Development and initial application of an in vitro model of apoptosis in rodent cholangiocytes

1997 ◽  
Vol 272 (1) ◽  
pp. G106-G115 ◽  
Author(s):  
F. G. Que ◽  
G. J. Gores ◽  
N. F. LaRusso
Keyword(s):  
In Vitro Model ◽  
Interleukin 1 ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Initial Application ◽  
Histological Data ◽  
Vitro Model ◽  
Induced Apoptosis ◽  
Intracellular Ions

Although histological data suggest that cholangiocytes die by apoptosis in human liver diseases, no information exists on the mechanisms of cholangiocyte apoptosis. Thus our aims were to establish an in vitro model of cholangiocyte apoptosis and to test the hypothesis that changes in intracellular ions would cause apoptosis in cholangiocytes by a protease-sensitive pathway. A large number of proapoptotic agents were ineffective in inducing apoptosis in rat or human cholangiocytes in culture; in contrast, beauvericin, a K+ ionophore, caused apoptosis in both cell lines, despite their expression of Bcl-2. Although beauvericin decreased intracellular K+ and increased intracellular Ca2+, abolishing the K+ gradient did not prevent beauvericin-induced apoptosis; in contrast, omission of extracellular Ca2+ inhibited apoptosis by 42%. The interleukin-1 beta-converting enzyme (ICE) family protease inhibitor, Z-Val-Ala-Asp chloromethylketone, inhibited apoptosis in a concentration-dependent manner. By Northern blot analysis, cholangiocytes expressed the mRNA for three members of the ICE protease family: ICE, ICE/ CED-3 homologue-1 (ICH-1), and cysteine protease P-32 (CPP-32). Cleavage of a substrate for CPP-32-like protease activity, but not a substrate for ICE and ICH-1, increased after beauvericin treatment. In summary, we have established an in vitro model of apoptosis in cholangiocytes. Our data suggest that beauvericin-induced apoptosis occurs by a Ca(2+)-dependent CPP-32 protease-sensitive pathway despite cholangiocyte expression of Bcl-2.

Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix

1997 ◽  
Vol 110 (7) ◽  
pp. 861-870 ◽  
Author(s):  
D. Greiling ◽  
R.A. Clark
Keyword(s):  
Wound Healing ◽  
In Vitro Model ◽  
Collagen Gel ◽  
Fibrin Clot ◽  
Dependent Manner ◽  
Fibrin Gel ◽  
Concentration Dependent Manner ◽  
Vitro Model ◽  
Early Wound

After injury, the wound space is filled with a fibrin/fibronectin clot containing growth factors released by platelets and monocytes. In response to these factors, fibroblasts migrate into the fibrin clot and contribute to the formation of granulation tissue. The functional mechanisms allowing fibroblasts to leave the collagenous matrix of normal connective tissue and invade the provisional matrix of the fibrin clot have not been fully defined. To investigate these mechanisms we established a new in vitro model which simulates specific aspects of early wound healing, that is, the migration of fibroblasts from a three-dimensional collagen matrix into a fibrin clot. This transmigration could be induced by physiological concentrations of platelet releasate or platelet-derived growth factor BB (PDGF-BB) in a concentration-dependent manner. At 24 hours irradiated fibroblasts invaded the fibrin gel almost as well as non-irradiated cells, indicating that transmigration was independent of proliferation. Plasminogen and its activators appear to be necessary for invasion of the fibrin clot since protease inhibitors decreased the amount of migration. These serine proteases, however, were not necessary for exit from the collagen gel as fibroblasts migrated out of the collagen gel onto a surface coated with fibrin fibrils even in the presence of inhibitors. Removal of fibronectin (FN) from either the collagen gel or the fibrin gel markedly decreased the number of migrating cells, suggesting that FN provides a conduit for transmigration. Cell movement in the in vitro model was inhibited by RGD peptide, and by monoclonal antibodies against the subunits of the alpha5 beta1 and alpha v beta3 integrin receptor. Thus, the functional requirements for fibroblast transmigration from collagen-rich to fibrin-rich matrices, such as occurs in early wound healing, have been partially defined using an in vitro paradigm of this important biologic process.

scholarly journals Olive Oil-Supplemented Lipid Emulsion Induces CELF1 Expression and Promotes Apoptosis in Caco-2 Cells

2017 ◽  
Vol 41 (2) ◽  
pp. 711-721 ◽  
Author(s):  
Jun-Kai Yan ◽  
Jie Zhu ◽  
Zi-Zhen Gong ◽  
Jie Wen ◽  
Yong-Tao Xiao ◽  
...  
Keyword(s):  
Olive Oil ◽  
Lipid Emulsion ◽  
In Vitro Model ◽  
Protein Translation ◽  
Dependent Manner ◽  
Enterocyte Apoptosis ◽  
Vitro Model ◽  
Induced Apoptosis ◽  
Apoptosis Inducing Factor

Background and Aims: Parenterally-administered lipid emulsion (LE) is a key cause of enterocyte apoptosis under total parenteral nutrition, yet the pathogenesis has not been fully understood. CUGBP, Elav-like family member 1 (CELF1) has been recently identified as a crucial modulator of apoptosis, and thus this study sought to investigate its role in the LE-induced apoptosis in vitro. Methods: Caco-2 cells were used as an in vitro model. The cells were treated with varying LEs derived from soybean oil, olive oil or fish oil, and changes in the apoptosis and CELF1 expression were assessed. Rescue study was performed using transient knockdown of CELF1 with specific siRNA prior to LE treatment. Regulation of CELF1 by LE treatment was studied using quantitative real-time PCR and Western blotting. Results: All the LEs up-regulated CELF1expression and induced apoptosis, but only olive oil-supplemented lipid emulsion (OOLE)-induced apoptosis was attenuated by depletion of CELF1. Up-regulation of apoptosis-inducing factor (AIF) was involved in OOLE-induced CELF1 dependent apoptosis. The protein expression of CELF1 was up-regulated by OOLE in a dose- and time-dependent manner, but the mRNA expression of CELF1 was unchanged. Analysis by polysomal profiling and nascent protein synthesis revealed that the regulation of CELF1 by OOLE treatment was mediated by directly accelerating its protein translation. Conclusion: OOLE-induces apoptosis in Caco-2 cells partially through up-regulation of CELF1.

In vitro study of placental trophoblast calcium uptake using JEG-3 human choriocarcinoma cells

1991 ◽  
Vol 98 (3) ◽  
pp. 333-342
Author(s):  
R.S. Tuan ◽  
C.J. Moore ◽  
J.W. Brittingham ◽  
J.J. Kirwin ◽  
R.E. Akins ◽  
...  
Keyword(s):  
Vitamin D3 ◽  
Calcium Uptake ◽  
In Vitro Model ◽  
Dependent Manner ◽  
Choriocarcinoma Cells ◽  
Vitro Model ◽  
Human Choriocarcinoma Cells ◽  
Functional Components ◽  
1,25 Dihydroxy Vitamin D3

During human fetal development, placental syncytiotrophoblasts actively transport calcium from the maternal to the fetal circulation. Two functional components, a cytosolic Ca2(+)-binding protein (CaBP) and a Ca2(+)-ATPase have been identified in the syncytiotrophoblasts of the chorionic villi. We report here the calcium uptake properties of a human choriocarcinoma cell line, JEG-3, which was used as an in vitro model cell system for the syncytiotrophoblasts. In culture, JEG-3 proliferated as large syncytial aggregates expressing typical syncytiotrophoblast markers. 45Ca uptake by JEG-3 was a substrate- and temperature-dependent, membrane-mediated active process that exhibited linear kinetics for up to 7 min. Both the CaBP and the Ca2(+)-ATPase were expressed by JEG-3, on the basis of biochemical, histochemical, immunochemical and or mRNA assays. Immunohistochemistry and in situ hybridization revealed that JEG-3 cells were heterogeneous with respect to the expression of the CaBP. The Ca2(+)-ATPase activity of JEG-3 was similar to the placental enzyme in terms of sensitivity to specific inhibitors, and was detected histochemically along the cell membrane. Fura-2 Ca2+ imaging revealed that calcium uptake by JEG-3 was not accompanied by a concomitant increase in cytosolic [Ca2+], suggesting a specific Ca2+ sequestration mechanism. The involvement of calciotropic hormonal regulation was evaluated by studying the response of JEG-3 to 1,25-dihydroxy vitamin D3. Calcium uptake was significantly stimulated in a dose-dependent manner by a 24-h treatment of the cells with 1,25-dihydroxy vitamin D3 (optimal dose approximately 0.5 nM); the CaBP level doubled whereas steady-state CaBP mRNA did not, suggesting that CaBP expression was regulated by 1,25-dihydroxy vitamin D3. These observations strongly suggest that the JEG-3 human choriocarcinoma cells should serve as a convenient in vitro model system for studying the cellular mechanism and regulation of transplacental calcium transport.

The Activity of Urolithin A and M4 Valerolactone, Colonic Microbiota Metabolites of Polyphenols, in a Prostate Cancer In Vitro Model

Planta Medica ◽  
2018 ◽  
Vol 85 (02) ◽  
pp. 118-125 ◽  
Author(s):  
Iwona Stanisławska ◽  
Sebastian Granica ◽  
Jakub Piwowarski ◽  
Joanna Szawkało ◽  
Krzysztof Wiązecki ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
In Vitro Model ◽  
Specific Antigen ◽  
Lncap Cells ◽  
Isobolographic Analysis ◽  
Human System ◽  
Urolithin A ◽  
Vitro Model ◽  
Induced Apoptosis

AbstractThe gut microbiota-derived metabolites of ellagitannins and green tea catechins, urolithin A (uroA) and 5-(3′,4′,5′-trihydroxyphenyl)-γ-valerolactone (M4), respectively, are among the main compounds absorbed into human system after ingestion of these polyphenols. The aim of this study was to establish the effects of M4, uroA, and their combinations on LNCaP cells, an androgen dependent prostate cancer in vitro model.. The LNCaP cells were incubated with increasing concentrations of tested metabolites. The cell proliferation was determined by measurement of DNA-bisbenzimide H 33 258 complexes fluorescence. The isobolographic analysis was used to establish the type of interaction between metabolites. The apoptosis, androgen receptor (AR) localization, and phosphorylation of Akt kinase were measured by flow cytometry. Prostate-specific antigen (PSA) secretion was determined by ELISA. M4 showed modest antiproliferative activity in LNCaP cells (IC50 = 117 µM; CI: 81 – 154). UroA decreased proliferation (IC50 = 32.7 µM; CI: 24.3 – 41.1) and induced apoptosis of LNCaP cells. The mixture of M4 with uroA had synergistic antiproliferative effect. Moreover, M4 potentiated inhibition of PSA secretion and enhanced retention of AR in cytoplasm caused by uroA. Interestingly, uroA increased levels of pSer473 Akt in LNCaP cells. These results show that colonic metabolites may contribute to chemoprevention of prostate cancer by varied polyphenol-rich diet or composite polyphenol preparations.

scholarly journals T-2 Toxin Exposure Induces Apoptosis in TM3 Cells by Inhibiting Mammalian Target of Rapamycin/Serine/Threonine Protein Kinase(mTORC2/AKT) to Promote Ca2+Production

2018 ◽  
Vol 19 (11) ◽  
pp. 3360 ◽  
Author(s):  
Ji Wang ◽  
Chenglin Yang ◽  
Zhihang Yuan ◽  
Jine Yi ◽  
Jing Wu
Keyword(s):  
Cell Injury ◽  
Mammalian Target Of Rapamycin ◽  
Annexin V ◽  
Target Of Rapamycin ◽  
Dependent Manner ◽  
Toxin Exposure ◽  
Concentration Changes ◽  
Vitro Model ◽  
Induced Apoptosis

Although mTOR (the mammalian target of rapamycin) can regulate intracellular free Ca2+concentration in normal cultured podocytes, it remains elusive as to how mTORC2/AKT-mediated Ca2+participates in the process of T-2 toxin-induced apoptosis. The potential signaling responsible for intracellular Ca2+ concentration changes was investigated using immunoblot assays in an in vitro model of TM3 cell injury induced by T-2 toxin. Changes in Ca2+ were assessed using the Ca2+-sensitive fluorescent indictor dye Fura 2-AM. The cytotoxicity of TM3 cells was assessed with an MTT bioassay, and apoptosis was measured using Annexin V-FITC staining. Following T-2 toxin treatment, the growth of cells, phospho-mTORSer2481, phospho-mTORSer2448, and phospho-AktSer473 were significantly decreased in a time-dependent manner, whereas Ca2+ and apoptosis were increased. T-2 toxin-induced apoptosis was prevented by BAPTA-AM (a Ca2+chelator) and MHY1485 (an mTOR activator), and the application of mTOR activator MHY1485 also prevented the increase of intracellular free Ca2+concentration in TM3 cells. Our results strongly suggest that T-2 toxin exposure induces apoptosis in TM3 cells by inhibiting mTORC2/AKT to promote Ca2+ production.

219 EFFECT OF THE ENDOCRINE DISRUPTING CHEMICALS ON PLACENTAL TRANSPORT IN BeWo CELLS AS AN IN VITRO MODEL

2015 ◽  
Vol 27 (1) ◽  
pp. 199
Author(s):  
J.-H. Lee ◽  
E.-B. Jeung
Keyword(s):  
In Vitro Model ◽  
Endocrine Disrupting Chemicals ◽  
Reproductive Hormones ◽  
Dependent Manner ◽  
Endocrine Disrupting ◽  
Calcium Cations ◽  
Vitro Model ◽  
Dose Dependent ◽  
Iron And Calcium

The placenta exchanges vital factors, including oxygen, carbon dioxide, copper, iron, calcium cations, and glucose, which are essential to fetal growth. Each molecule is transferred by specific receptors that are located at the cell membrane or in the cytoplasm. Copper, iron, calcium cations, and glucose transfer genes are regulated by estrogens, vitamin D, and human placental lactogen. Regulations of these receptors depend on pregnancy time length and maternal and fetal nutrient environment with various pathways. Some synthetic plastics known as endocrine disrupting chemicals (EDC) have a similar structure to reproductive hormones such as estrogens. Thus, these substances have a potential effect on the expression of genes which are regulated by estrogens or progesterone by interfering their pathways. Having an estrogenic property, EDC interact with oestrogen receptors and elevate or decrease the expression of target genes which are responsible for transporting essential molecules such as copper, iron, and calcium. To examine the effects of EDC exposure during pregnancy, we conducted an in vitro model study using the BeWo human trophoblast cell line. The BeWo cell was treated with well-known EDC, octyl-phenol (OP), nonyl-phenol (NP), and bisphenol A (BPA) in a dose-dependent manner (10–7, 10–6, and 10–5 M) for 24 h. The expression of copper (CTR1, ATP7A), iron (IREG1, HEPH), and calcium transporting genes (PMCA1, TRPV6), were measured by real-time RT–PCR and Western blot. The expression of copper, iron, and calcium transporting genes were elevated in a dose-dependent manner by all well-known EDC, including OP, NP, and BPA, as well as E2. To unveil the mechanism of these elevations of ionic transporting genes, an ERE promoter study will be needed. Taken together, essential cation transporting genes in placenta are modulated by EDC.

scholarly journals Novel Chrysin-De-Allyl PAC-1 Hybrid Analogues as Anticancer Compounds: Design, Synthesis, and Biological Evaluation

Molecules ◽  
2020 ◽  
Vol 25 (13) ◽  
pp. 3063
Author(s):  
Buthina A. Al-Oudat ◽  
Hariteja Ramapuram ◽  
Saloni Malla ◽  
Suaad A. Audat ◽  
Noor Hussein ◽  
...  
Keyword(s):  
Mammary Epithelial Cells ◽  
Biological Evaluation ◽  
Mammary Epithelial ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Anticancer Compounds ◽  
Design Synthesis ◽  
Apoptotic Pathways ◽  
Induced Apoptosis

New chrysin-De-allyl-Pac-1 hybrid analogues, tethered with variable heterocyclic systems (4a–4o), were rationally designed and synthesized. The target compounds were screened for in vitro antiproliferative efficacy in the triple-negative breast cancer (TNBC) cell line, MDA-MB-231, and normal human mammary epithelial cells (HMECs). Two compounds, 4g and 4i, had the highest efficacy and selectivity towards MDA-MB-231 cells, and thus, were further evaluated by mechanistic experiments. The results indicated that both compounds 4g and 4i induced apoptosis by (1) inducing cell cycle arrest at the G2 phase in MDA-MB-231 cells, and (2) activating the intrinsic apoptotic pathways in a concentration-dependent manner. Physicochemical characterizations of these compounds suggested that they can be further optimized as potential anticancer compounds for TNBC cells. Overall, our results suggest that 4g and 4i could be suitable leads for developing novel compounds to treat TNBC.

scholarly journals CdsH Contributes to the Replication of Salmonella Typhimurium inside Epithelial Cells in a Cysteine-Supplemented Medium

2020 ◽  
Vol 8 (12) ◽  
pp. 2019
Author(s):  
Fernando Díaz-Yáñez ◽  
Ricardo Álvarez ◽  
Iván L. Calderón ◽  
Juan A. Fuentes ◽  
Fernando Gil
Keyword(s):  
Epithelial Cells ◽  
Salmonella Typhimurium ◽  
Intestinal Lumen ◽  
Dependent Manner ◽  
Cell Infection ◽  
Concentration Dependent Manner ◽  
Intestinal Phase ◽  
Vitro Model ◽  
Bacterial Replication

Salmonella Typhimurium is a facultative, intracellular pathogen whose products range from self-limited gastroenteritis to systemic diseases. Food ingestion increases biomolecules’ concentration in the intestinal lumen, including amino acids such as cysteine, which is toxic in a concentration-dependent manner. When cysteine’s intracellular concentration reaches toxic levels, S. Typhimurium expresses a cysteine-inducible enzyme (CdsH), which converts cysteine into pyruvate, sulfide, and ammonia. Despite this evidence, the biological context of cdsH’s role is not completely clear, especially in the infective cycle. Since inside epithelial cells both cdsH and its positive regulator, ybaO, are overexpressed, we hypothesized a possible role of cdsH in the intestinal phase of the infection. To test this hypothesis, we used an in vitro model of HT-29 cell infection, adding extra cysteine to the culture medium during the infective process. We observed that, at 6 h post-invasion, the wild type S. Typhimurium proliferated 30% more than the ΔcdsH strain in the presence of extra cysteine. This result shows that cdsH contributes to the bacterial replication in the intracellular environment in increased concentrations of extracellular cysteine, strongly suggesting that cdsH participates by increasing the bacterial fitness in the intestinal phase of the S. Typhimurium infection.

Sign in / Sign up

Export Citation Format

Share Document