with siRNAs targeting either CREB or CBP blunts the induction of IL-6 gene expression. PGE(2 promotes the binding of pCREB to its target DNA sequence which is substantially greater in orbital fibroblasts. CONCLUSION/SIGNIFICANCE: These results identify the mechanism underlying the exaggerated induction of IL-6 in orbital fibroblasts and tie together two proinflammatory pathways involved in the pathogenesis of TAO. Moreover, they might therefore define an attractive therapeutic target for the treatment of TAO.

  11. Transitory FGF treatment results in the long-lasting suppression of the proliferative response to repeated FGF stimulation.

    Science.gov (United States)

    Poole, Ashleigh; Knowland, Nicholas; Cooper, Emily; Cole, Rebecca; Wang, Hongchuan; Booth, Lucas; Kacer, Doreen; Tarantini, Francesca; Friesel, Robert; Prudovsky, Igor

    2014-05-01

    FGF applied as a single growth factor to quiescent mouse fibroblasts induces a round of DNA replication, however continuous stimulation results in arrest in the G1 phase of the next cell cycle. We hypothesized that FGF stimulation induces the establishment of cell memory, which prevents the proliferative response to repeated or continuous FGF application. When a 2-5 days quiescence period was introduced between primary and repeated FGF treatments, fibroblasts failed to efficiently replicate in response to secondary FGF application. The establishment of "FGF memory" during the first FGF stimulation did not require DNA synthesis, but was dependent on the activity of FGF receptors, MEK, p38 MAPK and NFκB signaling, and protein synthesis. While secondary stimulation resulted in strongly decreased replication rate, we did not observe any attenuation of morphological changes, Erk1/2 phosphorylation and cyclin D1 induction. However, secondary FGF stimulation failed to induce the expression of cyclin A, which is critical for the progression from G1 to S phase. Treatment of cells with a broad range histone deacetylase inhibitor during the primary FGF stimulation rescued the proliferative response to the secondary FGF treatment suggesting that the establishment of "FGF memory" may be based on epigenetic changes. We suggest that "FGF memory" can prevent the hyperplastic response to cell damage and inflammation, which are associated with an enhanced FGF production and secretion. "FGF memory" may present a natural obstacle to the efficient application of recombinant FGFs for the treatment of ulcers, ischemias, and wounds.

  12. UVB-irradiated human keratinocytes and interleukin-1αindirectly increase MAP kinase/AP-1 activation and MMP-1 production in UVA-irradiated dermal fibroblasts

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-yong; BI Zhi-gang

    2006-01-01

    Background Solar ultraviolet (UV) irradiation induces the production of matrix metalloproteinases (MMPs) by activating cellular signalling transduction pathways. MMPs are responsible for the degradation and/or inhibition of synthesis of collagenous extracellular matrix in connective tissues. We mimicked the action of environmental ultraviolet on skin and investigated the effects of UVB-irradiated human keratinocytes HaCaT and IL-1α on mitogen activated protein (MAP) kinase activation, c-Jun and c-Fos (AP-1 is composed of Jun and Fos proteins)mRNA expression and MMP-1 production in UVA-irradiated dermal fibroblasts.Methods Following UVA irradiation, the culture medium of fibroblasts was replaced by culture medium from UVB-irradiated HaCaT, or replaced by the complete culture medium with interleukin (IL)-1α. MAP kinase activity expression in fibroblasts was detected by Western blot. c-Jun and c-Fos mRNA expressions were determined by reverse transcriptional polymerase chain reaction (RT-PCR); MMP-1 production in culture medium was detected by enzyme-linked immunosorbent assay (ELISA).Results Culture medium from UVB-irradiated keratinocytes increased MAP kinase activity and c-Jun mRNA expression in UVA-irradiated fibroblasts. IL-1α increased MAP kinase activity and c-Jun mRNA expression,IL-1 α also increased c-Fos mRNA expression. Both culture media from UVB-irradiated human keratinocytes and externally applied IL-1 α increased MMP-1 production in UVA-irradiated fibroblasts.Conclusions UVB-irradiated keratinocytes and IL-1α indirectly promote MMP-1 production in UVA-irradiated fibroblasts by increasing MAP kinase/AP-1 activity. IL-1 may play an important role in the paracrine activation and dermal collagen excessive degradation leading to skin photoaging.

  13. S100A8 and S100A9 Are Induced by Decreased Hydration in the Epidermis and Promote Fibroblast Activation and Fibrosis in the Dermis.

    Science.gov (United States)

    Zhong, Aimei; Xu, Wei; Zhao, Jingling; Xie, Ping; Jia, Shengxian; Sun, Jiaming; Galiano, Robert D; Mustoe, Thomas A; Hong, Seok J

    2016-01-01

    The most critical function of the epidermis is to prevent water loss and maintain skin homeostasis. Disruption of the functional skin barrier causes delayed wound healing, hypertrophic scarring, and many skin diseases. Herein, we show that reduced hydration increases the expression of S100 protein family members, S100A8/S100A9, in stratified keratinocyte culture and human ex vivo skin culture. Immunohistological analyses show that S100A8/A9 are highly expressed in the epidermis of human hypertrophic scar and keloid tissues. Reduced hydration demonstrates activation of fibroblasts in the keratinocyte-fibroblast co-culture. In contrast, knockdown of S100A8 or S100A9 by RNA interference in keratinocytes failed to activate fibroblasts. Pretreatment with pharmacological blockers of S100A8/A9 receptors, Toll-like receptor 4 and receptor for advanced glycation end products, inhibits fibroblast activation induced by recombinant S100A8/A9 proteins. Moreover, we observe that local delivery of S100A8 protein results in a marked increase in hypertrophic scarring in the in vivo rabbit ear scar model. Our results indicate that hydration status promotes fibroblast activation and fibrosis by directly affecting the expression of inflammatory signaling in keratinocytes, thereby strongly suggesting S100A8/A9 to be novel targets in preventing scarring.

  14. Loureirin B inhibits fibroblast proliferation and extracellular matrix deposition in hypertrophic scar via TGF-β/Smad pathway.

    Science.gov (United States)

    Bai, Xiaozhi; He, Ting; Liu, Jiaqi; Wang, Yunchuan; Fan, Lei; Tao, Ke; Shi, Jihong; Tang, Chaowu; Su, Linlin; Hu, Dahai

    2015-05-01

    The ethanolic extract of Resina Draconis (RDEE) has been reported beneficial to normal wound healing yielding more regularly arranged collagen fibres. Loureirin B, a major component in RDEE, has been supposed to be effective on the prevention and treatment of pathological scars. To investigate the therapeutic effects of loureirin B on hypertrophic scar (HS), fibroblasts from human HS and normal skin (NS) were isolated. Results showed that loureirin B dose-dependently downregulated both mRNA and protein levels of type I collagen (ColI), type III collagen (ColIII) and α-smooth muscle actin (α-SMA) in HS fibroblasts. Loureirin B also suppressed fibroblast proliferative activity and redistributed cell cycle, but did not affect cell apoptosis. In vivo rabbit ear scar model, loureirin B significantly improved the arrangement and deposition of collagen fibres, decreased protein levels of ColI, ColIII and α-SMA and suppressed myofibroblast differentiation and scar proliferative activity. In NS fibroblasts, loureirin B effectively inhibited TGF-β1-induced upregulation of ColI, ColIII and α-SMA levels, myofibroblast differentiation and the activation of Smad2 and Smad3. Loureirin B also affected mRNA levels of major MMPs and TIMPs in TGF-β1-stimulated fibroblasts. Taken together, this study demonstrates that loureirin B could downregulate the expression of fibrosis-related molecules by regulating MMPs and TIMPs levels, inhibit scar fibroblast proliferation and suppress TGF-β1-induced fibrosis, during which TGF-β1/Smad2/3 pathway is likely involved. These findings suggest that loureirin B is a potential therapeutic compound for HS treatment.

  15. Hypoxia-Induced Collagen Synthesis of Human Lung Fibroblasts by Activating the Angiotensin System

    Directory of Open Access Journals (Sweden)

    Shan-Shan Liu

    2013-12-01

    Full Text Available The exact molecular mechanism that mediates hypoxia-induced pulmonary fibrosis needs to be further clarified. The aim of this study was to explore the effect and underlying mechanism of angiotensin II (Ang II on collagen synthesis in hypoxic human lung fibroblast (HLF cells. The HLF-1 cell line was used for in vitro studies. Angiotensinogen (AGT, angiotensin converting enzyme (ACE, angiotensin II type 1 receptor (AT1R and angiotensin II type 2 receptor (AT2R expression levels in human lung fibroblasts were analysed using real-time polymerase chain reaction (RT-PCR after hypoxic treatment. Additionally, the collagen type I (Col-I, AT1R and nuclear factor κappaB (NF-κB protein expression levels were detected using Western blot analysis, and NF-κB nuclear translocation was measured using immunofluorescence localization analysis. Ang II levels in HLF-1 cells were measured with an enzyme-linked immunosorbent assay (ELISA. We found that hypoxia increased Col-I mRNA and protein expression in HLF-1 cells, and this effect could be inhibited by an AT1R or AT2R inhibitor. The levels of NF-κB, RAS components and Ang II production in HLF-1 cells were significantly increased after the hypoxia exposure. Hypoxia or Ang II increased NF-κB-p50 protein expression in HLF-1 cells, and the special effect could be inhibited by telmisartan (TST, an AT1R inhibitor, and partially inhibited by PD123319, an AT2R inhibitor. Importantly, hypoxia-induced NF-κB nuclear translocation could be nearly completely inhibited by an AT1R or AT2R inhibitor. Furthermore pyrrolidine dithiocarbamate (PDTC, a NF-κB blocker, abolished the expression of hypoxia-induced AT1R and Col-I in HLF-1 cells. Our results indicate that Ang II-mediated NF-κB signalling via ATR is involved in hypoxia-induced collagen synthesis in human lung fibroblasts.

  16. Topically Delivered Adipose Derived Stem Cells Show an Activated-Fibroblast Phenotype and Enhance Granulation Tissue Formation in Skin Wounds

    Science.gov (United States)

    Hong, Seok Jong; Xu, Wei; Leung, Kai P.; Mustoe, Thomas A.; Galiano, Robert D.

    2013-01-01

    Multipotent mesenchymal stem cells (MSCs) are found in various tissues and can proliferate extensively in vitro. MSCs have been used in preclinical animal studies and clinical trials in many fields. Adipose derived stem cells (ASCs) have several advantages compared to other MSCs for use in cell-based treatments because they are easy to isolate with relative abundance. However, quantitative approaches for wound repair using ASCs have been limited because of lack of animal models which allow for quantification. Here, we addressed the effect of topically delivered ASCs in wound repair by quantitative analysis using the rabbit ear model. We characterized rabbit ASCs, and analyzed their multipotency in comparison to bone marrow derived-MSCs (BM-MSCs) and dermal fibroblasts (DFs) in vitro. Topically delivered ASCs increased granulation tissue formation in wounds when compared to saline controls, whereas BM-MSCs or DFs did not. These studies suggest that ASCs and BM-MSCs are not identical, though they have similar surface markers. We found that topically delivered ASCs are engrafted and proliferate in the wounds. We showed that transplanted ASCs exhibited activated fibroblast phenotype, increased endothelial cell recruitment, and enhanced macrophage recruitment in vivo. PMID:23383253

  17. Cytotoxicity of MEIC chemicals Nos. 11-30 in 3T3 mouse fibroblasts with and without microsomal activation

    DEFF Research Database (Denmark)

    Rasmussen, Eva

    1999-01-01

    The cytotoxicity of MEIC chemicals Nos, 11-30 was evaluated by determination of neutral red uptake in Balb/c 3T3 mouse fibroblasts with and without the addition of a microsomal activation mixture. The use of microsomes significantly decreased the cytotoxicity of malathion, 2,4-dichlorophenoxyacetic...... acid, propranolol, thioridazine, lithium sulfate, copper sulfate and thallium sulfate, whereas the cytotoxicity of 1,1,1-trichloroethylene, phenol, nicotine, and paraquat was significantly increased by use of the microsomal activation mixture. These cytotoxicity data are in line with observations...... in other studies on microsomal modulation of the cytotoxicity of the test substances. Moderate to good correlations were found between the cytotoxicity data and rodent lethality data, and the addition of microsomes slightly improved the in vitro/in vivo concordance. The evidence to support the relevance...

  18. [Study on characteristic spectrum of ingredients from different species Cordyceps and its anti-fibrotic activity on human embryonic fibroblasts].

    Science.gov (United States)

    Si, Nan; Peng, Bo; Zhao, Hai-Yu; Wang, Hong-Jie; Yang, Jian; Li, Jian-Rong; Sun, Wei; Bian, Bao-Lin

    2016-07-01

    In this study, 10 samples of parasites, cursive, and the whole from six different species of Cordyceps were determined and compared by HPLC and LC-MS methods. Uridine, adenosine, and cordycepin were selected as the main evaluation index. The anti-fibrotic activity of different species Cordyceps extracts was observed using in vitro TGF-β1-induced ECM accumulation in human embryonic fibroblasts CCC-ESF-1. The results demonstrated that the number of atoms and hyphae ingredients of different species showed little difference, however, the content distribution of each component has obvious significance. The in vitro anti-fibrotic activities of different species were as follow: Cordyceps flower > Cicada Cordyceps (Cicada flower)> Silkworm Cordyceps> Tussah Cordyceps>natural Cordyceps. Our preliminary data could serve as reference for the discovery of artificial alternatives of natural Cordyceps. Copyright© by the Chinese Pharmaceutical Association.

  19. Mechanical tension increases CCN2/CTGF expression and proliferation in gingival fibroblasts via a TGFβ-dependent mechanism.

    Directory of Open Access Journals (Sweden)

    Fen Guo

    Full Text Available Unlike skin, oral gingival do not scar in response to tissue injury. Fibroblasts, the cell type responsible for connective tissue repair and scarring, are exposed to mechanical tension during normal and pathological conditions including wound healing and fibrogenesis. Understanding how human gingival fibroblasts respond to mechanical tension is likely to yield valuable insights not only into gingival function but also into the molecular basis of scarless repair. CCN2/connective tissue growth factor is potently induced in fibroblasts during tissue repair and fibrogenesis. We subjected gingival fibroblasts to cyclical strain (up to 72 hours using the Flexercell system and showed that CCN2 mRNA and protein was induced by strain. Strain caused the rapid activation of latent TGFβ, in a fashion that was reduced by blebbistatin and FAK/src inhibition, and the induction of endothelin (ET-1 mRNA and protein expression. Strain did not cause induction of α-smooth muscle actin or collagen type I mRNAs (proteins promoting scarring; but induced a cohort of pro-proliferative mRNAs and cell proliferation. Compared to dermal fibroblasts, gingival fibroblasts showed reduced ability to respond to TGFβ by inducing fibrogenic mRNAs; addition of ET-1 rescued this phenotype. Pharmacological inhibition of the TGFβ type I (ALK5 receptor, the endothelin A/B receptors and FAK/src significantly reduced the induction of CCN2 and pro-proliferative mRNAs and cell proliferation. Controlling TGFβ, ET-1 and FAK/src activity may be useful in controlling responses to mechanical strain in the gingiva and may be of value in controlling fibroproliferative conditions such as gingival hyperplasia; controlling ET-1 may be of benefit in controlling scarring in response to injury in the skin.

  20. Fibroblast activation protein increases metastatic potential of fibrosarcoma line HT1080 through upregulation of integrin-mediated signaling pathways.

    Science.gov (United States)

    Baird, Sarah K; Allan, Laura; Renner, Christoph; Scott, Fiona E; Scott, Andrew M

    2015-06-01

    The serine protease fibroblast activation protein (FAP) is selectively expressed on tumour-associated fibroblasts in most human epithelial tumours, as well as on some mesenchymal tumours such as sarcoma. High FAP expression is most often associated with poor outcome and increased metastasis. Here, we compare the in vitro metastatic potential of HT1080 fibrosarcoma cells with and without FAP expression in order to elucidate the mechanism by which FAP may influence metastasis. In the presence of FAP, cells were more adhesive to extracellular matrix proteins and migrated and invaded through Matrigel to a greater degree. The anti-FAP antibody ESC11, which caused internalization of FAP, decreased adhesion and migration, but only when cells expressed FAP. It was also found that blocking activity of integrins β1 and αvβ3 reduced both cell adhesion and migration and this effect was much more marked in FAP-expressing HT1080 cells than mock-transfected HT1080 cells. The expression or activation of intracellular proteins that form part of the downstream signaling of integrins, including integrin-linked kinase, Rac1 and focal adhesion kinase, was also upregulated when FAP was expressed, suggesting that FAP not only upregulates metastatic-like cell behaviours through interaction with integrins, but also influences the intracellular signaling of integrins. This was confirmed using both PI3 kinase and Src kinase inhibitors, which decreased adhesion and migration in FAP-expressing cells, but did not affect mock-transfected HT1080 cells. FAP is therefore a useful target for anti-cancer therapy, as not only is its expression tumour-selective, but its downregulation has the potential to reduce incidence of metastasis.

  1. Diffuse proliferative glomerulonephritis after bone marrow transplantation.

    Science.gov (United States)

    Suehiro, T; Masutani, K; Yokoyama, M; Tokumoto, M; Tsuruya, K; Fukuda, K; Kanai, H; Katafuchi, R; Nagatoshi, Y; Hirakata, H

    2002-09-01

    A 15-year-old boy developed nephrotic syndrome and acute renal failure 4 years after allogenic bone marrow transplantation (BMT) for lymphoid crisis of chronic myelocytic leukemia. On admission, he presented with clinical features of chronic GVHD including transient exacerbation of cholestatic liver injury. Renal biopsy showed diffuse proliferative glomerulonephritis with cellular crescents. The patient was treated with methylprednisolone pulse therapy (1 g/day, for 3 days) followed by oral prednisolone. Renal function gradually improved but nephrotic state was persistent. A second renal biopsy showed improvement of acute tubular necrosis and endocapillary proliferation and transformation of crescents into a fibrous form. After tapering of oral prednisolone, cyclophosphamide was started, which resulted in a gradual improvement of proteinuria. Several cases of nephrotic syndrome occurring after BMT have already been reported, but most cases had membranous nephropathy. In our case, renal biopsy revealed diffuse proliferative glomerulonephritis with findings of active cellular immunity, and aggressive treatment resulted in attenuation of these findings. Moreover, chronic GVHD-related liver injury was noted at the time of this episode. Our findings suggest that chronic GVHD may be complicated with diffuse proliferative glomerulonephritis through unknown cellular immune mechanism.

  2. Function, Activity, and Membrane Targeting of Cytosolic Phospholipase A2ζ in Mouse Lung Fibroblasts*S

    OpenAIRE

    Ghosh, Moumita; Loper, Robyn; Ghomashchi, Farideh; Tucker, Dawn E.; Bonventre, Joseph V.; Gelb, Michael H.; Leslie, Christina C.

    2007-01-01

    Group IVA cytosolic phospholipase A2 (cPLA2α) initiates eicosanoid production; however, this pathway is not completely ablated in cPLA2α−/− lung fibroblasts stimulated with A23187 or serum. cPLA2α+/+ fibroblasts preferentially released arachidonic acid, but A23187-stimulated cPLA2α−/− fibroblasts non-specifically released multiple fatty acids. Arachidonic acid release from cPLA2α−/− fibroblasts was inhibited by the cPLA2α inhibitors pyrrolidine-2 (IC50, 0.03 μM) and Wyeth-1 (IC50, 0.1 μM), im...

  3. Neurite outgrowth induced by a synthetic peptide ligand of neural cell adhesion molecule requires fibroblast growth factor receptor activation

    DEFF Research Database (Denmark)

    Rønn, L C; Doherty, P; Holm, A;

    2000-01-01

    The neural cell adhesion molecule NCAM is involved in axonal outgrowth and target recognition in the developing nervous system. In vitro, NCAM-NCAM binding has been shown to induce neurite outgrowth, presumably through an activation of fibroblast growth factor receptors (FGFRs). We have recently...... identified a neuritogenic ligand, termed the C3 peptide, of the first immunoglobulin (lg) module of NCAM using a combinatorial library of synthetic peptides. Here we investigate whether stimulation of neurite outgrowth by this synthetic ligand of NCAM involves FGFRs. In primary cultures of cerebellar neurons...... from wild-type mice, the C3 peptide stimulated neurite outgrowth. This response was virtually absent in cultures of cerebellar neurons from transgenic mice expressing a dominant-negative form of the FGFR1. Likewise, in PC12E2 cells transiently expressing a dominant-negative form of the mouse FGFR1...

  4. Kaempferol inhibits fibroblast collagen synthesis, proliferation and activation in hypertrophic scar via targeting TGF-β receptor type I.

    Science.gov (United States)

    Li, Hongwei; Yang, Liu; Zhang, Yuebing; Gao, Zhigang

    2016-10-01

    Hypertrophic scar (HPS) formation is a debilitating condition that results in pain, esthetic symptom and loss of tissue function. So far, no satisfactory therapeutic approach has been available for HPS treatment. In this study, we discovered that a natural small molecule, kaempferol, could significantly inhibit HPS formation in a mechanical load-induced mouse model. Our results also demonstrated that kaempferol remarkably attenuated collagen synthesis, proliferation and activation of fibroblasts in vitro and in vivo. Western blot analysis further revealed that kaempferol significantly down-regulated Smad2 and Smad3 phosphorylation in a dose-dependent manner. At last, we found that such bioactivity of kaempferol which resulted from the inhibition of TGF-β1/Smads signaling was induced by the selective binding of kaempferol to TGF-β receptor type I (TGFβRI). These findings suggest that kaempferol could be developed into a promising agent for the treatment of HPS or other fibroproliferative disorders.

  5. Targeted Epigenetic Remodeling of Endogenous Loci by CRISPR/Cas9-Based Transcriptional Activators Directly Converts Fibroblasts to Neuronal Cells.

    Science.gov (United States)

    Black, Joshua B; Adler, Andrew F; Wang, Hong-Gang; D'Ippolito, Anthony M; Hutchinson, Hunter A; Reddy, Timothy E; Pitt, Geoffrey S; Leong, Kam W; Gersbach, Charles A

    2016-09-01

    Overexpression of exogenous fate-specifying transcription factors can directly reprogram differentiated somatic cells to target cell types. Here, we show that similar reprogramming can also be achieved through the direct activation of endogenous genes using engineered CRISPR/Cas9-based transcriptional activators. We use this approach to induce activation of the endogenous Brn2, Ascl1, and Myt1l genes (BAM factors) to convert mouse embryonic fibroblasts to induced neuronal cells. This direct activation of endogenous genes rapidly remodeled the epigenetic state of the target loci and induced sustained endogenous gene expression during reprogramming. Thus, transcriptional activation and epigenetic remodeling of endogenous master transcription factors are sufficient for conversion between cell types. The rapid and sustained activation of endogenous genes in their native chromatin context by this approach may facilitate reprogramming with transient methods that avoid genomic integration and provides a new strategy for overcoming epigenetic barriers to cell fate specification. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Quantitative study on morphologic features and proliferative activity during DEN-induced hepatocarcinogenesis in rats%DEN诱发大鼠肝癌变的病理形态与细胞增殖活性的定量研究

    Institute of Scientific and Technical Information of China (English)

    张新立; 史景泉; 卞修武

    2001-01-01

    目的 探讨大鼠肝癌变的形态演变和细胞增殖活性的关系。方法 通过DEN诱发启动模型和肝癌模型,采用图像分析技术,对病变细胞的形态进行测量,以PCNA和BrdU免疫组化结果反映细胞增殖活性。结果 形态测量证实卵圆细胞是一大小仅为正常肝细胞的1/8、核浆比却大6倍、形态不规则的增生小细胞;增生灶/结节嗜碱性肝细胞形态参数与肝癌细胞相似;PCNA和BrdU阳性细胞主要位于癌前增生灶及肝癌组织中;并且病变形态与细胞增殖活性有较好的一致性。结论 大鼠肝癌变的形态演变与细胞增殖活性密切相关。%Objective To explore the relationship between morphologic evolution and proliferative activity during hepatocarcinogenesis in rats. Methods Imaging analysis technique was used to detect the morphologic parameters of cells in hepatic lesions in both Solt-Farber model and diethylnitrosamine (DEN)-induced liver cancer model. Immunohistochemistry was employed to detect proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU). Results The oval cells were identified as irregular small proliferating cells in size of one-eighth of and with a nucleus/cytoplasm ratio of 6 times of the normal hepatocyte by image analysis. The morphometric parameters of basophil hepatocyte in precancerous foci and nodule were similar to those of the liver cancer cell. PCNA and BrdU positive cells were mainly localized within the proliferative foci and hepatocellular carcinoma (HCC) tissues. There was a better consistency between the development of hepatic lesions and cellular proliferative activity. Conclusion The morphologic evolution is closely related to proliferative activity during hepatocarcinogenesis in rats.

  7. Enhanced anti-apoptosis and gut epithelium protection function of acidic fibroblast growth factor after cancelling of its mitogenic activity

    Institute of Scientific and Technical Information of China (English)

    Xiao-Bing Fu; Xiao-Kun Li; Tong Wang; Biao Cheng; Zhi-Yong Sheng

    2004-01-01

    AIM: Mitogenic and non-mitogenic activities of fibroblast growth factor (FGF) are coupled to a range of biological functions, from cell proliferation and differentiation to the onset of many diseases. Recent reports have shown that acidic fibroblast growth factor (aFGF) has a powerful antiapoptosis function, which may have potentially therapeutical effect on gut ischemia and reperfusion injuries. However,whether this function depends on its mitogenic or nonmitogenic activity remains unclear. In this study, we identified the source of its anti-apoptosis function with a mutant,aFGF28-154 and observed its effect on reducing gut ischemia and reperfusion injury.METHODS:aFGF28-154 was generated by amplification of appropriate DNA fragments followed by subcloning the products into pET-3c vectors, then they were expressed in BL21 (DE3) cells and purified on an M2 agarose affinity column.This mutant aFGF28-154 maintained its non-mitogenic activity and lost its mitogenic activity. With a dexamethasone (DEX)-induced mouse thymocyte apoptosis model in vitro and in vivo, we studied the anti-apoptotic function of aFGF28-154. Also, in vivo study was performed to further confirm whether aFGF28-154 could significantly reduce apoptosis in gut epithelium after gut ischemia-reperfusion injury in rats. Based on these studies, the possible signal transduction pathways involved were studied.RESULTS: With a dexamethasone (DEX)-induced mouse thymocyte apoptosis model in vitro and in vivo, we found that the anti-apoptotic function of aFGF28-154 was significantly enhanced when compared with the wild type aFGF. In vivo study further confirmed that aFGF28-154 significantly reduced apoptosis in gut epithelium after gut ischemiareperfusion injury in rats. The mechanisms of anti-apoptosis function of aFGF28-154 did not depend on its mitogenic activity and were mainly associated with its non-mitogenic activities, including the intracellular calcium ion balance protection, ERK1/2 activation

  8. AZD-4547 exerts potent cytostatic and cytotoxic activities against fibroblast growth factor receptor (FGFR)-expressing colorectal cancer cells.

    Science.gov (United States)

    Yao, Ting-Jing; Zhu, Jin-Hai; Peng, De-Feng; Cui, Zhen; Zhang, Chao; Lu, Pei-hua

    2015-07-01

    Colorectal cancer (CRC) causes significant mortalities worldwide. Fibroblast growth factor (FGF) receptor (FGFR) signaling is frequently dysregulated and/or constitutively activated in CRCs, contributing to cancer carcinogenesis and progression. Here, we studied the activity of AZD-4547, a novel and potent FGFR kinase inhibitor, on CRC cells. AZD-4547 inhibited CRC cell growth in vitro, and its activity correlated with the FGFR-1/2 expression level. AZD-4547 was cytotoxic and pro-apoptotic in FGFR-1/2-expressed CRC cell lines (NCI-H716 and HCT-116), but not in FGFR-1/2 null HT-29 cells. Further, AZD-4547 inhibited cell cycle progression and attenuated the activation of FGFR1-FGFR substrate 2 (FRS-2), ERK/mitogen-activated protein kinase (MAPK), and AKT/mammalian target of rapamycin (AKT/mTOR) signalings in NCI-H716 and HCT-116 cells. In vivo, AZD-4547 oral administration at effective doses inhibited NCI-H716 (high FGFR-1/2 expression) xenograft growth in nude mice. Phosphorylation of FGFR-1, AKT, and ERK1/2 in xenograft specimens was also inhibited by AZD-4547 administration. Thus, our preclinical studies strongly support possible clinical investigations of AZD-4547 for the treatment of CRCs harboring deregulated FGFR signalings.

  9. Low-level laser therapy activates NF-kB via generation of reactive oxygen species in mouse embryonic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Aaron C-H Chen

    Full Text Available BACKGROUND: Despite over forty years of investigation on low-level light therapy (LLLT, the fundamental mechanisms underlying photobiomodulation at a cellular level remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we isolated murine embryonic fibroblasts (MEF from transgenic NF-kB luciferase reporter mice and studied their response to 810 nm laser radiation. Significant activation of NF-kB was observed at fluences higher than 0.003 J/cm(2 and was confirmed by Western blot analysis. NF-kB was activated earlier (1 hour by LLLT compared to conventional lipopolysaccharide treatment. We also observed that LLLT induced intracellular reactive oxygen species (ROS production similar to mitochondrial inhibitors, such as antimycin A, rotenone and paraquat. Furthermore, we observed similar NF-kB activation with these mitochondrial inhibitors. These results, together with inhibition of laser induced NF-kB activation by antioxidants, suggests that ROS play an important role in the laser induced NF-kB signaling pathways. However, LLLT, unlike mitochondrial inhibitors, induced increased cellular ATP levels, which indicates that LLLT also upregulates mitochondrial respiration. CONCLUSION: We conclude that LLLT not only enhances mitochondrial respiration, but also activates the redox-sensitive NFkB signaling via generation of ROS. Expression of anti-apoptosis and pro-survival genes responsive to NFkB could explain many clinical effects of LLLT.

  10. Proliferative periostitis: a case report.

    Science.gov (United States)

    Zand, Vahid; Lotfi, Mehrdad; Vosoughhosseini, Sepideh

    2008-04-01

    Proliferative periostitis of Garré represents a periosteal reaction to the presence of infection or other irritants. This can be odontogenic or nonodontogenic. This is a case report of an odontogenic periostitis resulting from endodontic origin. It was successfully treated by nonsurgical root canal therapy without using antibiotic therapy during the treatment of this case.

  11. A hemagglutinin from northeast red beans with immunomodulatory activity and anti-proliferative and apoptosis-inducing activities toward tumor cells.

    Science.gov (United States)

    Chan, Yau Sang; Wong, Jack Ho; Fang, Evandro Fei; Pan, Wenliang; Ng, Tzi Bun

    2013-10-01

    A 64-kDa hemagglutinin from a Phaseolus vulgaris cultivar, the northeast red bean, was purified by a protocol composed of three chromatographic steps involving affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose and FPLC-gel filtration on Superdex 75. The purified hemagglutinin appeared as a single 32-kDa band in SDS-PAGE indicating its dimeric nature. The N-terminal amino acid sequence of the hemagglutinin resembled the sequences of lectins and hemagglutinins from a number of Phaseolus species. The hemagglutinin manifested moderate thermostability and pH stability. It retained full activity up to 65 °C and in the pH range 2-12. It did not interact with simple sugars such as glucose, mannose and galactose. The hemagglutinin exerted immunostimulatory effects by upregulating the expression of cytokines like interferon-γ and tumor necrosis factor-α. It also exhibited antiproliferative activity on a number of tumor cells including MCF7 (breast cancer), HepG2 (liver cancer), CNE1 and CNE2 (nasopharyngeal cancer) cells, with stronger activity toward MCF7 and CNE1 cells. The hemagglutinin induced phophatidylserine externalization, mitochondrial depolarization and DNA condensation in MCF7 cells, indicating initiation of apoptosis. However, at high hemagglutinin concentrations, severe damage to the MCF7 cells was detected.

  12. Effects of Sipgeondaebo-tang Pharmacopuncture Extracts on the Collagenase Activity and Procollagen Synthesis in HS68 Human Fibroblasts and Tyrosinase Activity Original Articles

    Directory of Open Access Journals (Sweden)

    Lee Sena

    2011-03-01

    Full Text Available Objectives: This study was designed to investigate the collagen metabolism and tyrosinase activity of Sipgeondaebo-tang Pharmacopuncture extracts (SP. Methods: The effect of SP on type I procollagen production and collagenase activity in human normal fibroblasts HS68 after UVB (312 nm irradiation was measured by ELISA method. The tyrosinase activity after treatment of SP was measured as well. Results: Type I procollagen production was recovered by SP in UVB damaged HS68 cells. The increased collagenase activity after UVB damage was significantly recovered by SP. The tyrosinase activity was significantly reduced as well. However, the L-DOPA oxidation was not changed. Conclusion: SP showed the anti-wrinkle effects and whitening effects in vitro. These results suggest that SP may be a potential pharmacopuncture as an anti-aging pharmacopuncture treatments.

  13. Effects of RWJ 67657, a p38 mitogen activated protein kinase (MAPK) inhibitor, on the production of inflammatory mediators by rheumatoid synovial fibroblasts

    NARCIS (Netherlands)

    Westra, J; Limburg, PC; de Boer, Peter; van Rijswijk, Martin

    2004-01-01

    Objective: To investigate the effect of the p38 mitogen activated protein kinase ( MAPK) inhibitor RWJ 67657 on inflammatory mediator production by rheumatoid synovial fibroblasts (RSF). Methods: RSF were pretreated with RWJ 67657 and stimulated with TNFalpha and/or IL-1beta. Protein levels and mRNA

  14. Antioxidant and potential anti-inflammatory activity of extracts and formulations of white tea, rose, and witch hazel on primary human dermal fibroblast cells

    Directory of Open Access Journals (Sweden)

    Hili Pauline

    2011-10-01

    Full Text Available Abstract Background Numerous reports have identified therapeutic roles for plants and their extracts and constituents. The aim of this study was to assess the efficacies of three plant extracts for their potential antioxidant and anti-inflammatory activity in primary human skin fibroblasts. Methods Aqueous extracts and formulations of white tea, witch hazel and rose were subjected to assays to measure anti-collagenase, anti-elastase, trolox equivalent and catalase activities. Skin fibroblast cells were employed to determine the effect of each extract/formulation on IL-8 release induced by the addition of hydrogen peroxide. Microscopic examination along with Neutral Red viability testing was employed to ascertain the effects of hydrogen peroxide directly on cell viability. Results Considerable anti-collagenase, anti-elastase, and antioxidant activities were measured for all extracts apart from the witch hazel distillate which showed no activity in the collagenase assay or in the trolox equivalence assay. All of the extracts and products tested elicited a significant decrease in the amount of IL-8 produced by fibroblast cells compared to the control (p Conclusions These data show that the extracts and products tested have a protective effect on fibroblast cells against hydrogen peroxide induced damage. This approach provides a potential method to evaluate the claims made for plant extracts and the products in which these extracts are found.

  15. Ultrasonic promoted synthesis of novel s-triazine-Schiff base derivatives; molecular structure, spectroscopic studies and their preliminary anti-proliferative activities

    Science.gov (United States)

    El-Faham, Ayman; Soliman, Saied M.; Ghabbour, Hazem A.; Elnakady, Yasser A.; Mohaya, Talal A.; Siddiqui, Mohammed R. H.; Albericio, Fernando

    2016-12-01

    Novel series of s-triazine-Schiff base derivatives were synthesized employing ultrasonic irradiation and characterized by NMR (1H and 13C), FT-IR, and elemental analysis. The use of ultrasonic irradiation has allowed the preparation of the target products with better yields in shorter reaction time and excellent purities compared to the conventional heating. X-ray single crystal diffraction experiments verified the molecular structure of four from the new prepared s-triaizne-Schiff base derivatives. The molecular structures of the studied compounds are computerized using DFT/B3LYP method. The effects of substituent at the triazine and phenyl ring on the electronic and spectroscopic properties of the studied compounds were also investigated. The natural atomic charges showed that pipridino-s-triazine derivatives are richer in electrons than those having morpholino derivatives. The anti-proliferative effects for the prepared compounds were tested against three different cancer cell lines.

  16. Anti-proliferative activity of oral anti-hyperglycemic agents on human vascular smooth muscle cells: thiazolidinediones (glitazones have enhanced activity under high glucose conditions

    Directory of Open Access Journals (Sweden)

    de Dios Stephanie T

    2007-10-01

    Full Text Available Abstract Background Inhibition of vascular smooth muscle cell (vSMC proliferation by oral anti-hyperglycemic agents may have a role to play in the amelioration of vascular disease in diabetes. Thiazolidinediones (TZDs inhibit vSMC proliferation but it has been reported that they anomalously stimulate [3H]-thymidine incorporation. We investigated three TZDs, two biguanides and two sulfonylureas for their ability of inhibit vSMC proliferation. People with diabetes obviously have fluctuating blood glucose levels thus we determined the effect of media glucose concentration on the inhibitory activity of TZDs in a vSMC preparation that grew considerably more rapidly under high glucose conditions. We further explored the mechanisms by which TZDs increase [3H]-thymidine incorporation. Methods VSMC proliferation was investigated by [3H]-thymidine incorporation into DNA and cell counting. Activation and inhibition of thymidine kinase utilized short term [3H]-thymidine uptake. Cell cycle events were analyzed by FACS. Results VSMC cells grown for 3 days in DMEM with 5% fetal calf serum under low (5 mM glucose and high (25 mM glucose increased in number by 2.5 and 4.7 fold, respectively. Rosiglitazone and pioglitazone showed modest but statistically significantly greater inhibitory activity under high versus low glucose conditions (P 3H]-thymidine into DNA but did not increase cell numbers. Troglitazone inhibited serum mediated thymidine kinase induction in a concentration dependent manner. FACS analysis showed that troglitazone and rosiglitazone but not pioglitazone placed a slightly higher percentage of cells in the S phase of a growing culture. Of the biguanides, metformin had no effect on proliferation assessed as [3H]-thymidine incorporation or cell numbers whereas phenformin was inhibitory in both assays albeit at high concentrations. The sulfonylureas chlorpropamide and gliclazide had no inhibitory effect on vSMC proliferation assessed by either [3H

  17. Biological responses of three-dimensional cultured fibroblasts by sustained compressive loading include apoptosis and survival activity.

    Directory of Open Access Journals (Sweden)

    Toshiki Kanazawa

    Full Text Available Pressure ulcers are characterized by chronicity, which results in delayed wound healing due to pressure. Early intervention for preventing delayed healing due to pressure requires a prediction method. However, no study has reported the prediction of delayed healing due to pressure. Therefore, this study focused on biological response-based molecular markers for the establishment of an assessment technology to predict delayed healing due to pressure. We tested the hypothesis that sustained compressive loading applied to three dimensional cultured fibroblasts leads to upregulation of heat shock proteins (HSPs, CD44, hyaluronan synthase 2 (HAS2, and cyclooxygenase 2 (COX2 along with apoptosis via disruption of adhesion. First, sustained compressive loading was applied to fibroblast-seeded collagen sponges. Following this, collagen sponge samples and culture supernatants were collected for apoptosis and proliferation assays, gene expression analysis, immunocytochemistry, and quantification of secreted substances induced by upregulation of mRNA and protein level. Compared to the control, the compressed samples demonstrated that apoptosis was induced in a time- and load- dependent manner; vinculin and stress fiber were scarce; HSP90α, CD44, HAS2, and COX2 expression was upregulated; and the concentrations of HSP90α, hyaluronan (HA, and prostaglandin E2 (PGE2 were increased. In addition, the gene expression of antiapoptotic Bcl2 was significantly increased in the compressed samples compared to the control. These results suggest that compressive loading induces not only apoptosis but also survival activity. These observations support that HSP90α, HA, and, PGE2 could be potential molecular markers for prediction of delayed wound healing due to pressure.

  18. The disintegrin/metalloprotease ADAM 10 is essential for Notch signalling but not for alpha-secretase activity in fibroblasts.

    Science.gov (United States)

    Hartmann, Dieter; de Strooper, Bart; Serneels, Lutgarde; Craessaerts, Katleen; Herreman, An; Annaert, Wim; Umans, Lieve; Lübke, Torben; Lena Illert, Anna; von Figura, Kurt; Saftig, Paul

    2002-10-01

    The metalloprotease ADAM 10 is an important APP alpha-secretase candidate, but in vivo proof of this is lacking. Furthermore, invertebrate models point towards a key role of the ADAM 10 orthologues Kuzbanian and sup-17 in Notch signalling. In the mouse, this function is, however, currently attributed to ADAM 17/TACE, while the role of ADAM 10 remains unknown. We have created ADAM 10-deficient mice. They die at day 9.5 of embryogenesis with multiple defects of the developing central nervous system, somites, and cardiovascular system. In situ hybridization revealed a reduced expression of the Notch target gene hes-5 in the neural tube and an increased expression of the Notch ligand dll-1, supporting an important role for ADAM 10 in Notch signalling in the vertebrates as well. Since the early lethality precluded the establishment of primary neuronal cultures, APPs alpha generation was analyzed in embryonic fibroblasts and found to be preserved in 15 out of 17 independently generated ADAM 10-deficient fibroblast cell lines, albeit at a quantitatively more variable level than in controls, whereas a severe reduction was found in only two cases. The variability was not due to differences in genetic background or to variable expression of the alternative alpha-secretase candidates ADAM 9 and ADAM 17. These results indicate, therefore, either a regulation between ADAMs on the post-translational level or that other, not yet known, proteases are able to compensate for ADAM 10 deficiency. Thus, the observed variability, together with recent reports on tissue-specific expression patterns of ADAMs 9, 10 and 17, points to the existence of tissue-specific 'teams' of different proteases exerting alpha-secretase activity.

  19. Inflammatory Gene Expression Upon TGF-β1-Induced p38 Activation in Primary Dupuytren's Disease Fibroblasts

    Science.gov (United States)

    Bujak, Maro; Ratkaj, Ivana; Markova-Car, Elitza; Jurišić, Davor; Horvatić, Anita; Vučinić, Srđan; Lerga, Jonatan; Baus-Lončar, Mirela; Pavelić, Krešimir; Kraljević Pavelić, Sandra

    2015-01-01

    Objectives: Inflammation is an underlying mechanism behind fibrotic processes and differentiation of cells into myofibroblasts. Presented study therefore provides new data on activation of autoimmune and inflammatory immune response genes that accompany activation of p38 and cell differentiation in primary cells derived from Dupuytren's disease (DD) patients. Methods: Primary non-Dupuytren's disease cells (ND) were isolated from macroscopically unaffected palmar fascia adjacent to diseased tissue obtained from patients diagnosed with the last stage of DD and cultured in vitro. Gene expression, collagen gel contraction assay and analysis of secreted proteins were performed in ND cells treated with TGF-β1 and/or inhibitor of p38 phosphorylation. Results: During differentiation of ND fibroblasts, increased expression of immune response genes PAI-1, TIMP-1, CCL11, and IL-6 was found. These changes were accompanied by increased cell contractility and activation of p38 and its target kinase MK2. Inhibition of p38 phosphorylation reversed these processes in vitro. Conclusions: TGF-β1 induced p38 phosphorylation in ND cells grown from macroscopically unaffected palmar fascia adjacent to diseased tissue from DD patients. This was accompanied by activation of the cytokine genes CCL-11 and IL-6 and secretion of extracellular matrix regulatory proteins PAI-1 and TIMP-1. A combined approach directed toward inflammation and p38 MAPK-mediated processes in DD might be considered for improving management of DD patients and prevention of recurrence. PMID:26697433

  20. Inflammatory gene expression upon TGF-β1- induced p38 activation in primary Dupuytren’s disease fibroblasts

    Directory of Open Access Journals (Sweden)

    Maro eBujak

    2015-12-01

    Full Text Available Objectives: Inflammation is an underlying mechanism behind fibrotic processes and differentiation of cells into myofibroblasts. Presented study therefore provides new data on activation of autoimmune and inflammatory immune response genes that accompany activation of p38 and cell differentiation in primary cells derived from Dupuytren's disease (DD patients. Methods: Primary non-Dupuytren's disease cells (ND were isolated from macroscopically unaffected palmar fascia adjacent to diseased tissue obtained from patients diagnosed with the last stage of DD and cultured in vitro. Gene expression, collagen gel contraction assay and analysis of secreted proteins were performed in ND cells treated with TGF-β1 and/or inhibitor of p38 phosphorylation.Results: During differentiation of ND fibroblasts, increased expression of immune response genes PAI-1, TIMP-1, CCL11 and IL-6 was found. These changes were accompanied by increased cell contractility and activation of p38 and its target kinase MK2. Inhibition of p38 phosphorylation reversed these processes in vitro.Conclusions: TGF-β1 induced p38 phosphorylation in ND cells grown from macroscopically unaffected palmar fascia adjacent to diseased tissue from DD patients. This was accompanied by activation of the cytokine genes CCL-11 and IL-6 and secretion of extracellular matrix regulatory proteins PAI-1 and TIMP-1. A combined approach directed towards inflammation and p38 MAPK-mediated processes in DD might be considered for improving management of DD patients and prevention of recurrence.

  1. Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment.

    Science.gov (United States)

    Clark, R A; Wikner, N E; Doherty, D E; Norris, D A

    1988-08-25

    Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell

  2. Evaluation of the effects of ethinylestradiol on sexual differentiation in the olvas-GFP/STII-YI medaka (transgenic Oryzias latipes) strain as estimated by proliferative activity of germ cells

    Energy Technology Data Exchange (ETDEWEB)

    Hano, Takeshi [National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, 2-17-5 Maruishi, Hatsukaichi, Hiroshima 739-0452 (Japan); Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581 (Japan); Oshima, Yuji, E-mail: yoshima@agr.kyushu-u.ac.jp [Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581 (Japan); Kinoshita, Masato [Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502 (Japan); Tanaka, Minoru [Laboratory of Molecular Genetics for Reproduction, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki 444-8585 Aichi (Japan); Mishima, Noriko; Wakamatsu, Yuko; Ozato, Kenjiro [Laboratory of Freshwater Fish Stocks, Bioscience and Biotechnology Center, Nagoya University Furo-cho, Chikusa-ku, Nagoya 464-8601 (Japan); Shimasaki, Yohei; Honjo, Tsuneo [Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581 (Japan)

    2011-08-15

    We evaluated the effects of 17(-ethinylestradiol (EE{sub 2}) on sexual differentiation in transgenic olvas-GFP/STII-YI medaka (Oryzias latipes) in terms of the proliferative activity of germ cells. This strain contains the green fluorescent protein (GFP) gene fused to the regulatory region of the medaka vasa gene, and germ cell-specific expression of GFP can be visualized in living (transparent) individuals. From 0 days post-hatch (0 dph) onwards, juveniles were exposed to graded concentrations of EE{sub 2} (25.2-1710 ng/L) for 35 days. The gonads of live specimens were monitored by measuring their size and calculating their GFP-fluorescence area. GFP-fluorescent area in control females was about 10 times that in control males at 10 days posthatch (dph) whereas the gonadal size of 10 dph males that had been exposed to 158 ng/L of EE{sub 2} significantly increased up to twice the size of control males, indicating that abnormal sexual differentiation towards female might occur in these individuals. Histological examination and identification of the sex-linked marker SL1 indicated that male to female sex reversal occurred at EE{sub 2} exposure {>=}45.1 ng/L at 35 dph. These results suggest that observation of proliferative activity of germ cells in the olvas-GFP/STII-YI strain could be applied to facilitated screening fish model to detect adverse effects on sexual differentiation as early as 10 dph juveniles.

  3. Smoothened Regulates Migration of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis via Activation of Rho GTPase Signaling

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    Peng, Wei-xiang; Zhu, Shang-ling; Zhang, Bai-yu; Shi, Yi-ming; Feng, Xiao-xue; Liu, Fang; Huang, Jian-lin; Zheng, Song Guo

    2017-01-01

    Fibroblast-like synoviocytes (FLSs) acquire aggressive phenotypes characterized with enhanced migration abilities and inherent invasive qualities in rheumatoid arthritis (RA). Smoothened (Smo) is a key component of sonic hedgehog (Shh) signaling and contributes to tumor cell invasion and metastasis. The objective of this study is to investigate the role of Smo in the modulation of cell migration and explore the underlying molecular mechanism(s). FLSs were isolated from RA synovium. Shh levels were regulated by a Smo agonist (purmorphamine), Smo antagonist (KAAD-cyclopamine), or small interfering RNA targeting the Smo gene (Smo-siRNA) in RA-FLSs. Expression of Smo was detected by real-time PCR and western blot analysis. Cell migration was examined by Transwell assay and activation of Rho GTPases was measured by pull-down assays. Incubation with purmorphamine resulted in a significant increase of cell migration and activation of Rho GTPase signaling compared to controls (P < 0.05). However, treatment with KAAD-cyclopamine or transfection with Smo-siRNA suppressed migration of RA-FLSs and showed an inhibitory effect of Rho GTPase signaling. Together, these results suggest that Smo plays an important role in RA-FLSs migration through activation of Rho GTPase signaling and may contribute to progression of RA, thus, targeting Shh signal may have a therapeutic potential in patients with RA. PMID:28261216

  4. Fibroblast Growth Factor-9 Activates c-Kit Progenitor Cells and Enhances Angiogenesis in the Infarcted Diabetic Heart

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    Dinender Singla

    2016-01-01

    Full Text Available We hypothesized that fibroblast growth factor-9 (FGF-9 would enhance angiogenesis via activating c-kit positive stem cells in the infarcted nondiabetic and diabetic heart. In brief, animals were divided into three groups: Sham, MI, and MI+FGF-9. Two weeks following MI or sham surgery, our data suggest that treatment with FGF-9 significantly diminished vascular apoptosis compared to the MI group in both C57BL/6 and db/db mice (p<0.05. Additionally, the number of c-kit+ve/SM α-actin+ve cells and c-kit+ve/CD31+ve cells were greatly enhanced in the MI+FGF-9 groups relative to the MI suggesting FGF-9 enhances c-Kit cell activation and their differentiation into vascular smooth muscle cells and endothelial cells, respectively (p<0.05. Histology shows that the total number of vessels were quantified for all groups and our data suggest that the FGF-9 treated groups had significantly more vessels than their MI counterparts (p<0.05. Finally, echocardiographic data suggests a significant improvement in left ventricular output, as indicated by fractional shortening and ejection fraction in both nondiabetic and diabetic animals treated with FGF-9 (p<0.05. Overall, our data suggests FGF-9 has the potential to attenuate vascular cell apoptosis, activate c-Kit progenitor cells, and enhance angiogenesis and neovascularization in C57BL/6 and db/db mice leading to improved cardiac function.

  5. Fibroblast growth factor-2 induces osteogenic differentiation through a Runx2 activation in vascular smooth muscle cells

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    Nakahara, Takehiro; Sato, Hiroko; Shimizu, Takehisa; Tanaka, Toru; Matsui, Hiroki; Kawai-Kowase, Keiko; Sato, Mahito; Iso, Tatsuya; Arai, Masashi [Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511 (Japan); Kurabayashi, Masahiko, E-mail: mkuraba@med.gunma-u.ac.jp [Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511 (Japan)

    2010-04-02

    Expression of bone-associated proteins and osteoblastic transcription factor Runx2 in arterial cells has been implicated in the development of vascular calcification. However, the signaling upstream of the Runx2-mediated activation of osteoblastic program in vascular smooth muscle cells (VSMC) is poorly understood. We examined the effects of fibroblast growth factor-2 (FGF-2), an important regulator of bone formation, on osteoblastic differentiation of VSMC. Stimulation of cultured rat aortic SMC (RASMC) with FGF-2 induced the expression of the osteoblastic markers osteopontin (OPN) and osteocalcin. Luciferase assays showed that FGF-2 induced osteocyte-specific element (OSE)-dependent transcription. Downregulation of Runx2 by siRNA repressed the basal and FGF-2-stimulated expression of the OPN gene in RASMC. FGF-2 produced hydrogen peroxide in RASMC, as evaluated by fluorescent probe. Induction of OPN expression by FGF-2 was inhibited not only by PD98059 (MEK1 inhibitor) and PP1 (c-Src inhibitor), but also by an antioxidant, N-acetyl cysteine. Nuclear extracts from FGF-2-treated RASMC exhibited increased DNA-binding of Runx2 to its target sequence. Immunohistochemistry of human coronary atherectomy specimens and calcified aortic tissues showed that expression of FGF receptor-1 and Runx2 was colocalized. In conclusion, these results suggest that FGF-2 plays a role in inducing osteoblastic differentiation of VSMC by activating Runx2 through mitogen-activated protein kinase (MAPK)-dependent- and oxidative stress-sensitive-signaling pathways.

  6. Gastrodia elata Blume Extract Modulates Antioxidant Activity and Ultraviolet A-Irradiated Skin Aging in Human Dermal Fibroblast Cells.

    Science.gov (United States)

    Song, Eunju; Chung, Haeyon; Shim, Eugene; Jeong, Jung-Ky; Han, Bok-Kyung; Choi, Hyuk-Joon; Hwang, Jinah

    2016-11-01

    Gastrodia elata Blume (GEB), a traditional herbal medicine, has been used to treat a wide range of neurological disorders (e.g., paralysis and stroke) and skin problems (e.g., atopic dermatitis and eczema) in oriental medicine. This study was designed to investigate the antioxidant ability of GEB and its antiaging effect on human dermal fibroblast cells (HDF). The total phenolic and flavonoid contents of GEB were 21.8 and 0.43 mg/g dry weight (DW), respectively. The ergothioneine content of GEB was 0.41 mg/mL DW. The DPPH and ABTS radical scavenging activities of GEB at 5 and 10 mg/mL approximately ranged between 31% and 44%. The superoxide dismutase activity of GEB at 10 and 25 mg/mL was 57% and 76%, respectively. GEB increased procollagen type 1 (PC1) production and inhibited matrix metalloproteinase-1 (MMP-1) production and elastase-1 activity in UVA-irradiated HDF. PC1 messenger RNA (mRNA) levels decreased upon UVA irradiation, but recovered in response to high doses of GEB in HDF. On the contrary, GEB significantly decreased MMP-1 and elastase-1 mRNA levels, which were markedly induced in UVA-irradiated HDF. Collectively, these results suggest that GEB has sufficient antioxidant ability to prevent the signs of skin aging in UVA-irradiated human skin cells, suggesting its potential as a natural antiaging product.

  7. Studying the Anti-aging Effect of Human Growth Hormone on Human Fibroblast Cells via Telomerase Activity

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    Nader Chaparzadeh

    2010-01-01

    Full Text Available Objective: In recent years, studies have focused on the telomerase for cancer treatmentby repressing telomerase in cancerous cells or prevent cell aging by activating it in theaged cells. Thus, in these studies natural and synthetic agents have been used to repressor activate telomerase. In this research, we investigated the effects of human growth hormone(hGH on aging via evaluation of telomerase activity.Materials and Methods: Primary human foreskin fibroblast cells were isolated, culturedand treated with different concentrations of hGH. BrdU and MTT cell proliferation assaysand cells number counting. Cell aging was assayed by the senescence sensitivegalactosidase staining method. Telomerase activity was measured with a telomerasePCR ELISA kit.Data were analyzed with SPSS software (one-way ANOVA and univariateANOVA.Results: Our results indicated that cells treated with a lower concentration (0.1, 1 ng/mlof hGH had more green color cells (aged cells. Furthermore, cell proliferation increasedwith increasing hGH concentrations (10 to 100 ng/ml which was significant in comparisonwith untreated control cells. TRAP assay results indicated that telomerase activityincreased with increasing hGH concentration, but there was no significant difference. Additionally,more rapid cell growth and telomerase activity was noted in the absence of H2O2when compared with the presence of H2O2, which was significantly different.Conclusion: Although increasing cell proliferation along with increasing hGH concentrationwas confirmed by all cell proliferation assays, only the cell counting test was statisticallysignificant. Thus, it is inconclusive that hGH (up to 100 ng/ml has an anti-agingeffect. Also, because there was no significant difference in the telomerase activity results(in spite of increasing progress along with increasing hGH concentration we can not certainlyconclude that hGH (up to 100 ng/ml impacts telomerase activity.

  8. Hsc70 facilitates TGF-β-induced activation of Smad2/3 in fibroblastic NRK-49F cells.

    Science.gov (United States)

    Ikezaki, Midori; Higashimoto, Natsuki; Matsumura, Ko; Ihara, Yoshito

    2016-08-26

    Heat-shock cognate protein 70 (Hsc70), a molecular chaperone constitutively expressed in the cell, is involved in the regulation of several cellular signaling pathways. In this study, we found that TGF-β-induced phosphorylation and nuclear translocation of Smad2/3 were suppressed in fibroblastic NRK-49F cells treated with small interfering RNA (siRNA) for Hsc70. In the cells underexpressing Hsc70, transcriptional induction of connective tissue growth factor (CTGF), a target gene of the TGF-β signaling, was also suppressed in the early phase of TGF-β stimulation. Upon stimulation with TGF-β, Hsc70 interacted with Smad2/3, suggesting functional interactions of Hsc70 and Smad2/3 for the activation of TGF-β-induced Smad signaling. Although the expression of heat-shock protein 70 (Hsp70) was upregulated in the cells treated with Hsc70 siRNA, TGF-β-induced Smad activation was not affected in the cells overexpressing Hsp70. Collectively, these results indicate that Hsc70, but not Hsp70, supportively regulates TGF-β-induced Smad signaling in NRK-49F cells.

  9. Extracellular matrix elasticity modulates TGF-β-induced p38 activation and myofibroblast transdifferentiation in human tenon fibroblasts.

    Science.gov (United States)

    Meyer-ter-Vehn, Tobias; Han, Hong; Grehn, Franz; Schlunck, Günther

    2011-11-25

    Extracellular matrix and the cytokine TGF-β influence scar formation in an interdependent fashion. In this study, the impact of extracellular matrix elasticity on TGF-β-induced signal transduction and myofibroblast transdifferentiation was examined. Primary human tenon fibroblasts were seeded on collagen-coated glass coverslips (rigid environment) or collagen or polyacrylamide gels (elastic environment) of different compliance and stimulated with TGF-β. Myofibroblast transdifferentiation was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis for the marker gene α-smooth muscle actin (SMA), and SMA incorporation into stress fibers was determined by confocal immunofluorescence microscopy. CTGF transcription was assessed by RT-qPCR. Signaling pathways were examined by Western blot using phosphospecific antibodies and by immunofluorescence microscopy. TGF-β-dependent myofibroblast transdifferentiation was enhanced in a stiff environment. Increasing matrix elasticity attenuated TGF-β-induced myofibroblast transdifferentiation and the associated CTGF expression. TGF-β-induced p38 activation was reduced on elastic substrates. The results suggest that matrix elasticity influences TGF-β-dependent activation of p38 signaling and subsequent myofibroblast transdifferentiation. Biomechanical cues represent an important determinant of scarring processes. Therefore, cellular signals elicited by mechanotransduction deserve consideration in the design of novel antifibrotic strategies.

  10. Matrix metalloproteinase-1 inhibitory activities of Morinda citrifolia seed extract and its constituents in UVA-irradiated human dermal fibroblasts.

    Science.gov (United States)

    Masuda, Megumi; Murata, Kazuya; Naruto, Shunsuke; Uwaya, Akemi; Isami, Fumiyuki; Matsuda, Hideaki

    2012-01-01

    The objective of this study was to examine whether a 50% ethanolic extract (MCS-ext) of the seeds of Morinda citrifolia (noni) and its constituents have matrix metalloproteinase-1 (MMP-1) inhibitory activity in UVA-irradiated normal human dermal fibroblasts (NHDFs). The MCS-ext (10 μg/mL) inhibited MMP-1 secretion from UVA-irradiated NHDFs, without cytotoxic effects, at 48 h after UV exposure. The ethyl acetate-soluble fraction of MCS-ext was the most potent inhibitor of MMP-1 secretion. Among the constituents of the fraction, a lignan, 3,3'-bisdemethylpinoresinol (1), inhibited the MMP-1 secretion at a concentration of 0.3 μM without cytotoxic effects. Furthermore, 1 (0.3 μM) reduced the level of intracellular MMP-1 expression. Other constituents, namely americanin A (2), quercetin (3) and ursolic acid (4), were inactive. To elucidate inhibition mechanisms of MMP-1 expression and secretion, the effect of 1 on mitogen-activated protein kinases (MAPKs) phosphorylation was examined. Western blot analysis revealed that 1 (0.3 μM) reduced the phosphorylations of p38 and c-Jun-N-terminal kinase (JNK). These results suggested that 1 suppresses intracellular MMP-1 expression, and consequent secretion from UVA-irradiated NHDFs, by down-regulation of MAPKs phosphorylation.

  11. Excitation-Contraction Coupling between Human Atrial Myocytes with Fibroblasts and Stretch Activated Channel Current: A Simulation Study

    Directory of Open Access Journals (Sweden)

    Heqing Zhan

    2013-01-01

    Full Text Available Myocytes have been regarded as the main objectives in most cardiac modeling studies and attracted a lot of attention. Connective tissue cells, such as fibroblasts (Fbs, also play crucial role in cardiac function. This study proposed an integrated myocyte-Isac-Fb electromechanical model to investigate the effect of Fbs and stretch activated ion channel current (Isac on cardiac electrical excitation conduction and mechanical contraction. At the cellular level, an active Fb model was coupled with a human atrial myocyte electrophysiological model (including Isac and a mechanical model. At the tissue level, electrical excitation conduction was coupled with an elastic mechanical model, in which finite difference method (FDM was used to solve the electrical excitation equations, while finite element method (FEM was used for the mechanics equations. The simulation results showed that Fbs and Isac coupling caused diverse effects on action potential morphology during repolarization, depolarized the resting membrane potential of the human atrial myocyte, slowed down wave propagation, and decreased strains in fibrotic tissue. This preliminary simulation study indicates that Fbs and Isac have important implications for modulating cardiac electromechanical behavior and should be considered in future cardiac modeling studies.

  12. Synovial chondromatosis of the right side temporomandibular joint extending to the middle cranial fossa: A case report with 7-year postoperative follow up and expression of a biomarker of cell proliferative activity

    Science.gov (United States)

    Yoshitake, Hiroyuki; Kayamori, Kou; Wake, So; Sato, Fumiaki; Kino, Koji; Harada, Kiyoshi

    2016-01-01

    Introduction Synovial chondromatosis of the temporomandibular joint (TMJ) with cranial extension is rare. Here, we report 7-year follow-up of a case with immunohistochemical examination of cell proliferative activity. Presentation of case The patient was a 72-year-old man. Severe bone resorption of the glenoid fossa was apparent on CT images. Pathological findings by biopsy led to diagnosis of synovial chondromatosis of the right side TMJ. Extirpation of the tumor was performed via temporopreauricular incision under general anesthesia. PCNA expression was examined by immunohistochemical analysis. The lesion had penetrated into the middle cranial fossa, but the cranial dura mater was intact. Expression of PCNA was confirmed. Discussion The PCNA expression suggested that growth activity caused expansion of the lesion to the skull base. Conclusion We were able to follow up this case for a long period without recurrence postoperatively. PMID:26855075

  13. The hypoxia-inducible factor-1α activates ectopic production of fibroblast growth factor 23 in tumor-induced osteomalacia

    Science.gov (United States)

    Zhang, Qian; Doucet, Michele; Tomlinson, Ryan E; Han, Xiaobin; Quarles, L Darryl; Collins, Michael T; Clemens, Thomas L

    2016-01-01

    Tumor-induced osteomalacia (TIO) is a rare paraneoplastic syndrome in which ectopic production of fibroblast growth factor 23 (FGF23) by non-malignant mesenchymal tumors causes phosphate wasting and bone fractures. Recent studies have implicated the hypoxia-inducible factor-1α (HIF-1α) in other phosphate wasting disorders caused by elevated FGF23, including X-linked hypophosphatemic rickets and autosomal dominant hypophosphatemia. Here we provide evidence that HIF-1α mediates aberrant FGF23 in TIO by transcriptionally activating its promoter. Immunohistochemical studies in phosphaturic mesenchymal tumors resected from patients with documented TIO showed that HIF-1α and FGF23 were co-localized in spindle-shaped cells adjacent to blood vessels. Cultured tumor tissue produced high levels of intact FGF23 and demonstrated increased expression of HIF-1α protein. Transfection of MC3T3-E1 and Saos-2 cells with a HIF-1α expression construct induced the activity of a FGF23 reporter construct. Prior treatment of tumor organ cultures with HIF-1α inhibitors decreased HIF-1α and FGF23 protein accumulation and inhibited HIF-1α-induced luciferase reporter activity in transfected cells. Chromatin immunoprecipitation assays confirmed binding to a HIF-1α consensus sequence within the proximal FGF23 promoter, which was eliminated by treatment with a HIF-1α inhibitor. These results show for the first time that HIF-1α is a direct transcriptional activator of FGF23 and suggest that upregulation of HIF-1α activity in TIO contributes to the aberrant FGF23 production in these patients. PMID:27468359

  14. Fibroblasts in fibrosis: novel roles and mediators

    Directory of Open Access Journals (Sweden)

    Ryan Thomas Kendall

    2014-05-01

    Full Text Available Fibroblasts are the most common cell type of the connective tissues found throughout the body and the principal source of the extensive extracellular matrix (ECM characteristic of these tissues. They are also the central mediators of the pathological fibrotic accumulation of ECM and the cellular proliferation and differentiation that occurs in response to prolonged tissue injury and chronic inflammation. The transformation of the fibroblast cell lineage involves classical developmental signaling programs and includes a surprisingly diverse range of precursor cell types—most notably, myofibroblasts that are the apex of the fibrotic phenotype. Myofibroblasts display exaggerated ECM production; constitutively secrete and are hypersensitive to chemical signals such as cytokines, chemokines, and growth factors; and are endowed with a contractile apparatus allowing them to manipulate the ECM fibers physically to close open wounds. In addition to ECM production, fibroblasts have multiple concomitant biological roles, such as in wound healing, inflammation, and angiogenesis, which are each interwoven with the process of fibrosis. We now recognize many common fibroblast-related features across various physiological and pathological protracted processes. Indeed, a new appreciation has emerged for the role of noncancerous fibroblast interactions with tumors in cancer progression. Although the predominant current clinical treatments of fibrosis involve nonspecific immunosuppressive and anti-proliferative drugs, a variety of potential therapies under investigation specifically target fibroblast biology.

  15. Activation of Robo1 signaling of breast cancer cells by Slit2 from stromal fibroblast restrains tumorigenesis via blocking PI3K/Akt/β-catenin pathway.

    Science.gov (United States)

    Chang, Po-Hao; Hwang-Verslues, Wendy W; Chang, Yi-Cheng; Chen, Chun-Chin; Hsiao, Michael; Jeng, Yung-Ming; Chang, King-Jen; Lee, Eva Y-H P; Shew, Jin-Yuh; Lee, Wen-Hwa

    2012-09-15

    Tumor microenvironment plays a critical role in regulating tumor progression by secreting factors that mediate cancer cell growth. Stromal fibroblasts can promote tumor growth through paracrine factors; however, restraint of malignant carcinoma progression by the microenvironment also has been observed. The mechanisms that underlie this paradox remain unknown. Here, we report that the tumorigenic potential of breast cancer cells is determined by an interaction between the Robo1 receptor and its ligand Slit2, which is secreted by stromal fibroblasts. The presence of an active Slit2/Robo1 signal blocks the translocation of β-catenin into nucleus, leading to downregulation of c-myc and cyclin D1 via the phosphoinositide 3-kinase (PI3K)/Akt pathway. Clinically, high Robo1 expression in the breast cancer cells correlates with increased survival in patients with breast cancer, and low Slit2 expression in the stromal fibroblasts is associated with lymph node metastasis. Together, our findings explain how a specific tumor microenvironment can restrain a given type of cancer cell from progression and show that both stromal fibroblasts and tumor cell heterogeneity affect breast cancer outcomes.

  16. Hsc70 facilitates TGF-β-induced activation of Smad2/3 in fibroblastic NRK-49F cells

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    Ikezaki, Midori; Higashimoto, Natsuki; Matsumura, Ko; Ihara, Yoshito, E-mail: y-ihara@wakayama-med.ac.jp

    2016-08-26

    Heat-shock cognate protein 70 (Hsc70), a molecular chaperone constitutively expressed in the cell, is involved in the regulation of several cellular signaling pathways. In this study, we found that TGF-β-induced phosphorylation and nuclear translocation of Smad2/3 were suppressed in fibroblastic NRK-49F cells treated with small interfering RNA (siRNA) for Hsc70. In the cells underexpressing Hsc70, transcriptional induction of connective tissue growth factor (CTGF), a target gene of the TGF-β signaling, was also suppressed in the early phase of TGF-β stimulation. Upon stimulation with TGF-β, Hsc70 interacted with Smad2/3, suggesting functional interactions of Hsc70 and Smad2/3 for the activation of TGF-β-induced Smad signaling. Although the expression of heat-shock protein 70 (Hsp70) was upregulated in the cells treated with Hsc70 siRNA, TGF-β-induced Smad activation was not affected in the cells overexpressing Hsp70. Collectively, these results indicate that Hsc70, but not Hsp70, supportively regulates TGF-β-induced Smad signaling in NRK-49F cells. - Highlights: • Hsc70 siRNA treatment suppressed the expression of Hsc70 but induced the expression of Hsp70 in NRK-49F cells. • Hsc70 siRNA treatment suppressed the activation of Smad2/3 in the cells treated with TGF-β. • Hsc70 interacted with Smad2/3 on stimulation with TGF-β in the cells. • Hsp70 did not influence the TGF-β-induced activation of Smad2/3 in the cells overexpressing Hsp70.

  17. Vitronectin-binding PAI-1 protects against the development of cardiac fibrosis through interaction with fibroblasts.

    Science.gov (United States)

    Zhong, Jianyong; Yang, Hai-Chun; Kon, Valentina; Fogo, Agnes B; Lawrence, Daniel A; Ma, Ji

    2014-06-01

    Plasminogen activator inhibitor-1 (PAI-1) promotes or abates fibrotic processes occurring in different organs. Binding of PAI-1 to vitronectin, an extracellular matrix component, may inhibit vitronectin-integrin complex-mediated cellular responses in pathophysiological conditions. To investigate the importance of plasmin suppression vs vitronectin-binding pathways of PAI-1 in cardiac fibrosis, we studied uninephrectomized mice fed a high salt diet and infused with angiotensin II (Ang II) together with different PAI-1 variants, including PAI-1AK (AK) that inhibits plasminogen activators but does not bind vitronectin, PAI-1RR (RR) that binds vitronectin but does not have protease inhibitory effects or control PAI-1 (CPAI), the control mutant that has similar molecular backbone and half-life as AK and RR while retaining all functions of native PAI-1. Compared with RR and CPAI, non-vitronectin-binding AK significantly increased expression of cardiac fibroblast marker, periostin (Ang+AK 8.40±3.55 vs Ang+RR 2.23±0.44 and Ang+CPAI 2.33±0.12% positive area, both PPAI-1 against fibrosis, fibroblasts from normal adult human ventricles were stimulated with Ang and different PAI-1 variants. Protease inhibitory AK and CPAI increased supernatant fibronectin, while decreasing plasminogen activator/plasmin activities and matrix metalloproteinase. RR and CPAI variants significantly reduced fibroblast expression of integrin β3, vitronectin level in the supernatant and fibroblast adhesion to vitronectin compared with the non-vitronectin-binding AK. Further, RR and CPAI preserved apoptotic, decreased anti-apoptotic and proliferative activities in fibroblasts. Thus, PAI-1 promotes or protects against development of cardiac fibrosis differentially through the protease inhibitory pathway or through its binding to vitronectin.

  18. Synthesis and in vitro anti-proliferative activity of some novel isatins conjugated with quinazoline/phthalazine hydrazines against triple-negative breast cancer MDA-MB-231 cells as apoptosis-inducing agents.

    Science.gov (United States)

    Eldehna, Wagdy M; Almahli, Hadia; Al-Ansary, Ghada H; Ghabbour, Hazem A; Aly, Mohamed H; Ismael, Omnia E; Al-Dhfyan, Abdullah; Abdel-Aziz, Hatem A

    2017-12-01

    Treatment of patients with triple-negative breast cancer (TNBC) is challenging due to the absence of well- defined molecular targets and the heterogeneity of such disease. In our endeavor to develop potent isatin-based anti-proliferative agents, we utilized the hybrid-pharmacophore approach to synthesize three series of novel isatin-based hybrids 5a-h, 10a-h and 13a-c, with the prime goal of developing potent anti-proliferative agents toward TNBC MDA-MB-231 cell line. In particular, compounds 5e and 10g were the most active hybrids against MDA-MB-231 cells (IC50 = 12.35 ± 0.12 and 12.00 ± 0.13 μM), with 2.37- and 2.44-fold increased activity than 5-fluorouracil (5-FU) (IC50 = 29.38 ± 1.24 μM). Compounds 5e and 10g induced the intrinsic apoptotic mitochondrial pathway in MDA-MB-231; evidenced by the reduced expression of the anti-apoptotic protein Bcl-2, the enhanced expression of the pro-apoptotic protein Bax and the up-regulated active caspase-9 and caspase-3 levels. Furthermore, 10g showed significant increase in the percent of annexin V-FITC positive apoptotic cells from 3.88 to 31.21% (8.4 folds compared to control).

  19. Activation of homologous recombination DNA repair in human skin fibroblasts continuously exposed to X-ray radiation.

    Science.gov (United States)

    Osipov, Andreyan N; Grekhova, Anna; Pustovalova, Margarita; Ozerov, Ivan V; Eremin, Petr; Vorobyeva, Natalia; Lazareva, Natalia; Pulin, Andrey; Zhavoronkov, Alex; Roumiantsev, Sergey; Klokov, Dmitry; Eremin, Ilya

    2015-09-29

    Molecular and cellular responses to protracted ionizing radiation exposures are poorly understood. Using immunofluorescence microscopy, we studied the kinetics of DNA repair foci formation in normal human fibroblasts exposed to X-rays at a dose rate of 4.5 mGy/min for up to 6 h. We showed that both the number of γH2AX foci and their integral fluorescence intensity grew linearly with time of irradiation up to 2 h. A plateau was observed between 2 and 6 h of exposure, indicating a state of balance between formation and repair of DNA double-strand breaks. In contrast, the number and intensity of foci formed by homologous recombination protein RAD51 demonstrated a continuous increase during 6 h of irradiation. We further showed that the enhancement of the homologous recombination repair was not due to redistribution of cell cycle phases. Our results indicate that continuous irradiation of normal human cells triggers DNA repair responses that are different from those elicited after acute irradiation. The observed activation of the error-free homologous recombination DNA double-strand break repair pathway suggests compensatory adaptive mechanisms that may help alleviate long-term biological consequences and could potentially be utilized both in radiation protection and medical practices.

  20. Fibroblast growth factor receptor 3 interacts with and activates TGFβ-activated kinase 1 tyrosine phosphorylation and NFκB signaling in multiple myeloma and bladder cancer.

    Directory of Open Access Journals (Sweden)

    Lisa Salazar

    Full Text Available Cancer is a major public health problem worldwide. In the United States alone, 1 in 4 deaths is due to cancer and for 2013 a total of 1,660,290 new cancer cases and 580,350 cancer-related deaths are projected. Comprehensive profiling of multiple cancer genomes has revealed a highly complex genetic landscape in which a large number of altered genes, varying from tumor to tumor, impact core biological pathways and processes. This has implications for therapeutic targeting of signaling networks in the development of treatments for specific cancers. The NFκB transcription factor is constitutively active in a number of hematologic and solid tumors, and many signaling pathways implicated in cancer are likely connected to NFκB activation. A critical mediator of NFκB activity is TGFβ-activated kinase 1 (TAK1. Here, we identify TAK1 as a novel interacting protein and target of fibroblast growth factor receptor 3 (FGFR3 tyrosine kinase activity. We further demonstrate that activating mutations in FGFR3 associated with both multiple myeloma and bladder cancer can modulate expression of genes that regulate NFκB signaling, and promote both NFκB transcriptional activity and cell adhesion in a manner dependent on TAK1 expression in both cancer cell types. Our findings suggest TAK1 as a potential therapeutic target for FGFR3-associated cancers, and other malignancies in which TAK1 contributes to constitutive NFκB activation.

  1. Activating the expression of human K-rasG12D stimulates oncogenic transformation in transgenic goat fetal fibroblast cells.

    Science.gov (United States)

    Gong, Jianhua; Wang, Zhongde; Polejaeva, Irina; Salgia, Ravi; Kao, Chien-Min; Chen, Chin-Tu; Chen, Guangchun; Chen, Liaohai

    2014-01-01

    Humane use of preclinical large animal cancer models plays a critical role in understanding cancer biology and developing therapeutic treatments. Among the large animal candidates, goats have great potentials as sustainable sources for large animal cancer model development. Goats are easier to handle and cheaper to raise. The genome of the goats has been sequenced recently. It has been known that goats develop skin, adrenal cortex, breast and other types of cancers. Technically, goats are subject to somatic cell nuclear transfer more efficiently and exhibit better viability through the cloning process. Towards the development of a goat cancer model, we created a transgenic goat fetal fibroblast (GFF) cell as the donor cell for SCNT. Human mutated K-ras (hK-rasG12D) was chosen as the transgene, as it is present in 20% of cancers. Both hK-rasG12D and a herpes simplex viral thymidine kinase (HSV1-tk) reporter genes, flanked by a pair of LoxP sites, were knocked in the GFF endogenous K-ras locus through homologous recombination. Following Cre-mediated activation (with a 95% activation efficiency), hK-rasG12D and HSV1-tk were expressed in the transgenic GFF cells, evidently through the presence of corresponding mRNAs, and confirmed by HSV1-tk protein function assay. The hK-rasG12D expressing GFF cells exhibited enhanced proliferation rates and an anchorage-independent growth behavior. They were able to initiate tumor growth in athymic nude mice. In conclusion, after activating hK-rasG12D gene expression, hK-rasG12D transgenic GFF cells were transformed into tumorgenesis cells. Transgenic goats via SCNT using the above-motioned cells as the donor cells have been established.

  2. Activating the expression of human K-rasG12D stimulates oncogenic transformation in transgenic goat fetal fibroblast cells.

    Directory of Open Access Journals (Sweden)

    Jianhua Gong

    Full Text Available Humane use of preclinical large animal cancer models plays a critical role in understanding cancer biology and developing therapeutic treatments. Among the large animal candidates, goats have great potentials as sustainable sources for large animal cancer model development. Goats are easier to handle and cheaper to raise. The genome of the goats has been sequenced recently. It has been known that goats develop skin, adrenal cortex, breast and other types of cancers. Technically, goats are subject to somatic cell nuclear transfer more efficiently and exhibit better viability through the cloning process. Towards the development of a goat cancer model, we created a transgenic goat fetal fibroblast (GFF cell as the donor cell for SCNT. Human mutated K-ras (hK-rasG12D was chosen as the transgene, as it is present in 20% of cancers. Both hK-rasG12D and a herpes simplex viral thymidine kinase (HSV1-tk reporter genes, flanked by a pair of LoxP sites, were knocked in the GFF endogenous K-ras locus through homologous recombination. Following Cre-mediated activation (with a 95% activation efficiency, hK-rasG12D and HSV1-tk were expressed in the transgenic GFF cells, evidently through the presence of corresponding mRNAs, and confirmed by HSV1-tk protein function assay. The hK-rasG12D expressing GFF cells exhibited enhanced proliferation rates and an anchorage-independent growth behavior. They were able to initiate tumor growth in athymic nude mice. In conclusion, after activating hK-rasG12D gene expression, hK-rasG12D transgenic GFF cells were transformed into tumorgenesis cells. Transgenic goats via SCNT using the above-motioned cells as the donor cells have been established.

  3. NH2-terminal cleavage of xenopus fibroblast growth factor 3 is necessary for optimal biological activity and receptor binding.

    Science.gov (United States)

    Antoine, M; Daum, M; Köhl, R; Blecken, V; Close, M J; Peters, G; Kiefer, P

    2000-11-01

    Fibroblast growth factor 3 (FGF3) was originally identified as the mouse proto-oncogene Int-2, which is activated by proviral insertion in tumors induced by mouse mammary tumor virus. To facilitate the biological characterization of the ligand, we have analyzed its homologue in Xenopus laevis, XFGF3. Here we confirm that the X. laevis genome contains two distinct FGF3 alleles, neither of which is capable of encoding the NH2-terminally extended forms specified by the mouse and human FGF3 genes. Unlike the mammalian proteins, XFGF3 is efficiently secreted as a Mr 31,000 glycoprotein, gp31, which undergoes proteolytic cleavage to produce an NH2-terminally truncated product, gp27. Processing removes a segment of 18 amino acids immediately distal to the signal peptide that is not present in the mammalian homologues. By inserting an epitope-tag adjacent to the cleavage site, we show that a substantial amount of the gp27 is generated intracellularly, although processing can also occur in the extracellular matrix. Two residues are also removed from the COOH terminus. To compare the biological properties of the different forms, cDNAs were constructed that selectively give rise to the larger, gp31, or smaller, gp27, forms of XFGF3. As judged by their ability to cause morphological transformation of NIH3T3 cells, their mitogenicity on specific cell types, and their affinity for the IIIb and IIIc isoforms of Xenopus FGF receptors, gp27 has a much higher biological activity than gp31. Sequence comparison revealed an intriguing similar cleavage motif immediately downstream of the signal peptide cleavage site in the NH2-terminus of mouse and human FGF3. Analysis of secreted mutant mouse FGF3 confirmed an additional NH2-terminal processing at the corresponding sequence motif. NH2-terminal trimming of Xenopus and mammalian FGF3s may therefore be a prerequisite of optimal biological activity.

  4. Epidermal growth factor inhibits hormone- and fibroblast growth factor-induced activation of phospholipase C in rat pancreatic acini.

    Science.gov (United States)

    Stryjek-Kaminska, D; Piiper, A; Caspary, W F; Zeuzem, S

    1995-01-01

    Epidermal growth factor (EGF) inhibits cholecystokinin-octapeptide-stimulated amylase release and inositol 1,4,5-trisphosphate (1,4,5-IP3) production in isolated rat pancreatic acini. In the present study, pancreatic acini were used to investigate the effect of EGF on amylase release and 1,4,5-IP3 production induced by secretagogues that activate either phospholipase C-beta (carbachol, bombesin) or phospholipase C-gamma [basic fibroblast growth factor (bFGF)]. The results show that EGF (100 ng/ml) inhibited bombesin (0.1 nM-1 microM)-induced amylase release almost completely. Similarly, the effect of EGF on carbachol-stimulated amylase release was substantial at submaximal (0.1 microM: 44% inhibition), maximal (1 microM: 75% inhibition), and supramaximal (100 microM: 33% inhibition) carbachol concentrations. EGF reduced amylase release at submaximal bFGF concentrations (0.1 nM: 40% inhibition), but not at supramaximal bFGF concentrations (1 and 10 nM). EGF decreased the peak increase of 1,4,5-IP3 in response to bombesin and carbachol (5 s after beginning of the incubation) and bFGF (15 s after beginning of the incubation) by 81 +/- 19%, 65 +/- 15%, and 56 +/- 18%, respectively. Receptor binding characteristics for secretagogues that activate phospholipase C were not influenced by coincubation with EGF excluding heterologous transmembrane receptor modulation. These results suggest that EGF inhibits the action of phospholipase C-beta- and gamma-isoenzyme-activating secretagogues in the exocrine pancreas by a postreceptor mechanism.

  5. Extended Synaptotagmin Interaction with the Fibroblast Growth Factor Receptor Depends on Receptor Conformation, Not Catalytic Activity.

    Science.gov (United States)

    Tremblay, Michel G; Herdman, Chelsea; Guillou, François; Mishra, Prakash K; Baril, Joëlle; Bellenfant, Sabrina; Moss, Tom

    2015-06-26

    We previously demonstrated that ESyt2 interacts specifically with the activated FGF receptor and is required for a rapid phase of receptor internalization and for functional signaling via the ERK pathway in early Xenopus embryos. ESyt2 is one of the three-member family of Extended Synaptotagmins that were recently shown to be implicated in the formation of endoplasmic reticulum (ER)-plasma membrane (PM) junctions and in the Ca(2+) dependent regulation of these junctions. Here we show that ESyt2 is directed to the ER by its putative transmembrane domain, that the ESyts hetero- and homodimerize, and that ESyt2 homodimerization in vivo requires a TM adjacent sequence but not the SMP domain. ESyt2 and ESyt3, but not ESyt1, selectively interact in vivo with activated FGFR1. In the case of ESyt2, this interaction requires a short TM adjacent sequence and is independent of receptor autophosphorylation, but dependent on receptor conformation. The data show that ESyt2 recognizes a site in the upper kinase lobe of FGFR1 that is revealed by displacement of the kinase domain activation loop during receptor activation.

  6. Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts

    DEFF Research Database (Denmark)

    Friis, Martin Barfred; Vorum, Katrine Gribel; Lambert, Ian Henry

    2008-01-01

    +-mobilizing agonist ATP (10 microM) potentiates the release of taurine but has no effect on ROS production under hypotonic conditions. On the other hand, addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) or the lipid messenger lysophosphatidic acid (LPA, 10 n...

  7. Altered membrane NTPase activity in Lesch-Nyhan disease fibroblasts: comparison with HPRT knockout mice and HPRT-deficient cell lines.

    Science.gov (United States)

    Pinto, Cibele S; Jinnah, Hyder A; Shirley, Thomas L; Nyhan, William L; Seifert, Roland

    2005-06-01

    Lesch-Nyhan disease (LND) is a rare disorder caused by a defect of an enzyme in the purine salvage pathway, hypoxanthine phosphoribosyl transferase (HPRT). It is still unknown how the metabolic defect translates into the complex neuropsychiatric phenotype characterized by self-injurious behavior, dystonia and mental retardation. There are abnormalities in purine and pyrimidine nucleotide content in HPRT-deficient cells. We hypothesized that altered nucleotide concentrations in HPRT deficiency change G-protein-mediated signal transduction. Therefore, our original study aim was to examine the high-affinity GTPase activity of G-proteins in membranes from primary human skin and immortalized mouse skin fibroblasts, rat B103 neuroblastoma cells and mouse Neuro-2a neuroblastoma cells. Unexpectedly, in membranes from human fibroblasts, B103- and Neuro-2a cells, V(max) of low-affinity nucleoside 5'-triphosphatase (NTPase) activities was decreased up to 7-fold in HPRT deficiency. In contrast, in membranes from mouse fibroblasts, HPRT deficiency increased NTPase activity up to 4-fold. The various systems analyzed differed from each other in terms of K(m) values for NTPs, absolute V(max) values and K(i) values for nucleoside 5'-[beta,gamma-imido]triphosphates. Our data show that altered membrane NTPase activity is a biochemical hallmark of HPRT deficiency, but species and cell-type differences have to be considered. Thus, future studies on biochemical changes in LND should be conducted in parallel in several HPRT-deficient systems.

  8. AMP-Activated Protein Kinase Alleviates Extracellular Matrix Accumulation in High Glucose-Induced Renal Fibroblasts through mTOR Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Xia Luo

    2015-01-01

    Full Text Available Background/Aims: Extracellular matrix accumulation contributes significantly to the pathogenesis of diabetic nephropathy. Although AMP-activated protein kinase (AMPK has been found to inhibit extracellular matrix synthesis by experiments in vivo and vitro, its role in alleviating the deposition of extracellular matrix in renal interstitial fibroblasts has not been well defined. Methods: Currently, we conducted this study to investigate the effects of AMPK on high glucose-induced extracellular matrix synthesis and involved intracellular signaling pathway by using western blot in the kidney fibroblast cell line (NRK-49f. Results: Collagen IV protein levels were significantly increased by high glucose in a time-dependent manner. This was associated with a decrease in Thr72 phosphorylation of AMPK and an increase in phosphorylation of mTOR on Ser2448. High glucose-induced extracellular matrix accumulation and mTOR activation were significantly inhibited by the co-treatment of rAAV-AMPKα1312 (encoding constitutively active AMPKα1 whereas activated by r-AAV-AMPKα1D157A (encoding dominant negative AMPKα1. In cultured renal fibroblasts, overexpression of AMPKα1D157A upregulated mTOR signaling and matrix synthesis, which were ameliorated by co-treatment with the inhibitor of mTOR, rapamycin. Conclusion: Collectively, these findings indicate that AMPK exerts renoprotective effects by inhibiting the accumulation of extracellular matrix through mTOR signaling pathway.

  9. Dlg-1 Interacts With and Regulates the Activities of Fibroblast Growth Factor Receptors and EphA2 in the Mouse Lens

    OpenAIRE

    Lee, Sungkyoung; Shatadal, Shalini; Griep, Anne E.

    2016-01-01

    Purpose We previously showed that Discs large-1 (Dlg-1) regulates lens fiber cell structure and the fibroblast growth factor receptor (Fgfr) signaling pathway, a pathway required for fiber cell differentiation. Herein, we investigated the mechanism through which Dlg-1 regulates Fgfr signaling. Methods Immunofluorescence was used to measure levels of Fgfr1, Fgfr2, and activated Fgfr signaling intermediates, pErk and pAkt, in control and Dlg-1–deficient lenses that were haplodeficient for Fgfr1...

  10. Activation of cellular oncogenes by chemical carcinogens in Syrian hamster embryo fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Ebert, R.; Reiss, E.; Roellich, G.; Schiffmann, D. (Univ. of Wuerzburg (West Germany)); Barrett, J.C.; Wiseman, R.W. (National Institute of Environmental Health Sciences, Research Triangle Park, NC (USA)); Pechan, R.

    1990-08-01

    Carcinogen-induced point mutations resulting in activation of ras oncogenes have been demonstrated in various experimental systems such as skin carcinogenesis, mammary, and liver carcinogenesis. In many cases, the data support the conclusion that these point mutations are critical changes in the initiation of these tumors. The Syrian hamster embryo (SHE) cell transformation model system has been widely used to study the multistep process of chemically induced neoplastic transformation. Recent data suggest that activation of the Ha-ras gene via point mutation is one of the crucial events in the transformation of these cells. The authors have now cloned the c-Ha-ras proto-oncogene from SHE cDNA-libraries, and we have performed polymerase chain reaction and direct sequencing to analyze tumor cell lines induced by different chemical carcinogens for the presence of point mutations. No changes were detectable at codons 12, 13, 59, 61, and 117 or adjacent regions in tumor cell lines induced by diethylstilbestrol, asbestos, benzo(a)pyrene, trenbolone, or aflatoxin B{sub 1}. Thus, it is not known whether point mutations in the Ha-ras proto-oncogene are essential for the acquisition of the neoplastic phenotype of SHE cells. Activation of other oncogenes or inactivation of tumor suppressor genes may be responsible for the neoplastic progression of these cells. However, in SHE cells neoplastically transformed by diethylstilbestrol or trenbolone, a significant elevation of the c-Ha-ras expression was observed. Enhanced expression of c-myc was detected in SHE cells transformed by benzo(a)pyrene or trenbolone.

  11. Bispecific single-chain diabody-immunoliposomes targeting endoglin (CD105) and fibroblast activation protein (FAP) simultaneously.

    Science.gov (United States)

    Rabenhold, Markus; Steiniger, Frank; Fahr, Alfred; Kontermann, Roland E; Rüger, Ronny

    2015-03-10

    Liposomes are well-established drug delivery systems with cancer chemotherapy as main focus. To increase the cellular drug delivery, liposomes can be endowed with ligands, e.g. recombinant antibody fragments, which ensure specific cell interaction. Multispecific immunoliposomes can be prepared to improve the liposome to cell interaction by targeting multiple different targets at the same time, for instance by coupling two or more different ligands to the liposomal surface, resulting in a synergistic or additive increase in binding. An alternative approach is the use of bispecific ligands to address at least two different targets. For this purpose we cloned a single-chain diabody fragment (scDb`), a bispecific molecule targeting two antigens, endoglin (CD105) and fibroblast activation protein (FAP), expressed on cells of the tumor microenvironment. As model cell system, a human fibrosarcoma cell line was used expressing endoglin and FAP simultaneously. Monospecific immunoliposomes directed either against endoglin or FAP were compared in vitro for cell binding and cytotoxic activity with bispecific dual-targeted scFv`-IL (bispecific scFv`FAP/CD105-IL) and bispecific single-chain diabody`-IL (scDb`CD105/FAP-IL) targeting endoglin and FAP simultaneously. In the underlying study, bispecific scFv`FAP/CD105-IL interacted stronger with cells expressing FAP and endoglin (both targets simultaneously) compared to the monospecific immunoliposomes. Furthermore, bispecific scDb`-immunoliposomes increased the cell interaction massively and showed enhanced cytotoxicity against target cells using doxorubicin-loaded immunoliposomes. The use of recombinant bispecific ligands as scDb`-molecules facilitates the generation of bispecific immunoliposomes by using the established post-insertion technique, enabling an extension of the ligand specificity spectrum via genetic modification.

  12. Fibroblast growth factors 7 and 10 are involved in ameloblastoma proliferation via the mitogen-activated protein kinase pathway.

    Science.gov (United States)

    Nakao, Yu; Mitsuyasu, Takeshi; Kawano, Shintaro; Nakamura, Norifumi; Kanda, Shiori; Nakamura, Seiji

    2013-11-01

    Ameloblastoma is an epithelial benign tumor of the odontogenic apparatus and its growth mechanisms are not well understood. Fibroblast growth factor (FGF) 3, FGF7 and FGF10, which are expressed by the neural crest-derived ectomesenchymal cells, induce the proliferation of odontogenic epithelial cells during tooth development. Therefore, we examined the expression and function of these FGFs in ameloblastoma. We examined 32 cases of ameloblastoma as well as AM-1 cells (an ameloblastoma cell line) and studied the expression of FGF3, FGF7, FGF10 and their specific receptors, namely, FGF receptor (FGFR) 1 and FGFR2. Proliferation, mitogen-activated protein kinase (MAPK) signaling and PI3K signaling were examined in AM-1 cells after the addition of FGF7, FGF10 and these neutralizing antibodies. The expression of FGF7, FGF10, FGFR1 and FGFR2 was detected in ameloblastoma cells and AM-1 cells, while that of FGF3 was not. FGF7 and FGF10 stimulated AM-1 cell proliferation and phosphorylation of p44/42 MAPK. However, Akt was not phosphorylated. Blocking the p44/42 MAPK pathway by using a specific mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor (U0126) completely neutralized the effects of FGF7 and FGF10 on AM-1 cell proliferation. However, Anti FGF7 and FGF10 neutralizing antibodies did not decrease cell proliferation and MAPK phosphorylation of AM-1 cells. These results suggested that FGF7 and FGF10 are involved in the proliferation of ameloblastoma cells through the MAPK pathway.

  13. Interactions of the alveolar macrophage and the fibroblast: regulation of basal and silica-activated eicosanoid production

    Energy Technology Data Exchange (ETDEWEB)

    Kuhn, D.C.; Demers, L.M. [Pennsylvania State University College of Medicine, Hershey, PA (United States). Dept. of Pathology

    1996-07-01

    Intercellular communication between lung cells has been shown to modulate physiology and pathophysiology in the lung. In particular, it is apparent that the alveolar macrophage (AM) and the fibroblast (Fb) interact via soluble signalling molecules to mediate inflation and fibrosis in response to a variety of noxious substances. To characterize the interaction of the AM and Fb in the relative production of proinflammatory eicosanoids in the lung, we evaluated basal and silica-activated production of prostaglandin E{sub 2} (PGE{sub 2}), thromboxane A{sub 2} (TXA{sub 2}) and leukotriene B{sub 4} (LTB{sub 4}) by human AM and Fb alone and when cultured together. Unstimulated AM and Fb cultured alone produced equal amounts of PGE{sub 2}. However, AM produced 20-fold more TXA{sub 2} and 3-fold more LTB{sub 4} than Fb. When Am and Fb were cultured together, either separated by a membrane or in physical contact with one another, the total production of TXA{sub 2} and LTB{sub 4} by both cell types was reduced by 70 to 90% while production of PGE{sub 2} was unaffected. When AM were first exposed to silica dust and then cocultured with Fb, the total production of PGE{sub 2} was decreased while that of TXA{sub 2} and LTB{sub 4} was unchanged compared to coculture in the absence of silica. The results of these studies suggest that basal eicosanoid synthesis may be modulated by the interaction of the AM and the Fb and that this communication is via soluble substances. In addition, dust-activated AM produce soluble mediators that may further alter lung eicosanoid production as part of a mechanism to modulate dust-induced pathophysiology. 20 refs., 3 figs.

  14. 黑布药膏对兔耳增生性瘢痕成纤维细胞增殖与凋亡的影响%Effect of Heibu Ointment on Proliferative Activity and Apoptosis of Fibroblats in Hypertrophic Scarring Model in Rabbit Ears

    Institute of Scientific and Technical Information of China (English)

    赵丽; 王莹; 关洪全

    2011-01-01

    Objective:To investigate the effect of Heibu Ointment on proliferative activity and apoptosis of fibroblasts in the hypetrrophic scarring model in rabbit ears. Method: Twenty-four white rabbits were used to establish the model of hypertrophic scar in ears. After twenty-one days, the model rabbits were randomly divided into Heibu Ointment treatment group and model group. In Heibu Ointment treatment group, Heibu Ointment was plastered on the hypetrrophic scars once in every three days. The treatment course lasted for fifty-six days. The scar tissue was sampled on the second, the fourth, the sixth and the eighth week for the two groups. The changes in fibroblasts treated with Heibu Ointment were compared with that of the controls during the forming of hypertrophic scars. The expression of proliferation cell nuclear antigen (PCNA) was measured by immunohistochemistry technique, and the apoptosis was measured by TUNEL. Result: The expression of PCNA in the Heibu Ointment treatment group was obviously weaker than that of the controls ( P < 0.05 ), and the apoptosis was increased in Heibu Ointment treatment group (P < 0. 05 ). Conclusion: Heibu Ointment can inhibit the proliferation of fibroblasts and accelerate cell apoptosis in hypertrophic scars in rabbit ears.%目的:探讨黑布药膏对兔耳增生性瘢痕成纤维细胞增殖与凋亡的影响.方法:成年大耳白兔24只,建立兔耳增生性瘢痕动物模型,21 d后,将瘢痕动物模型随机分为黑布药膏治疗组和瘢痕模型组,在黑布药膏治疗组瘢痕局部涂抹黑布药膏,每3 d 1次,连续用药56 d.在用药第2,4,6,8周分别切取两组瘢痕组织,对比研究在瘢痕形成过程中黑布药膏对兔耳瘢痕成纤维细胞的影响,采用免疫组化方法检测增殖细胞核抗原(PCNA)蛋白表达和细胞凋亡的原位检测.结果:按不同时间段取材进行免疫组化染色,高倍镜下观察,结果显示:黑布药膏治疗组和模型对照组比

  15. Proliferative verrucous leukoplakia: An update.

    Science.gov (United States)

    Munde, Anita; Karle, Ravindra

    2016-01-01

    Proliferative verrucous leukoplakia (PVL) is a rare form of oral leukoplakia, which was first described in 1985 by Hansen et al. Since then, various published case series have presented PVL as a disease with aggressive biological behavior due to its high probability of recurrence and a high rate of malignant transformation, usually higher than 70%. PVL is a long-term progressive condition, which is observed more frequently in elderly women, over 60 years at the time of diagnosis. The buccal mucosa and tongue are the most frequently involved sites. It develops initially as a white plaque of hyperkeratosis that eventually becomes a multifocal disease with confluent, exophytic and proliferative features with a progressive deterioration of the lesions, making it more and more difficult to control. Tobacco use does not seem to have a significant influence on the appearance or progression of PVL and may occur both in smokers and nonsmokers. Prognosis is poor for this seemingly harmless-appearing white lesion of the oral mucosa. At present, the etiology of PVL remains unclear as well as its management and diagnosis, which is still retrospective, late and poorly defined, lacking consensus criteria. This short review discusses the clinical and histopathological features, diagnosis, traditional treatment and the current management of the disease.

  16. Agent based modelling helps in understanding the rules by which fibroblasts support keratinocyte colony formation.

    Directory of Open Access Journals (Sweden)

    Tao Sun

    Full Text Available BACKGROUND: Autologous keratincoytes are routinely expanded using irradiated mouse fibroblasts and bovine serum for clinical use. With growing concerns about the safety of these xenobiotic materials, it is desirable to culture keratinocytes in media without animal derived products. An improved understanding of epithelial/mesenchymal interactions could assist in this. METHODOLOGY/PRINCIPAL FINDINGS: A keratincyte/fibroblast o-culture model was developed by extending an agent-based keratinocyte colony formation model to include the response of keratinocytes to both fibroblasts and serum. The model was validated by comparison of the in virtuo and in vitro multicellular behaviour of keratinocytes and fibroblasts in single and co-culture in Greens medium. To test the robustness of the model, several properties of the fibroblasts were changed to investigate their influence on the multicellular morphogenesis of keratinocyes and fibroblasts. The model was then used to generate hypotheses to explore the interactions of both proliferative and growth arrested fibroblasts with keratinocytes. The key predictions arising from the model which were confirmed by in vitro experiments were that 1 the ratio of fibroblasts to keratinocytes would critically influence keratinocyte colony expansion, 2 this ratio needed to be optimum at the beginning of the co-culture, 3 proliferative fibroblasts would be more effective than irradiated cells in expanding keratinocytes and 4 in the presence of an adequate number of fibroblasts, keratinocyte expansion would be independent of serum. CONCLUSIONS: A closely associated computational and biological approach is a powerful tool for understanding complex biological systems such as the interactions between keratinocytes and fibroblasts. The key outcome of this study is the finding that the early addition of a critical ratio of proliferative fibroblasts can give rapid keratinocyte expansion without the use of irradiated mouse

  17. Peroxiredoxin 5 Protects TGF-β Induced Fibrosis by Inhibiting Stat3 Activation in Rat Kidney Interstitial Fibroblast Cells.

    Directory of Open Access Journals (Sweden)

    Hoon-In Choi

    Full Text Available Renal fibrosis is a common final pathway of end-stage kidney disease which is induced by aberrant accumulation of myofibroblasts. This process is triggered by reactive oxygen species (ROS and proinflammatory cytokines generated by various source of injured kidney cells. Peroxiredoxin 5 (Prdx5 is a thiol-dependent peroxidase that reduces oxidative stress by catalyzing intramolecular disulfide bonds. Along with its antioxidant effects, expression level of Prdx5 also was involved in inflammatory regulation by immune stimuli. However, the physiological effects and the underlying mechanisms of Prdx5 in renal fibrosis have not been fully characterized. Sprague-Dawley rats were subjected to unilateral ureteral obstruction (UUO for 1 or 7 days. For the in vitro model, NRK49F cells, a rat kidney interstitial fibroblast cell lines, were treated with transforming growth factor β (TGF-β for 0, 1, 3, or 5 days. To access the involvement of its peroxidase activity in TGF-β induced renal fibrosis, wild type Prdx5 (WT and double mutant Prdx5 (DM, converted two active site cysteines at Cys 48 and Cys 152 residue to serine, were transiently expressed in NRK49F cells. The protein expression of Prdx5 was reduced in UUO kidneys. Upregulation of fibrotic markers, such as fibronectin and alpha-smooth muscle actin (α-SMA, declined at 5 days in time point of higher Prdx5 expression in TGF-β treated NRK49F cells. The overexpression of wild type Prdx5 by transient transfection in NRK49F cells attenuated the TGF-β induced upregulation of fibronectin and α-SMA. On the other hand, the transient transfection of double mutant Prdx5 did not prevent the activation of fibrotic markers. Overexpression of Prdx5 also suppressed the TGF-β induced upregulation of Stat3 phosphorylation, while phosphorylation of Smad 2/3 was unchanged. In conclusion, Prdx5 protects TGF-β induced fibrosis in NRK49F cells by modulating Stat3 activation in a peroxidase activity dependent manner.

  18. Blocking the class I histone deacetylase ameliorates renal fibrosis and inhibits renal fibroblast activation via modulating TGF-beta and EGFR signaling.

    Science.gov (United States)

    Liu, Na; He, Song; Ma, Li; Ponnusamy, Murugavel; Tang, Jinhua; Tolbert, Evelyn; Bayliss, George; Zhao, Ting C; Yan, Haidong; Zhuang, Shougang

    2013-01-01

    Histone deacetylase (HDAC) inhibitors are promising anti-fibrosis drugs; however, nonselective inhibition of class I and class II HDACs does not allow a detailed elucidation of the individual HDAC functions in renal fibrosis. In this study, we investigated the effect of MS-275, a selective class I HDAC inhibitor, on the development of renal fibrosis in a murine model of unilateral ureteral obstruction (UUO) and activation of cultured renal interstitial fibroblasts. The UUO model was established by ligation of the left ureter and the contralateral kidney was used as a control. At seven days after UUO injury, kidney developed fibrosis as indicated by deposition of collagen fibrils and increased expression of collagen I, fibronectin and alpha-smooth muscle actin (alpha-SMA). Administration of MS-275 inhibited all these fibrotic responses and suppressed UUO-induced production of transforming growth factor-beta1 (TGF-beta), increased expression of TGF-beta receptor I, and phosphorylation of Smad-3. MS-275 was also effective in suppressing phosphorylation and expression of epidermal growth factor receptor (EGFR) and its downstream signaling molecule, signal transducer and activator of transcription-3. Moreover, class I HDAC inhibition reduced the number of renal tubular cells arrested in the G2/M phase of the cell cycle, a cellular event associated with TGF-beta1overproduction. In cultured renal interstitial fibroblasts, MS-275 treatment inhibited TGF-beta induced phosphorylation of Smad-3, differentiation of renal fibroblasts to myofibroblasts and proliferation of myofibroblasts. These results demonstrate that class I HDACs are critically involved in renal fibrogenesis and renal fibroblast activation through modulating TGF-beta and EGFR signaling and suggest that blockade of class I HDAC may be a useful treatment for renal fibrosis.

  19. Blocking the class I histone deacetylase ameliorates renal fibrosis and inhibits renal fibroblast activation via modulating TGF-beta and EGFR signaling.

    Directory of Open Access Journals (Sweden)

    Na Liu

    Full Text Available BACKGROUND: Histone deacetylase (HDAC inhibitors are promising anti-fibrosis drugs; however, nonselective inhibition of class I and class II HDACs does not allow a detailed elucidation of the individual HDAC functions in renal fibrosis. In this study, we investigated the effect of MS-275, a selective class I HDAC inhibitor, on the development of renal fibrosis in a murine model of unilateral ureteral obstruction (UUO and activation of cultured renal interstitial fibroblasts. METHODS/FINDINGS: The UUO model was established by ligation of the left ureter and the contralateral kidney was used as a control. At seven days after UUO injury, kidney developed fibrosis as indicated by deposition of collagen fibrils and increased expression of collagen I, fibronectin and alpha-smooth muscle actin (alpha-SMA. Administration of MS-275 inhibited all these fibrotic responses and suppressed UUO-induced production of transforming growth factor-beta1 (TGF-beta, increased expression of TGF-beta receptor I, and phosphorylation of Smad-3. MS-275 was also effective in suppressing phosphorylation and expression of epidermal growth factor receptor (EGFR and its downstream signaling molecule, signal transducer and activator of transcription-3. Moreover, class I HDAC inhibition reduced the number of renal tubular cells arrested in the G2/M phase of the cell cycle, a cellular event associated with TGF-beta1overproduction. In cultured renal interstitial fibroblasts, MS-275 treatment inhibited TGF-beta induced phosphorylation of Smad-3, differentiation of renal fibroblasts to myofibroblasts and proliferation of myofibroblasts. CONCLUSIONS AND SIGNIFICANCE: These results demonstrate that class I HDACs are critically involved in renal fibrogenesis and renal fibroblast activation through modulating TGF-beta and EGFR signaling and suggest that blockade of class I HDAC may be a useful treatment for renal fibrosis.

  20. Effect of transforming growth factor-beta on activity of connective tissue growth factor gene promoter in mouse NIH/3T3 fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Qing ZHAO; Nan CHEN; Wei-ming WANG; Jian LU; Bing-bing DAI

    2004-01-01

    AIM: To investigate the regulatory mechanism of transforming growth factor-beta on activity of connective tissue growth factor promoter in mouse NIH/3T3 fibroblasts. METHODS: The regulation fragment of the 5' flanking region of the human CTGF gene was linked to pGL3-Basic vector, a firefly luciferase reporter construct without promoter. The recombinant plasmid pCTGF-luc was transiently transfected to NIH/3T3 fibroblasts. The activity of CTGF promoter after treatment of TGF-β1 and MAPK pathway inhibitors were assayed with luciferase reporter gene assay system. RESULTS: TGF-β1-induced increase of CTGF promoter activity was concentration-dependent,with a plateau at 5 μg/L by 2.67-fold vs control (P<0.05). The TGF-β1 stimulation of CTGF promoter activity was time-dependent, too. After exposure to TGF-β1 (5 μg/L), the maximal level of luciferase activity was reached at 12 h and maintained to 24 h by 2.76- and 2.20-fold vs control, respectively (P<0.05). Blockade of mitogen-activated protein kinases (MAPK) pathway with PD98059 (10 μmnol/L), the MAP kinase kinase 1 inhibitor, and SB203580 (10μmol/L), the p38 MAP kinase inhibitor, decreased basal and TGF-β1-induced activation of CTGF promoter. However,inhibition of c-Jun-N-terminal kinase/stress-activated protein kinase by SP600125 (20 μmol/L) was without effect.CONCLUSION: TGF-β1 stimulated the transcriptional activity of CTGF gene promoter in NIH/3T3 fibroblasts in a dose- and time-dependent manner. MAPK pathway may play a role in the regulation of TGF-β1-induced CTGF expression.

  1. Estrogen signaling in the proliferative endometrium: implications in endometriosis

    Directory of Open Access Journals (Sweden)

    Rita de Cássia Pereira da Costa e Silva

    2016-02-01

    Full Text Available SUMMARY Even though the physiological role of estrogen in the female reproductive cycle and endometrial proliferative phase is well established, the signaling pathways by which estrogen exerts its action in the endometrial tissue are still little known. In this regard, advancements in cell culture techniques and maintenance of endometrial cells in cultures enabled the discovery of new signaling mechanisms activated by estrogen in the normal endometrium and in endometriosis. This review aims to present the recent findings in the genomic and non-genomic estrogen signaling pathways in the proliferative human endometrium specifically associated with the pathogenesis and development of endometriosis.

  2. Effects of basic fibroblast growth factor on superoxide dismutase activity and malondialdehyde content in the rat brain following intracerebral hemorrhage

    Institute of Scientific and Technical Information of China (English)

    Hongqiao Wei; Juen Huang; Junjie Huang; Bing Li; Qianming Li

    2008-01-01

    BACKGROUND: Studies have confirmed that basic fibroblast growth factor (bFGF) promotes neuronal survival and neurite outgrowth. OBJECTIVE: To compare and verify the effects of bFGF on superoxide dismutase activity and malondialdehyde content in rat brain tissues surrounding a hemorrhagic lesion, as well as the hippocampus at the hemorrhagic side. DESIGN, TIME AND SETTING: The randomized, controlled, neurobiological study was performed at the Science Experimental Center and Research Laboratory, Guangxi Medical University, China, from September to December 2006. MATERIALS: Ninety-two adult, healthy, Wistar rats of equal gender were used to establish intraeerebral hemorrhage by infusing type VII collagenase into the left internal capsule. Type Ⅶ collagenase (Sigma, USA), superoxide dismutase and malondialdehyde kits (Jiancheng, China), and bFGF (Institute of Bioengineering, Ji'nan University, China) were used for this study. METHODS: Ninety successfully lesioned rats were equally and randomly divided into three groups. Rats in the bFGF group were intramuscularly injected daily with bFGF (8μg/kg). Rats in the saline control group received an equal volume of saline. The rats in the model group did not receive other interventions. Superoxide dismutase activity was measured using the xanthine oxidase method. Malondialdehyde contents were detected using the thiobarbituric acid method. MAIN OUTCOME MEASURES: At 1, 3, and 7 days following intracerebral hemorrhage, superoxide dismutase and malondialdehyde were determined in the brain tissue surrounding the hematoma and in the hippocampus in the affected hemisphere. RESULTS: In brain tissue surrounding the hematoma, superoxide dismutase activity was significantly increased in the bFGF group at 3 and 7 days after intracerebral hemorrhage compared with the saline control group, whereas malondialdehyde content was significantly decreased (P 0.05). CONCLUSION: Increased superoxide dismutase activity and decreased

  3. HCV derived from sera of HCV-infected patients induces pro-fibrotic effects in human primary fibroblasts by activating GLI2

    Science.gov (United States)

    Granato, M.; Zompetta, C.; Vescarelli, E.; Rizzello, C.; Cardi, A.; Valia, S.; Antonelli, G.; Marchese, C.; Torrisi, M. R.; Faggioni, A.; Cirone, M.

    2016-01-01

    Hepatitis C virus (HCV) infection is a leading cause of liver fibrosis, especially in developing countries. The process is characterized by the excess accumulation of ECM that may lead, over time, to hepatic cirrhosis, liver failure and also to hepatocarcinoma. The direct role of HCV in promoting fibroblasts trans-differentiation into myofibroblasts, the major fibrogenic cells, has not been fully clarified. In this study, we found that HCV derived from HCV-infected patients infected and directly induced the trans-differentiation of human primary fibroblasts into myofibroblasts, promoting fibrogenesis. This effect correlated with the activation of GLI2, one of the targets of Hedgehog signaling pathway previously reported to be involved in myofibroblast generation. Moreover, GLI2 activation by HCV correlated with a reduction of autophagy in fibroblasts, that may further promoted fibrosis. GLI2 inhibition by Gant 61 counteracted the pro-fibrotic effects and autophagy inhibition mediated by HCV, suggesting that targeting HH/GLI2 pathway might represent a promising strategy to reduce the HCV-induced fibrosis. PMID:27476557

  4. Salvianolic Acid B Protects Normal Human Dermal Fibroblasts Against Ultraviolet B Irradiation-Induced Photoaging Through Mitogen-Activated Protein Kinase and Activator Protein-1 Pathways.

    Science.gov (United States)

    Sun, Zhengwang; Park, Sang-Yong; Hwang, Eunson; Zhang, Mengyang; Jin, Fengxie; Zhang, Baochun; Yi, Tae Hoo

    2015-01-01

    Exposure to ultraviolet (UV) light causes increased matrix metalloproteinase (MMP) activity and decreased collagen synthesis, leading to skin photoaging. Salvianolic acid B (SAB), a polyphenol, was extracted and purified from salvia miltiorrhiza. We assessed effects of SAB on UVB-induced photoaging and investigated its molecular mechanism of action in UVB-irradiated normal human dermal fibroblasts. Our results show that SAB significantly inhibited the UVB-induced expression of metalloproteinases-1 (MMP-1) and interleukin-6 (IL-6) while promoting the production of type I procollagen and transforming growth factor β1 (TGF-β1). Moreover, treatment with SAB in the range of 1-100 μg/mL significantly inhibited UVB-induced extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 phosphorylation, which resulted in decreasing UVB-induced phosphorylation of c-Fos and c-Jun. These results indicate that SAB downregulates UV-induced MMP-1 expression by inhibiting Mitogen-activated protein kinase (MAPK) signaling pathways and activator protein-1 (AP-1) activation. Our results suggest a potential use for SAB in skin photoprotection.

  5. Pirfenidone attenuates IL-1β-induced COX-2 and PGE2 production in orbital fibroblasts through suppression of NF-κB activity.

    Science.gov (United States)

    Choi, Youn-Hee; Back, Keum Ok; Kim, Hee Ja; Lee, Sang Yeul; Kook, Koung Hoon

    2013-08-01

    The aim of this study was to determine the effect of pirfenidone on interleukin (IL)-1β-induced cyclooxygenase (COX)-2 and prostaglandin (PG)E2 expression in orbital fibroblasts from patients with thyroid-associated ophthalmopathy (TAO). Primary cultures of orbital fibroblasts from patients with TAO (n = 4) and non-TAO subjects (n = 4) were prepared. The level of PGE2 in orbital fibroblasts treated with IL-1β in the presence or absence of pirfenidone was measured using an enzyme-linked immunosorbent assay. The effect of pirfenidone on IL-1β-induced COX-2 expression in orbital fibroblasts from patients with TAO was evaluated by reverse transcription-polymerase chain reaction (PCR) and quantitative real-time PCR analyses, and verified by Western blot. Activation of nuclear factor-κB (NF-κB) was evaluated by immunoblotting for inhibitor of κB (IκB)α and phosphorylated IκBα, and DNA-binding activity of p50/p65 NF-κB was analyzed by electrophoretic mobility shift assay. In addition, IL-1 receptor type 1 (IL-1R1) expression was assessed by RT-PCR in IL-1β-treated cells with or without pirfenidone. Pirfenidone significantly attenuated IL-1β-induced PGE2 release in both TAO and non-TAO cells. IL-1β-induced COX-2 mRNA and protein expression decreased significantly following co-treatment with pirfenidone. IL-1β-induced IκBα phosphorylation and degradation decreased in the presence of pirfenidone and led to decreased nuclear translocation and DNA binding of the active NF-κB complex. In our system, neither IL-1β nor pirfenidone co-treatment influenced IL-1R1 expression. Our results suggest that pirfenidone attenuates the IL-1β-induced PGE2/COX-2 production in TAO orbital fibroblasts, which is related with suppression of the NF-κB activation.

  6. Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts

    DEFF Research Database (Denmark)

    Su, J; Muranjan, M; Sap, J

    1999-01-01

    BACKGROUND: Fyn and c-Src are two of the most widely expressed Src-family kinases. Both are strongly implicated in the control of cytoskeletal organization and in the generation of integrin-dependent signalling responses in fibroblasts. These proteins are representative of a large family of tyros...

  7. Rapid mitogen-activated protein kinase by basic fibroblast growth factor in rat intestin after ischemia/reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Xiao-Bing Fu; Yin-Hui Yang; Tong-Zhu Sun; Wei Chen; Jun-You Li; Zhi-Yong Sheng

    2003-01-01

    AIM: Previous studies showed that exogenous basic fibroblast growth factor (bFGF or FGF-2) could improve physiological dysfunction after intestinal ischemia/reperfusion (I/R) injury. However, the mechanisms of this protective effect of bFGF are still unclear. The present study was to detect the effect of bFGF on the activities of mitogen-activated protein kinase (MlAPK) signaling pathway in rat intestine after I/R injury, and to investigate the protective mechanisms of bFGF on intestinal ischemia injury. METttODS: Rat intestinal I/R injury was produced by clamping the superior mesenteric artery (SMA) for 45minutes and followed by repeffusion for 48 hours. Seventyeight Wistar rats were used and divided randomly into sham-operated group (A), normal saline control group (B),bFGF antibody pre-treated group (C), and bFGF treated group (D). Tn group A, SMA was separated without occlusion. In groups B, C and D, SMA was separated and occluded for 45 minutes, then, released for reperfusion for 48 hours. After the animals were sacrificed, blood and tissue samples were taken from the intestine 45 minutes after ischemia in group A and 2, 6, 24, and 48 hours after reperfusion in the other groups. Phosphorylated forms of p42/p44 MAPK, p38 MAPK and stress activated protein kinase/C-Jun N-terminal kinase (SAPK/JNK) were measured by immunohistochemistry. Plasma levels of D-lactate were examined and histological changes were observed under the light microscope. RESULTS: Intestinal I/R injury induced the expression of p42/p44 MAPK, p38 MAPK, and SAPK/JNK pathways and exogenous bFGF stimulated the early activation of p42/p44 MAPK and p38 MlAPK pathways. The expression of phosphorylated forms of p42/p44 MAPK was primarily localized in the nuclei of crypt cells and in the cytoplasm and nuclei of villus cells. The positive expression of p38MAPK was localized mainly in the nuclei of crypt cells, very few in villus cells. The activities of p42/p44 MAPK and p38MAPK peaked 6 hours after

  8. Ligand-activated PPARδ upregulates α-smooth muscle actin expression in human dermal fibroblasts: A potential role for PPARδ in wound healing.

    Science.gov (United States)

    Ham, Sun Ah; Hwang, Jung Seok; Yoo, Taesik; Lee, Won Jin; Paek, Kyung Shin; Oh, Jae-Wook; Park, Chan-Kyu; Kim, Jin-Hoi; Do, Jung Tae; Kim, Jae-Hwan; Seo, Han Geuk

    2015-12-01

    The phenotypic changes that accompany differentiation of resident fibroblasts into myofibroblasts are important aspects of the wound healing process. Recent studies showed that peroxisome proliferator-activated receptor (PPAR) δ plays a critical role in wound healing. To determine whether the nuclear receptor PPARδ can modulate the differentiation of human dermal fibroblasts (HDFs) into myofibroblasts. These studies were undertaken in primary HDFs using Western blot analyses, small interfering (si)RNA-mediated gene silencing, reporter gene assays, chromatin immunoprecipitation (ChIP), migration assays, collagen gel contraction assays, and real-time PCR. Activation of PPARδ by GW501516, a specific ligand of PPARδ, specifically upregulated the myofibroblast marker α-smooth muscle actin (α-SMA) in a time- and concentration-dependent manner. This induction was significantly inhibited by the presence of siRNA against PPARδ, indicating that PPARδ is involved in myofibroblast transdifferentiation of HDFs. Ligand-activated PPARδ increased α-SMA promoter activity in a dual mode by directly binding a direct repeat-1 (DR1) site in the α-SMA promoter, and by inducing expression of transforming growth factor (TGF)-β, whose downstream effector Smad3 interacts with a Smad-binding element (SBE) in another region of the promoter. Mutations in these cis-elements totally abrogated transcriptional activation of the α-SMA gene by the PPARδ ligand; thus both sites represent novel types of PPARδ response elements. GW501516-activated PPARδ also increased the migration and contractile properties of HDFs, as demonstrated by Transwell and collagen lattice contraction assays, respectively. In addition, PPARδ-mediated upregulation of α-SMA was correlated with elevated expression of myofibroblast markers such as collagen I and fibronectin, with a concomitant reduction in expression of the epithelial marker E-cadherin. PPARδ plays pivotal roles in wound healing by promoting

  9. Induction of tissue inhibitor of matrix metalloproteinase-2 by cholesterol depletion leads to the conversion of proMMP-2 into active MMP-2 in human dermal fibroblasts.

    Science.gov (United States)

    Kim, Sangmin; Oh, Jang-Hee; Lee, Youngae; Lee, Jeongyoon; Cho, Kwang Hyun; Chung, Jin Ho

    2010-01-31

    Cholesterol is one of major components of cell membrane and plays a role in vesicular trafficking and cellular signaling. We investigated the effects of cholesterol on matrix metalloproteinase-2 (MMP-2) activation in human dermal fibroblasts. We found that tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) expression and active form MMP-2 (64 kD) were dose-dependently increased by methyl-beta-cyclodextrin (MbetaCD), a cholesterol depletion agent. In contrast, cholesterol depletion-induced TIMP-2 expression and MMP-2 activation were suppressed by cholesterol repletion. Then we investigated the regulatory mechanism of TIMP-2 expression by cholesterol depletion. We found that the phosphorylation of JNK as well as ERK was significantly increased by cholesterol depletion. Moreover, cholesterol depletion-induced TIMP-2 expression and MMP-2 activation was significantly decreased by MEK inhibitor U0126, and JNK inhibitor SP600125, respectively. While a low dose of recombinant TIMP-2 (100 ng/ml) increased the level of active MMP-2 (64 kD), the high dose of TIMP-2 (>or=200 ng/ml) decreased the level of active MMP-2 (64 kD). Taken together, we suggest that the induction of TIMP-2 by cholesterol depletion leads to the conversion of proMMP-2 (72 kD) into active MMP-2 (64 kD) in human dermal fibroblasts.

  10. Activation of α1A-adrenergic receptor promotes differentiation of rat-1 fibroblasts to a smooth muscle-like phenotype

    Directory of Open Access Journals (Sweden)

    Malik Kafait U

    2004-12-01

    Full Text Available Abstract Background Fibroblasts, as connective tissue cells, are able to transform into another cell type including smooth muscle cells. α1A-adrenergic receptor (α1A-AR stimulation in rat-1 fibroblasts is coupled to cAMP production. However, the significance of an increase in cAMP produced by α1A-AR stimulation on proliferation, hypertrophy and differentiation in these cells is not known. Results Activation of the α1A-AR in rat-1 fibroblasts by phenylephrine (PE inhibited DNA synthesis by 67% and blocked the re-entry of 81% of the cells into S phase of the cell cycle. This cell cycle blockage was associated with hypertrophy characterized by an increase in protein synthesis (64% and cell size. Elevation of cAMP levels decreased both DNA and protein synthesis. Inhibition of adenylyl cyclase or protein kinase A reversed the antiproliferative effect of cAMP analogs but not PE; the hypertrophic effect of PE was also not altered. The functional response of rat-1 cells to PE was accompanied by increased expression of cyclin-dependent kinase (Cdk inhibitors p27kip1 and p21cip1/waf1, which function as negative regulators of the cell cycle. Stimulation of α1A-AR also upregulated the cell cycle regulatory proteins pRb, cyclin D1, Cdk 2, Cdk 4, and proliferating cell nuclear antigen. The antiproliferative effect of PE was blocked by p27kip1 antisense but not sense oligonucleotide. PE also promoted expression of smooth muscle cell differentiation markers (smooth muscle alpha actin, caldesmon, and myosin heavy chain as well as the muscle development marker MyoD. Conclusions Stimulation of α1A-AR promotes cell cycle arrest, hypertrophy and differentiation of rat-1 fibroblasts into smooth muscle-like cells and expression of negative cell cycle regulators by a mechanism independent of the cAMP/PKA signaling pathway.

  11. Tissue-specific Expression of βKlotho and Fibroblast Growth Factor (FGF) Receptor Isoforms Determines Metabolic Activity of FGF19 and FGF21*†

    OpenAIRE

    Kurosu, Hiroshi; Choi, Mihwa; Ogawa, Yasushi; Dickson, Addie S.; Goetz, Regina; Eliseenkova, Anna V.; Mohammadi, Moosa; Rosenblatt, Kevin P.; Kliewer, Steven A.; Kuro-o, Makoto

    2007-01-01

    The fibroblast growth factor (FGF) 19 subfamily of ligands, FGF19, FGF21, and FGF23, function as hormones that regulate bile acid, fatty acid, glucose, and phosphate metabolism in target organs through activating FGF receptors (FGFR1–4). We demonstrated that Klotho and βKlotho, homologous single-pass transmembrane proteins that bind to FGFRs, are required for metabolic activity of FGF23 and FGF21, respectively. Here we show that, like FGF21, FGF19 also requires βKlotho. Both FGF19 and FGF21 c...

  12. Rheumatoid arthritis and pigmented villonodular synovitis: comparative analysis of cell polyploidy, cell cycle phases and expression of macrophage and fibroblast markers in proliferating synovial cells.

    Science.gov (United States)

    Berger, I; Weckauf, H; Helmchen, B; Ehemann, V; Penzel, R; Fink, B; Bernd, L; Autschbach, F

    2005-05-01

    Rheumatoid arthritis (RA) and pigmented villonodular synovitis (PVNS) are aggressive diseases with progressive joint destruction. The present study aims to define cell cycle phases, polyploidy and the immunophenotype of proliferating synovial cells in both diseases. Synovial tissues from patients with proliferative-active RA, localized and diffuse PVNS were analysed by DNA flow cytometry, immunohistochemistry and double immunofluorescence with confocal laser scan microscopy. Expression of macrophage markers (CD68/CD163), fibroblast markers (h4Ph/CD55) and Ki67 antigen was examined. Synovial cells positive for either macrophage or fibroblast markers as well as double-labelled cells were found in both RA and PVNS. In RA, CD68/CD163+ synoviocytes were preferentially located in the vicinity of the synovial lining layer, while they were more randomly distributed in PVNS. Of cases with diffuse PVNS, 20% showed an aneuploid cell pattern. All samples of localized PVNS and RA were diploid. Proliferative activity was significantly higher in aneuploid PVNS. In spite of their histologically homogeneous appearance, proliferating synovial cells display a heterogeneous immunophenotype in both RA and PVNS, indicating functional properties of both macrophages and fibroblasts. Aneuploidy seems to be a special feature of diffuse PVNS.

  13. Cell competition in mouse NIH3T3 embryonic fibroblasts is controlled by the activity of Tead family proteins and Myc.

    Science.gov (United States)

    Mamada, Hiroshi; Sato, Takashi; Ota, Mitsunori; Sasaki, Hiroshi

    2015-02-15

    Cell competition is a short-range communication originally observed in Drosophila. Relatively little is known about cell competition in mammals or in non-epithelial cells. Hippo signaling and its downstream transcription factors of the Tead family, control cell proliferation and apoptosis. Here, we established an in vitro model system that shows cell competition in mouse NIH3T3 embryo fibroblast cells. Co-culture of Tead-activity-manipulated cells with normal (wild-type) cells caused cell competition. Cells with reduced Tead activity became losers, whereas cells with increased Tead activity became super-competitors. Tead directly regulated Myc RNA expression, and cells with increased Myc expression also became super-competitors. At low cell density, cell proliferation required both Tead activity and Myc. At high cell density, however, reduction of either Tead activity or Myc was compensated for by an increase in the other, and this increase was sufficient to confer 'winner' activity. Collectively, NIH3T3 cells have cell competition mechanisms similar to those regulated by Yki and Myc in Drosophila. Establishment of this in vitro model system should be useful for analyses of the mechanisms of cell competition in mammals and in fibroblasts. © 2015. Published by The Company of Biologists Ltd.

  14. Number and proliferative capacity of osteogenic stem cells are maintained during aging and in patients with osteoporosis

    DEFF Research Database (Denmark)

    Dokkedahl, Karin Stenderup; Justesen, J; Eriksen, E F

    2001-01-01

    Decreased bone formation is an important pathophysiological mechanism responsible for bone loss associated with aging and osteoporosis. Osteoblasts (OBs), originate from mesenchymal stem cells (MSCs) that are present in the bone marrow and form colonies (termed colony-forming units-fibroblastic [......Decreased bone formation is an important pathophysiological mechanism responsible for bone loss associated with aging and osteoporosis. Osteoblasts (OBs), originate from mesenchymal stem cells (MSCs) that are present in the bone marrow and form colonies (termed colony-forming units......-fibroblastic [CFU-Fs]) when cultured in vitro. To examine the effect of aging and osteoporosis on the MSC population, we quantified the number of MSCs and their proliferative capacity in vitro. Fifty-one individuals were studied: 38 normal volunteers (23 young individuals [age, 22-44 years] and 15 old individuals...... [age, 66-74 years]) and 13 patients with osteoporosis (age, 58-83 years). Bone marrow was aspirated from iliac crest; mononuclear cells were enriched in MSCs by magnetic activated cell sorting (MACS) using STRO-1 antibody. Total CFU-F number, size distribution, cell density per CFU-F, number...

  15. Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor

    Directory of Open Access Journals (Sweden)

    Thiel Gerald

    2009-05-01

    Full Text Available Abstract Background The serine protease thrombin catalyzes fibrin clot formation by converting fibrinogen into fibrin. Additionally, thrombin stimulation leads to an activation of stimulus-responsive transcription factors in different cell types, indicating that the gene expression pattern is changed in thrombin-stimulated cells. The objective of this study was to analyze the signaling cascade leading to the expression of the zinc finger transcription factor Egr-1 in thrombin-stimulated lung fibroblasts. Results Stimulation of 39M1-81 fibroblasts with thrombin induced a robust and transient biosynthesis of Egr-1. Reporter gene analysis revealed that the newly synthesized Egr-1 was biologically active. The signaling cascade connecting thrombin stimulation with Egr-1 gene expression required elevated levels of cytosolic Ca2+, the activation of diacylgycerol-dependent protein kinase C isoenzymes, and the activation of extracellular signal-regulated protein kinase (ERK. Stimulation of the cells with thrombin triggered the phosphorylation of the transcription factor Elk-1. Expression of a dominant-negative mutant of Elk-1 completely prevented Egr-1 expression in stimulated 39M1-81 cells, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of protease-activated receptors with transcription of the Egr-1 gene. Lentiviral-mediated expression of MAP kinase phosphatase-1, a dual-specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, prevented Elk-1 phosphorylation and Egr-1 biosynthesis in thrombin stimulated 39M1-81 cells, confirming the importance of nuclear ERK and Elk-1 for the upregulation of Egr-1 expression in thrombin-stimulated lung fibroblasts. 39M1-81 cells additionally express M1 muscarinic acetylcholine receptors. A comparison between the signaling cascades induced by thrombin or carbachol showed no differences, except that signal transduction via M

  16. Proliferative and inflammatory factors in the vitreous of patients with proliferative diabetic retinopathy

    Directory of Open Access Journals (Sweden)

    V V Chernykh

    2015-01-01

    Full Text Available Purpose: The purpose was to measure the concentrations of various cytokines and growth factors (including vascular endothelial growth factor [VEGF] and pigment epithelium-derived factor [PEDF] in the vitreous of patients with proliferative diabetic retinopathy (PDR and to investigate interaction between inflammatory and proliferative factors in the genesis of PDR. Materials and Methods : Vitreous samples from 32 eyes with PDR and 25 eyes without diabetes mellitus and signs of DR (control were collected. Vitreous concentrations of VEGF, PEDF, monocyte chemotactic protein-1 (MCP-1, interleukin-4 (IL-4, IL-6, IL-8, IL-10, IL-17A, and secretory immunoglobulin A (sIgA were simultaneously measured using enzyme-linked immunoassay. Results : Vitreous levels of VEGF, PEDF, IL-17A, IL-6, IL-8, IL-4, and sIgA were significantly (Π < 0.05 higher in eyes with PDR compared to control. The concentration of VEGF was more than 17-times higher than in control, and the concentration of PEDF was not changed oppositely and was also higher (1.45-times compared to control, that may indicate disturbances of compensatory mechanisms in angiogenesis regulation in PDR. Significant (Π < 0.05 positive correlations were observed between vitreous concentrations of VEGF and IL-17ΐ (r = 0.45, VEGF and IL-8 (r = 0.48, VEGF and IL-4 (r = 0.51, PEDF and IL-17ΐ (r = 0.48, PEDF and IL-8 (r = 0.59, MCP-1 and PEDF (r = 0.72, MCP-1 and IL-8 (r0 = 0.45, IL-4 and IL-17ΐ (r = 0.65, IL-4 and IL-8 (r = 0.71, IL-8 and IL-17ΐ (r = 0.59. Conclusions: Significantly raised levels of inflammatory and proliferative factors and numerous positive correlations between them may demonstrate a significant role of activation of vascular proliferation and local inflammation in the pathogenesis of PDR.

  17. Constitutive Macropinocytosis in Oncogene-transformed Fibroblasts Depends on Sequential Permanent Activation of Phosphoinositide 3-Kinase and Phospholipase C

    OpenAIRE

    Amyere, Mustapha; Payrastre, Bernard; Krause, Ulrike; Van Der Smissen, Patrick; Veithen, Alex; Courtoy, Pierre J

    2000-01-01

    Macropinocytosis results from the closure of lamellipodia generated by membrane ruffling, thereby reflecting cortical actin dynamics. Both transformation of Rat-1 fibroblasts by v-Src or K-Ras and stable transfection for expression of dominant-positive, wild-type phosphoinositide 3-kinase (PI3K) regulatory subunit p85α constitutively led to stress fiber disruption, cortical actin recruitment, extensive ruffling, and macropinosome formation, as measured by a selecti...

  18. Constitutive macropinocytosis in oncogene-transformed fibroblasts depends on sequential permanent activation of phosphoinositide 3-kinase and phospholipase C.

    OpenAIRE

    Amyere, Mustapha; Payrastre, B.; Krause, U.; Van Der Smissen, Patrick; Veithen, A.; Courtoy, Pierre J

    2000-01-01

    Macropinocytosis results from the closure of lamellipodia generated by membrane ruffling, thereby reflecting cortical actin dynamics. Both transformation of Rat-1 fibroblasts by v-Src or K-Ras and stable transfection for expression of dominant-positive, wild-type phosphoinositide 3-kinase (PI3K) regulatory subunit p85 alpha constitutively led to stress fiber disruption, cortical actin recruitment, extensive ruffling, and macropinosome formation, as measured by a selective acceleration of flui...

  19. Cardiac fibroblast in development and wound healing.

    Science.gov (United States)

    Deb, Arjun; Ubil, Eric

    2014-05-01

    Cardiac fibroblasts are the most abundant cell type in the mammalian heart and comprise approximately two-thirds of the total number of cardiac cell types. During development, epicardial cells undergo epithelial-mesenchymal-transition to generate cardiac fibroblasts that subsequently migrate into the developing myocardium to become resident cardiac fibroblasts. Fibroblasts form a structural scaffold for the attachment of cardiac cell types during development, express growth factors and cytokines and regulate proliferation of embryonic cardiomyocytes. In post natal life, cardiac fibroblasts play a critical role in orchestrating an injury response. Fibroblast activation and proliferation early after cardiac injury are critical for maintaining cardiac integrity and function, while the persistence of fibroblasts long after injury leads to chronic scarring and adverse ventricular remodeling. In this review, we discuss the physiologic function of the fibroblast during cardiac development and wound healing, molecular mediators of activation that could be possible targets for drug development for fibrosis and finally the use of reprogramming technologies for reversing scar. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium." Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. The rapid immunosuppression in phytohemagglutinin-activated human T cells is inhibited by the proliferative Ca(2+) influx induced by progesterone and analogs.

    Science.gov (United States)

    Lin, Veronica Hui-Chen; Chen, Jiann-Jong; Liao, Chen-Chung; Lee, Shinn-Shing; Chien, Eileen Jea

    2016-07-01

    Progesterone, an endogenous immunomodulator, suppresses human T-cell activation during pregnancy. A sustained Ca(2 +) influx is an important signal for T-cell proliferation after crosslinking of T-cell receptor/CD3 complexes by anti-CD3 antibodies or phytohemagglutinin (PHA). Progesterone targets cell membrane sites inducing rapid responses including elevated intracellular free calcium concentration ([Ca(2+)]i) and suppressed T-cell PHA-activated proliferation. Interestingly, both PHA and progesterone induce [Ca(2+)]i elevation, but it remains unclear whether the PHA-induced Ca(2+) influx is affected by progesterone leading to T-cell immunosuppression. Primary T-cells were isolated from human peripheral blood and the quench effect on intracellular fura-2 fluorescence of Mn(2+) was used to explore the responses to Ca(2+) influx with cell proliferation being determined by MTT assay. PHA-stimulated Ca(2+) influx was dose-dependently suppressed by progesterone and its agonist R5020, which correlated with PHA-activated T-cell proliferation inhibition. A similar dose-dependent suppression effect on cellular Ca(2+) influx and proliferation occurred with the TRPC channel inhibitor BTP2 and selective TRPC3 channel inhibitor Pyr3. In addition, two progesterone analogs, Org OD 02-0 and 20α-hydroxyprogesterone (20α-OHP), also produced dose-dependent suppression of Ca(2+) influx, but had no effect on proliferation. Finally, inhibition of PHA-activated T-cell proliferation by progesterone is further suppressed by 20α-OHP, but not by Org OD 02-0. Overall, progesterone and R5020 are able to rapidly decrease PHA-stimulated sustained Ca(2+) influx, probably via blockade of TRPC3 channels, which suppresses T-cell proliferation. Taken together, the roles of progesterone and its analogs regarding the rapid response Ca(2+) influx need to be further explored in relation to cytokine secretion and proliferation in activated T-cells.

  1. GLD-4-Mediated Translational Activation Regulates the Size of the Proliferative Germ Cell Pool in the Adult C. elegans Germ Line

    OpenAIRE

    Sophia Millonigg; Ryuji Minasaki; Marco Nousch; Jakub Novak; Eckmann, Christian R.

    2014-01-01

    To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult Caenorhabditis elegans reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identifi...

  2. Vitrectomy for proliferative diabetic retinopathy

    Directory of Open Access Journals (Sweden)

    Chao Peng

    2013-10-01

    Full Text Available AIM:To observe the clinical effect of vitrectomy for proliferative diabetic retinopathy(PDR.METHODS: The clinical data of 55 cases(65 eyes, underwent vitrectomy, membrane peeling, endolaser photocoagulation and silicone oil or C3F8 injection, were retrospectively studied. During 6 months to 1 year follow-up period, visual acuity, intraocular pressure, retinal conditions and complications were observed.RESULTS: All 65 eyes received vitrectomy, of which silicone oil was tamponaded in 32 eyes, C3F8 was injected in 8 eyes, BBS was filled in 25 eyes. Visual improvement achieved in 42 eyes. Two eyes were manually vision, form count fingers to 0.05 in 18 eyes, >0.05-0.1 in 28 eyes, >0.1-0.3 in 12 eyes and >0.3 in 5 eyes. Retinal hole was occurred in 7 eyes, limitations fibrosis membrane remained in 8 eyes, retinal detachment appeared in 5 eyes, IOP increased in 18 eyes, vitreous hemorrhage relapsed in 12 eyes, 36 eyes received supplemental photocoagulation treatment 1-3 times after operation.CONCLUSION:Vitrectomy combined endophotocoagulation is an effective treatment for PDR. Silicone oil tamponade can limit the hemorrhage.

  3. Induction of cAMP-dependent protein kinase A activity in human skin fibroblasts and rat osteoblasts by extremely low-frequency electromagnetic fields

    Energy Technology Data Exchange (ETDEWEB)

    Thumm, S.; Glock, S.; Haemmerle, H. [Natural and Medical Sciences Institute Reutlingen, University of Tuebingen (NMI), Markwiesenstrasse 55, D-72770 Reutlingen (Germany); Loeschinger, M.; Rodemann, H.P. [Section of Radiobiology and Molecular Environmental Research, University of Tuebingen, Roentgenweg 11, D-72076 Tuebingen (Germany)

    1999-09-01

    Sinusoidal extremely low-frequency electromagnetic fields (ELF-EMF; 7-8 mT, 20 Hz) have already been shown to inhibit proliferation and to accelerate terminal differentiation of human skin fibroblasts in vitro. In order to elucidate the underlying processes of signal transduction, we analysed the activity of cAMP-dependent protein kinase (PKA). EMF exposure for 60 min resulted in an increased PKA activity in human skin fibroblasts (2-fold) and rat embryonic osteoblasts (1.7-fold). Long-term exposure for up to 7 days with a constant 1 h-on/1 h-off EMF exposure rhythm indicated a transient stimulation of PKA activity during the first two exposure rhythms followed by a decrease to the baseline levels of sham-exposed controls. Based on these results, we postulate that a modulation of proliferation and differentiation processes in cells of mesenchymal origin is triggered by an immediate and transient EMF-induced increase in PKA activity. (orig.)

  4. Regulation of metalloproteinases and NF-kappaB activation in rabbit synovial fibroblasts via E prostaglandins and Erk: contrasting effects of nabumetone and 6MNA.

    Science.gov (United States)

    Pillinger, Michael H; Dinsell, Victoria; Apsel, Beth; Tolani, Sonia N; Marjanovic, Nada; Chan, Edwin S L; Gomez, Paul; Clancy, Robert; Chang, Lih-Fan; Abramson, Steven B

    2004-07-01

    1 Nabumetone is a prodrug that is converted in vivo into 6-methoxy-2-naphthylacetic acid (6MNA), a cyclooxygenase inhibitor with anti-inflammatory properties. We tested the effects of nabumetone and 6MNA on the inflammatory responses of synovial fibroblasts (SFs). 2 Brief exposures to 6MNA (50-150 microm) had no effect on IL-1beta/TNF-alpha (each 20 ng ml(-1))-stimulated Erk activation. Longer exposures depleted prostaglandin E1 (PGE1) as much as 70%, and stimulated Erk as much as 300%. Nabumetone (150 microm) inhibited Erk activation by 60-80%. 6MNA (50-150 microm) stimulated (approximately 200%) and nabumetone (150 microm) inhibited (approximately 50%) matrix metalloproteinase (MMP)-1, but not MMP-13 secretion from SFs. 3 6MNA stimulation of MMP-1 secretion was inhibited approximately 30% by PGE1 (1 microm) and approximately 80% by the Erk pathway inhibitor UO126 (10 microm), confirming that PGE depletion and Erk activation mediate MMP-1 secretion by 6MNA. 4 Consistent with its role as an Erk inhibitor, nabumetone (150 microm) abrogated 6MNA enhancement of MMP-1 secretion. 5 UO126 (10 microm) and nabumetone (150 microm) inhibited (approximately 70 and 40%, respectively), but 6MNA (150 microm) enhanced (approximately 40%), NF-kappaB activation. 6 Our data indicate that 6MNA shares with other COX inhibitors several proinflammatory effects on synovial fibroblasts. In contrast, nabumetone demonstrates anti-inflammatory and potentially arthroprotective effects that have not been previously appreciated.

  5. Effects of osmotic stress on the activity of MAPKs and PDGFR-beta-mediated signal transduction in NIH-3T3 fibroblasts

    DEFF Research Database (Denmark)

    Nielsen, M-B; Christensen, Søren Tvorup; Hoffmann, E K

    2008-01-01

    Signaling in cell proliferation, cell migration, and apoptosis is highly affected by osmotic stress and changes in cell volume, although the mechanisms underlying the significance of cell volume as a signal in cell growth and death are poorly understood. In this study, we used NIH-3T3 fibroblasts...... in a serum- and nutrient-free inorganic medium (300 mosM) to analyze the effects of osmotic stress on MAPK activity and PDGF receptor (PDGFR)-beta-mediated signal transduction. We found that hypoosmolarity (cell swelling at 211 mosM) induced the phosphorylation and nuclear translocation of ERK1/2, most...

  6. Anti-proliferative effects of Salacia reticulata leaves hot-water extract on interleukin-1β-activated cells derived from the synovium of rheumatoid arthritis model mice

    Directory of Open Access Journals (Sweden)

    Sekiguchi Yuusuke

    2012-04-01

    Full Text Available Abstract Background Salacia reticulata (SR is a plant native to Sri Lanka. In ayurvedic medicine, SR bark preparations, taken orally, are considered effective in the treatment of rheumatism and diabetes. We investigated the ability of SR leaves (SRL to inhibit in vitro the interleukin-1β (IL-1β-activated proliferation of synoviocyte-like cells derived from rheumatoid arthritis model mice. Findings Inflammatory synovial tissues were harvested from type II collagen antibody-induced arthritic mice. From these tissues, a synoviocyte-like cell line was established and named MTS-C H7. To determine whether SRL can suppress cell proliferation and gene expression in MTS-C H7 cells, fractionation of the SRL hot-water extract was performed by high-performance liquid chromatography (HPLC, liquid-liquid extraction, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE, and protease digestion. The 50% inhibitory concentration of the SRL hot-water extract against MTS-C H7 cells proliferation was ~850 μg/mL. Treatment with a low dose (25 μg dry matter per millilitre of the extract inhibited IL-1β-induced cell proliferation and suppressed the expression of the matrix metalloproteinase (MMP genes in MTS-C H7 cells. Various polyphenolic fractions obtained from HPLC and the fractions from liquid-liquid extraction did not affect cell proliferation. Only the residual water sample from liquid-liquid extraction significantly affected cell proliferation and the expression of MMP genes. The results of SDS-PAGE and protease digestion experiment showed that low molecular weight proteins present in SRL inhibited the IL-1β-activated cell proliferation. Conclusions We surmised that the residual water fraction of the SRL extract was involved in the inhibition of IL-1β-activated cell proliferation and regulation of mRNA expression in MTS-C H7 cells. In addition, we believe that the active ingredients in the extract are low molecular weight proteins.

  7. Antileukemic activity of sulforaphane in primary blasts from patients affected by myelo- and lympho-proliferative disorders and in hypoxic conditions.

    Directory of Open Access Journals (Sweden)

    Carmela Fimognari

    Full Text Available Sulforaphane is a dietary isothiocyanate found in cruciferous vegetables showing antileukemic activity. With the purpose of extending the potential clinical impact of sulforaphane in the oncological field, we investigated the antileukemic effect of sulforaphane on blasts from patients affected by different types of leukemia and, taking into account the intrinsically hypoxic nature of bone marrow, on a leukemia cell line (REH maintained in hypoxic conditions. In particular, we tested sulforaphane on patients with chronic lymphocytic leukemia, acute myeloid leukemia, T-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, and blastic NK cell leukemia. Sulforaphane caused a dose-dependent induction of apoptosis in blasts from patients diagnosed with acute lymphoblastic or myeloid leukemia. Moreover, it was able to cause apoptosis and to inhibit proliferation in hypoxic conditions on REH cells. As to its cytotoxic mechanism, we found that sulforaphane creates an oxidative cellular environment that induces DNA damage and Bax and p53 gene activation, which in turn helps trigger apoptosis. On the whole, our results raise hopes that sulforaphane might set the stage for a novel therapeutic principle complementing our growing armature against malignancies and advocate the exploration of sulforaphane in a broader population of leukemic patients.

  8. Antileukemic activity of sulforaphane in primary blasts from patients affected by myelo- and lympho-proliferative disorders and in hypoxic conditions.

    Science.gov (United States)

    Fimognari, Carmela; Turrini, Eleonora; Sestili, Piero; Calcabrini, Cinzia; Carulli, Giovanni; Fontanelli, Giulia; Rousseau, Martina; Cantelli-Forti, Giorgio; Hrelia, Patrizia

    2014-01-01

    Sulforaphane is a dietary isothiocyanate found in cruciferous vegetables showing antileukemic activity. With the purpose of extending the potential clinical impact of sulforaphane in the oncological field, we investigated the antileukemic effect of sulforaphane on blasts from patients affected by different types of leukemia and, taking into account the intrinsically hypoxic nature of bone marrow, on a leukemia cell line (REH) maintained in hypoxic conditions. In particular, we tested sulforaphane on patients with chronic lymphocytic leukemia, acute myeloid leukemia, T-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, and blastic NK cell leukemia. Sulforaphane caused a dose-dependent induction of apoptosis in blasts from patients diagnosed with acute lymphoblastic or myeloid leukemia. Moreover, it was able to cause apoptosis and to inhibit proliferation in hypoxic conditions on REH cells. As to its cytotoxic mechanism, we found that sulforaphane creates an oxidative cellular environment that induces DNA damage and Bax and p53 gene activation, which in turn helps trigger apoptosis. On the whole, our results raise hopes that sulforaphane might set the stage for a novel therapeutic principle complementing our growing armature against malignancies and advocate the exploration of sulforaphane in a broader population of leukemic patients.

  9. Wound healing properties of ethyl acetate fraction of Moringa oleifera in normal human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    Sivapragasam Gothai

    2016-03-01

    Full Text Available Background/Aim: Wounds are the outcome of injuries to the skin that interrupt the soft tissue. Healing of a wound is a complex and long-drawn-out process of tissue repair and remodeling in response to injury. A large number of plants are used by folklore traditions for treatment of cuts, wounds and burns. Moringa oleifera is an herb used as traditional folk medicine for the treatment of various skin wounds and associated diseases. The underlying mechanisms of wound healing activity of ethyl acetate fraction of M. oleifera leaves extract are completely unknown. Methods: In the current study, ethyl acetate fraction of Moringa oleifera leaves was investigated for its efficacy on cell viability, proliferation and migration (wound closure rate in human normal dermal fibroblast cells. Results: Results revealed that lower concentration (12.5 and micro;g/ml, 25 and micro;g/ml, and 50 and micro;g/ml of ethyl acetate fraction of M. oleifera leaves showed remarkable proliferative and migratory effect on normal human dermal fibroblasts. Conclusion: The present study suggested that ethyl acetate fraction of M. oleifera leaves might be a potential therapeutic agent for skin wound healing by promoting fibroblast proliferation and migration through increasing the wound closure rate corroborating its traditional use. [J Intercult Ethnopharmacol 2016; 5(1.000: 1-6

  10. Synthesis, characterization and anti-proliferative activity of Cd(II) complexes with NNN type pyrazole-based ligand and pseudohalide ligands as coligand

    Science.gov (United States)

    Hopa, Cigdem; Yildirim, Hatice; Kara, Hulya; Kurtaran, Raif; Alkan, Mahir

    2014-03-01

    Cd(II) complexes of tridentate nitrogen donor ligand, 2,6-bis(3,4,5-trimethylpyrazolyl)pyridine (btmpp), Cd(btmpp)X2 (X:Cl, ONO or N(CN)2) have been synthesized and characterized by elemental and spectral (FT-IR, 1H NMR, 13C NMR, UV-Vis) analyses, differential thermal analysis and single crystal X-ray diffraction studies. The molecular structure of reported complex 1, revealed distorted square-pyramidal geometry around Cadmium. Complexes 1-3 and corresponding ligand were tested for cytotoxic activity against the human carcinoma cell lines HEP3B (hepatocellular carcinoma), PC3 (prostate adenocarcinoma), MCF7 (breast adenocarcinoma) and Saos2 (osteosarcoma). The results show that, complexes are more cytotoxic than the free ligand and complex 2 is the most cytotoxic complex for PC3.

  11. Wound healing and the role of fibroblasts.

    Science.gov (United States)

    Bainbridge, P

    2013-08-01

    Fibroblasts are critical in supporting normal wound healing, involved in key processes such as breaking down the fibrin clot, creating new extra cellular matrix (ECM) and collagen structures to support the other cells associated with effective wound healing, as well as contracting the wound. This article explores and summarises the research evidence on the role of fibroblasts, their origins and activation, and how they navigate the wound bed, as well as how their activity leads to wound contraction. This article also explores the local conditions at the wound site, which activate, regulate and ultimately reduce the fibroblast activity as the skin's integrity returns on healing.

  12. Brown Kidney Bean Bowman-Birk Trypsin Inhibitor is Heat and pH Stable and Exhibits Anti-proliferative Activity.

    Science.gov (United States)

    Chan, Yau Sang; Zhang, Yanbo; Ng, Tzi Bun

    2013-02-01

    A trypsin inhibitor with a molecular mass around 17 kDa was purified from the seeds of Phaseolus vulgaris cv. 'brown kidney bean'. The purification protocol involved, in sequence, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Q-Sepharose and Mono Q, and gel filtration on Superdex 75. The molecular size and N-terminal amino acid sequence of the trypsin inhibitor resembled leguminous Bowman-Birk protease inhibitors (BBIs), signifying that brown kidney bean trypsin inhibitor is a BBI. Brown kidney bean trypsin inhibitor exhibited pronounced thermostability and pH stability. Its trypsin inhibitory activity was retained at all pH values (0-14) and up to 90 °C. The trypsin inhibitor also inhibited the proliferation of human breast cancer MCF7 cells with an IC(50) of 71.52 μM. On the other hand, it only slightly inhibited proliferation of hepatoma HepG2 and embryonic liver WRL68 cells at a concentration over 110 μM.

  13. Fibroblast growth factor receptor 2 plays an essential role in telencephalic progenitors.

    Science.gov (United States)

    Ever, Leah; Zhao, Rui-Jing; Eswarakumar, Veraragavan P; Gaiano, Nicholas

    2008-01-01

    We used loss-of-function analysis to determine the role of fibroblast growth factor receptor 2 (FGFR2) in telencephalic progenitors, and also to examine interactions between FGFR and Notch signaling. While the telencephalon of FGFR2 mutants appears grossly normal, mutant telencephalic progenitors exhibit altered proliferative behavior in vivo and in vitro. Based upon our prior finding that Notch1 activation increased neurosphere frequency in FGF2, we tested whether this effect is mediated by FGFR1 or FGFR2. We found that Notch1 activation increased neurosphere frequency in cells mutant for either FGFR1 or FGFR2, but had no effect on the reduced size of neurospheres mutant for those receptors. Additional analyses revealed biochemical changes in the adult neocortex mutant for the IIIc isoform of FGFR2, and essential roles for FGFR2 in nasopharynx, eyelid, and cornea development.

  14. Assessment of p21, p53 expression, and Ki-67 proliferative activities in the gastric mucosa of children with Helicobacter pylori gastritis.

    Science.gov (United States)

    Saf, Coskun; Gulcan, Enver Mahir; Ozkan, Ferda; Cobanoglu Saf, Seyhan Perihan; Vitrinel, Ayca

    2015-02-01

    Helicobacter pylori that is generally acquired in childhood and infects the gastric mucosa is considered to be responsible for many pathobiological changes that are linked to the pathogenesis of gastric cancer. Although the majority of studies on the subject have been carried out in adults, there are a limited number of studies on children that reflect the early period of infection and may be of greater significance. We aimed to determine the role of H. pylori infection and/or gastritis in several histopathological changes, p53, p21, and cell proliferation-associated Ki-67 antigen expression in the gastric mucosa. We studied 60 patients with a mean age of 7.5 ± 4.5 years at referral. On the basis of endoscopic appearance and the evaluation of the gastric antral specimens, the patients were divided into three groups: patients without gastritis, patients with H. pylori-positive gastritis, and patients with H. pylori-negative gastritis. To determine the expression of p53, Ki-67, and p21 in gastric biopsy specimens, immunohistochemical stains were performed. The incidence of neutrophil activity, which was one of our histopathologic parameters, was significantly higher in the H. pylori-positive gastritis group than the other two groups. The presence of lymphoid aggregate was more frequent in H. pylori ± gastritis groups than the nongastritis group. p53 expression was found to be significantly higher in the H. pylori-positive gastritis group than the nongastritis group. Ki-67 and p21 expressions were significantly more frequent in the H. pylori-positive gastritis group than the other two groups. When we evaluated the density of H. pylori, as the density of bacteria increases, we found that the expressions of p53, p21, and Ki-67 increased significantly. Expression of the studied precancerous markers in significant amounts indicates the importance of childhood H. pylori infection in the constitution of gastric cancer in adulthood.

  15. Precursor of advanced glycation end products mediates ER-stress-induced caspase-3 activation of human dermal fibroblasts through NAD(PH oxidase 4.

    Directory of Open Access Journals (Sweden)

    Danielle T Loughlin

    Full Text Available BACKGROUND: The precursor for advanced glycation end products, 3-deoxyglucosone (3DG is highly upregulated in skin explants of diabetic cutaneous wounds, and has been shown to negatively impact dermal fibroblasts, which are crucial in wound remodeling. 3DG induces apoptosis however; the mechanisms involved in the apoptotic action of 3DG in the pathogenesis of diabetic chronic wounds are poorly understood. Therefore, we sought to delineate novel mechanisms involved with the 3DG-collagen induced apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: Using human dermal fibroblasts, we demonstrated that 3DG-modified collagen induces oxidative stress and caspase-3 activation. Oxidative stress was found to be dependent on the upregulation of NAD(PH oxidase 4 (Nox4, a reactive oxygen species (ROS Nox homologue, triggering endoplasmic reticulum (ER stress, as assessed by the ER stress-induced apoptosis marker Growth Arrest and DNA Damage-inducible gene 153 (GADD153. We demonstrated that 3DG-collagen activated GADD153 via phosphorylation of p38 mitogen activated protein kinase (MAPK, and this was dependent on upstream ROS. Inhibition of ROS and/or p38 MAPK abrogated 3DG-collagen induced caspase-3 activation. Our investigations also demonstrated that 3DG-collagen-induced caspase-3 activation did not signal through the canonical receptor for advanced glycation end products (RAGE but through integrin alpha1beta1. To further verify the role of integrins, neutralization of integrins alpha1beta1 prevented 3DG-collagen-induced upregulation of ROS, GADD153, and caspase-3 activation; suggesting that 3DG-collagen signaling to the fibroblast is dependent on integrins alpha1beta1. CONCLUSIONS/SIGNIFICANCE: Taken together, these findings demonstrate for the first time that a RAGE independent mechanism is involved in 3DG-collagen-induced apoptosis. Moreover, the ER stress pathway through activation of Nox4 by integrins alpha1beta1 plays a key role in 3DG-collagen-induced caspase

  16. AhR-dependent secretion of PDGF-BB by human classically activated macrophages exposed to DEP extracts stimulates lung fibroblast proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Jaguin, Marie [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 Avenue du Pr Léon Bernard, 35043 Rennes Cedex (France); Fardel, Olivier [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 Avenue du Pr Léon Bernard, 35043 Rennes Cedex (France); Pôle Biologie, Centre Hospitalier Universitaire (CHU) Rennes, 2 rue Henri Le Guilloux, 35033 Rennes Cedex (France); Lecureur, Valérie, E-mail: valerie.lecureur@univ-rennes1.fr [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 Avenue du Pr Léon Bernard, 35043 Rennes Cedex (France)

    2015-06-15

    Lung diseases are aggravated by exposure to diesel exhaust particles (DEPs) found in air pollution. Macrophages are thought to play a crucial role in lung immune response to these pollutants, even if the mechanisms involved remain incompletely characterized. In the present study, we demonstrated that classically and alternative human macrophages (MΦ) exhibited increased secretion of PDGF-B in response to DEP extract (DEPe). This occurred via aryl hydrocarbon receptor (AhR)-activation because DEPe-induced PDGF-B overexpression was abrogated after AhR expression knock-down by RNA interference, in both M1 and M2 polarizing MΦ. In addition, TCDD and benzo(a)pyrene, two potent AhR ligands, also significantly increased mRNA expression of PDGF-B in M1 MΦ, whereas some weak ligands of AhR did not. We next evaluated the impact of conditioned media (CM) from MΦ culture exposed to DEPe or of recombinant PDGF-B onto lung fibroblast proliferation. The tyrosine kinase inhibitor, AG-1295, prevents phosphorylations of PDGF-Rβ, AKT and ERK1/2 and the proliferation of MRC-5 fibroblasts induced by recombinant PDGF-B and by CM from M1 polarizing MΦ, strongly suggesting that the PDGF-BB secreted by DEPe-exposed MΦ is sufficient to activate the PDGF-Rβ pathway of human lung fibroblasts. In conclusion, we demonstrated that human MΦ, whatever their polarization status, secrete PDGF-B in response to DEPe and that PDGF-B is a target gene of AhR. Therefore, induction of PDGF-B by DEP may participate in the deleterious effects towards human health triggered by such environmental urban contaminants. - Highlights: • PDGF-B expression and secretion are increased by DEPe exposure in human M1 and M2 MΦ. • DEPe-induced PDGF-B expression is aryl-hydrocarbon-dependent. • DEPe-exposed M1 MΦ secrete sufficient PDGF-B to increase lung fibroblast proliferation.

  17. [Proliferative diabetic retinopathy -- therapeutic approach (clinical case)].

    Science.gov (United States)

    Burcea, M; Muşat, Ovidiu; Mahdi, Labib; Gheorghe, Andreea; Spulbar, F; Gobej, I

    2014-01-01

    We present the case of a 54 year old pacient diagnosed with neglected insulin dependent diabetes and proliferative diabetic retinopathy. Surgery was recommended and we practiced posterior vitrectomy, endolaser and heavy silicone oil endotamponade. Post-operative evolution was favorable.

  18. Transcriptional activation of p21(WAF¹/CIP¹) is mediated by increased DNA binding activity and increased interaction between p53 and Sp1 via phosphorylation during replicative senescence of human embryonic fibroblasts.

    Science.gov (United States)

    Kim, Hyun-Seok; Heo, Jee-In; Park, Seong-Hoon; Shin, Jong-Yeon; Kang, Hong-Jun; Kim, Min-Ju; Kim, Sung Chan; Kim, Jaebong; Park, Jae-Bong; Lee, Jae-Yong

    2014-01-01

    Although p21(WAF1/CIP1) is known to be elevated during replicative senescence of human embryonic fibroblasts (HEFs), the mechanism for p21 up-regulation has not been elucidated clearly. In order to explore the mechanism, we analyzed expression of p21 mRNA and protein and luciferase activity of full-length p21 promoter. The result demonstrated that p21 up-regulation was accomplished largely at transcription level. The promoter assay using serially-deleted p21 promoter constructs revealed that p53 binding site was the most important site and Sp1 binding sites were necessary but not sufficient for transcriptional activation of p21. In addition, p53 protein was shown to interact with Sp1 protein. The interaction was increased in aged fibroblasts and was regulated by phosphorylation of p53 and Sp1. DNA binding activity of p53 was significantly elevated in aged fibroblasts but that of Sp1 was not. DNA binding activities of p53 and Sp1 were also regulated by phosphorylation. Phosphorylation of p53 at serine-15 and of Sp1 at serines appears to be involved. Taken together, the result demonstrated that p21 transcription during replicative senescence of HEFs is up-regulated by increase in DNA binding activity and interaction between p53 and Sp1 via phosphorylation.

  19. Bryostatin-1 causes radiosensitization of BMG-1 malignant glioma cells through differential activation of protein kinase-Cδ not evident in the non-malignant AA8 fibroblasts.

    Science.gov (United States)

    Dagur, Raghubendra Singh; Hambarde, Shashank; Chandna, Sudhir

    2015-03-01

    Bryostatin-1 (bryo-1), a non-phorbol ester, is known to sensitize mammalian cells against certain chemotherapeutic drugs. We assessed its ability to modify radiation response of mammalian cells using Chinese hamster fibroblasts AA8 cells and human malignant glioma BMG-1 cells. In the malignant glioma BMG-1 cell line, bryo-1 pre-treatment significantly enhanced radiation-induced growth inhibition and cytogenetic damage, and further reduced the clonogenic cell survival as compared to cells irradiated at the clinically relevant dose of 2 Gy. PKCδ expression increased significantly when bryo-1 pre-treated BMG-1 glioma cells were irradiated at 2 Gy and induced prolonged ERK-1/2 activation associated with p21 overexpression. Silencing PKCδ resulted in inhibition of bryo-1-induced radiosensitization. In contrast, bryo-1 failed to alter radiosensitivity (cell survival; growth inhibition; cytogenetic damage) or activate ERK1/2 pathway in the AA8 fibroblasts despite PKCδ phosphorylation at its regulatory (Y155) domain, indicating alternate mechanisms in these non-malignant cells as compared to the glioma cells. This study suggests that bryo-1 may effectively enhance the radiosensitivity of malignant cells and warrants further in-depth investigations to evaluate its radiosensitizing potential in various cell types.

  20. Signaling pathway activation drift during aging: Hutchinson-Gilford Progeria Syndrome fibroblasts are comparable to normal middle-age and old-age cells.

    Science.gov (United States)

    Aliper, Alexander M; Csoka, Antonei Benjamin; Buzdin, Anton; Jetka, Tomasz; Roumiantsev, Sergey; Moskalev, Alexy; Zhavoronkov, Alex

    2015-01-01

    For the past several decades, research in understanding the molecular basis of human aging has progressed significantly with the analysis of premature aging syndromes. Progerin, an altered form of lamin A, has been identified as the cause of premature aging in Hutchinson-Gilford Progeria Syndrome (HGPS), and may be a contributing causative factor in normal aging. However, the question of whether HGPS actually recapitulates the normal aging process at the cellular and organismal level, or simply mimics the aging phenotype is widely debated. In the present study we analyzed publicly available microarray datasets for fibroblasts undergoing cellular aging in culture, as well as fibroblasts derived from young, middle-age, and old-age individuals, and patients with HGPS. Using GeroScope pathway analysis and drug discovery platform we analyzed the activation states of 65 major cellular signaling pathways. Our