Apoptosis is a major mechanism of deoxycholate-induced gastric mucosal cell death

2003 ◽  
Vol 285 (5) ◽  
pp. G870-G879 ◽  
Author(s):  
Maria J. Redlak ◽  
Miranda S. Dennis ◽  
Thomas A. Miller
Keyword(s):  
Cell Death ◽  
Prolonged Exposure ◽  
Lactic Dehydrogenase ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Bathing Solution ◽  
Mucosal Cell ◽  
Ags Cells ◽  
Cellular Death ◽  
Major Mechanism ◽  
Ldh Assay

This study was undertaken to determine whether necrosis or apoptosis was the predominant mechanism responsible for gastric mucosal cellular death using the cell line known as AGS cells. Cells were exposed to various concentrations of deoxycholate (DC; 50–500 μM) for periods ranging from 30 min to 24 h. Lactic dehydrogenase (LDH) activity was used as a marker for necrotic cell death, whereas apoptosis was characterized by 4′,6-diamidino-2 phenylindole staining, DNA gel electrophoresis, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and DNA-histone-associated complex formation. When cells were bathed in Hank's balanced salt solution, DC-induced necrosis was the predominant mechanism of cell death. In contrast, when cells were bathed in Ham's F-12 solution (a more physiologically relevant medium), no evidence of cytotoxicity (by LDH assay) was discernible when cells were exposed to DC (50–300 μM) for periods as long as 8 h; instead, clear evidence of apoptosis was noted that was time and dose dependent. When cells were exposed for 24 h to these DC concentrations, cytotoxicity was also present, indicating necrosis as well. Furthermore, acidification of the ambient environment also evoked a necrotic response when exposed to DC. We demonstrated that apoptosis induced by DC shows early activation of caspase-3 that is dependent on both receptor and mitochondrial pathways. Our results indicate that physiological concentrations of DC (50–300 μM) primarily induce cellular death through an apoptotic process. Only after prolonged exposure to DC or acidification of the bathing solution does necrosis also occur.

scholarly journals Inhibition of Growth and Induction of Apoptosis in Fibrosarcoma Cell Lines byEchinophora platylobaDC: In Vitro Analysis

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Fatemeh Zare Shahneh ◽  
Samira Valiyari ◽  
Abbas Azadmehr ◽  
Reza Hajiaghaee ◽  
Saeid Yaripour ◽  
...  
Keyword(s):  
Cell Death ◽  
Nuclear Dna ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Dependent Manner ◽  
Fibrosarcoma Cell ◽  
Major Mechanism ◽  
Methanolic Extracts ◽  
Induction Of Apoptosis ◽  
Vitro Analysis

Echinophora platylobaDC plant (Khousharizeh) is one of the indigenous medicinal plants which is used as a food seasoning and medicine in Iran. The objective of this study was to examine the in vitro cytotoxic activity and the mechanism of cell death of crude methanolic extracts prepared fromEchinophora platylobaDC, on mouse fibrosarcoma cell line (WEHI-164). Cytotoxicity and viability of methanolic extract was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dye exclusion assay. Cell death ELISA was employed to quantify the nucleosome production result from nuclear DNA fragmentation during apoptosis and determine whether the mechanism involves induction of apoptosis or necrosis. The cell death was identified as apoptosis using terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) assay. Our results demonstrated that the extract decreased cell viability, suppressed cell proliferation, and induced cell death in a time- and dose-dependent manner in WEHI-164 cells (IC50 = 196.673 ± 12.4 μg/mL) when compared with a chemotherapeutic anticancer drug, Toxol. Observation proved that apoptosis was the major mechanism of cell death. So theEchinophora platylobaDC extract was found to time- and dose-dependently inhibit the proliferation of fibrosarcoma cell possibly via an apoptosis-dependent pathway.

Single-chain Antibody Against Reg4 Suppresses Gastric Cancer Cell Growth and Enhances 5-FU-induced Cell Death in vitro

2019 ◽  
Vol 19 (5) ◽  
pp. 610-619 ◽  
Author(s):  
Xue-Qing Zhang ◽  
Lu-Ting Yu ◽  
Pei Du ◽  
Tian-Qi Yin ◽  
Zhi-Yuan Zhang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Cell Death ◽  
Cancer Cell ◽  
Gastric Cancer Cell ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Ags Cells ◽  
Thiazolyl Blue Tetrazolium Bromide

Background:Regenerating islet-derived gene family member 4 (Reg4), a well-investigated growth factor in the regenerative pancreas, has recently been reported to be highly associated with a majority of gastrointestinal cancers. Pathological hyper-expression or artificial over-expression of Reg4 causes acceleration of tumor growth, migration, and resistance to chemotherapeutic 5-Fluorouracil (5-FU). Until now, no method has been successfully established for eliminating the effects of Reg4 protein.Methods:This study reports the production of an engineered immunoglobin, a single-chain variable fragment (scFv-Reg4), to specifically bind Reg4 and block the bioactivity. The complementary-determining regions (CDRs) against Reg4 were assigned using MOE and ZDOCK servers. The binding affinity (KD) was determined by bio-layer interferometry (BLI). MKN45 and AGS cell proliferation was determined by Thiazolyl blue tetrazolium bromide (MTT) method and the cell apoptosis was detected by flow cytometry assay.Results:The KD of scFv-Reg4 to Reg4 was determined to be 1.91×10-8. In MKN45 and AGS cell lines, scFv- Reg4 depressed Reg4-stimulated cell proliferation and the inhibitory rates were 27.7±1.5% and 17.3±2.6%, respectively. Furthermore, scFv significantly enhanced 5-FU-induced cell death, from 23.0±1.0% to 28.4±1.2% in MKN45 and 28.2±0.7% to 36.6±0.6% in AGS cells. Treatment with scFv alone could lyse cancer cells to a certain extent, but no significance has been observed.Conclusion:The single-chain antibody (scFv-Reg4) significantly inhibited gastric cancer cell proliferation and synergistically enhanced the lethal effect of 5-FU. Thus, traditional chemo-/radio- therapeutics supplemented with scFv-Reg4 may provide advances in the strategy for gastrointestinal cancer treatment.

scholarly journals TUNEL Assay: A Powerful Tool for Kidney Injury Evaluation

2021 ◽  
Vol 22 (1) ◽  
pp. 412
Author(s):  
Christopher L. Moore ◽  
Alena V. Savenka ◽  
Alexei G. Basnakian
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Cell Injury ◽  
Kidney Injury ◽  
Cell Types ◽  
Tunel Assay ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Hypoxic Injury ◽  
Additional Information ◽  
Tissue Compartments

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay is a long-established assay used to detect cell death-associated DNA fragmentation (3’-OH DNA termini) by endonucleases. Because these enzymes are particularly active in the kidney, TUNEL is widely used to identify and quantify DNA fragmentation and cell death in cultured kidney cells and animal and human kidneys resulting from toxic or hypoxic injury. The early characterization of TUNEL as an apoptotic assay has led to numerous misinterpretations of the mechanisms of kidney cell injury. Nevertheless, TUNEL is becoming increasingly popular for kidney injury assessment because it can be used universally in cultured and tissue cells and for all mechanisms of cell death. Furthermore, it is sensitive, accurate, quantitative, easily linked to particular cells or tissue compartments, and can be combined with immunohistochemistry to allow reliable identification of cell types or likely mechanisms of cell death. Traditionally, TUNEL analysis has been limited to the presence or absence of a TUNEL signal. However, additional information on the mechanism of cell death can be obtained from the analysis of TUNEL patterns.

scholarly journals 3,4-dehydro-L-proline Induces Programmed Cell Death in the Roots of Brachypodium distachyon

2021 ◽  
Vol 22 (14) ◽  
pp. 7548
Author(s):  
Artur Pinski ◽  
Alexander Betekhtin ◽  
Jolanta Kwasniewska ◽  
Lukasz Chajec ◽  
Elzbieta Wolny ◽  
...  
Keyword(s):  
Cell Death ◽  
Brachypodium Distachyon ◽  
Root Hairs ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cell Wall Proteins ◽  
Root Epidermis ◽  
Root Hair Development ◽  
Transmission Electron ◽  
Hair Development

As cell wall proteins, the hydroxyproline-rich glycoproteins (HRGPs) take part in plant growth and various developmental processes. To fulfil their functions, HRGPs, extensins (EXTs) in particular, undergo the hydroxylation of proline by the prolyl-4-hydroxylases. The activity of these enzymes can be inhibited with 3,4-dehydro-L-proline (3,4-DHP), which enables its application to reveal the functions of the HRGPs. Thus, to study the involvement of HRGPs in the development of root hairs and roots, we treated seedlings of Brachypodium distachyon with 250 µM, 500 µM, and 750 µM of 3,4-DHP. The histological observations showed that the root epidermis cells and the cortex cells beneath them ruptured. The immunostaining experiments using the JIM20 antibody, which recognizes the EXT epitopes, demonstrated the higher abundance of this epitope in the control compared to the treated samples. The transmission electron microscopy analyses revealed morphological and ultrastructural features that are typical for the vacuolar-type of cell death. Using the TUNEL test (terminal deoxynucleotidyl transferase dUTP nick end labelling), we showed an increase in the number of nuclei with damaged DNA in the roots that had been treated with 3,4-DHP compared to the control. Finally, an analysis of two metacaspases’ gene activity revealed an increase in their expression in the treated roots. Altogether, our results show that inhibiting the prolyl-4-hydroxylases with 3,4-DHP results in a vacuolar-type of cell death in roots, thereby highlighting the important role of HRGPs in root hair development and root growth.

scholarly journals LDH as a prognostic marker in hypertensive pregnancy

2017 ◽  
Vol 6 (6) ◽  
pp. 2444
Author(s):  
Prathap Talwar ◽  
Triveni Kondareddy ◽  
Pranidha Shree C. A.
Keyword(s):  
Lactic Dehydrogenase ◽  
High Serum ◽  
Pregnancy Induced Hypertension ◽  
Intracellular Enzyme ◽  
Cellular Death ◽  
Severity Of Disease ◽  
Induced Hypertension ◽  
Prospective Comparative Study ◽  
Severity Of The Disease ◽  
Poor Outcomes

Background: Pregnancy induced hypertension (PIH) is a global problem with a 5-15% incidence rate in India and complicating 10-17% of all pregnancies. These are multisystem disorders and lead to a lot of cellular death. LDH is an intracellular enzyme and its level is increased in these women due to cellular death. So, serum LDH levels can be used to assess the extent of cellular death and thereby the severity of disease in this group of women. The objective of the study was to correlate the severity of the disease, maternal and perinatal outcome with Lactic Dehydrogenase (LDH) levels in serum in patients of preeclampsia and eclampsia.Methods: A prospective comparative study was conducted in the department of Obstetrics and Gynecology, JSS Medical Hospital, Mysore.Results: LDH levels were significantly elevated in women with preeclampsia and eclampsia (<0.001). Higher LDH levels had significant correlation with high blood pressure (P <0.10) as well as poor maternal and perinatal outcome.Conclusions: High serum LDH levels correlate well with the severity of the disease and poor outcomes in patients of preeclampsia and eclampsia.

Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase-3-independent pathway

2004 ◽  
Vol 287 (4) ◽  
pp. H1730-H1739 ◽  
Author(s):  
Ron Zohar ◽  
Baoqian Zhu ◽  
Peter Liu ◽  
Jaro Sodek ◽  
C. A. McCulloch
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Cardiac Fibroblasts ◽  
Chromatin Condensation ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tunel Staining ◽  
Wild Type ◽  
Nuclear Fragmentation ◽  
A Cell

Reperfusion-induced oxidative injury to the myocardium promotes activation and proliferation of cardiac fibroblasts and repair by scar formation. Osteopontin (OPN) is a proinflammatory cytokine that is upregulated after reperfusion. To determine whether OPN enhances fibroblast survival after exposure to oxidants, cardiac fibroblasts from wild-type (WT) or OPN-null (OPN−/−) mice were treated in vitro with H2O2to model reperfusion injury. Within 1 h, membrane permeability to propidium iodide (PI) was increased from 5 to 60% in OPN−/−cells but was increased to only 20% in WT cells. In contrast, after 1–8 h of treatment with H2O2, the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-stained cells was more than twofold higher in WT than OPN−/−cells. Electron microscopy of WT cells treated with H2O2showed chromatin condensation, nuclear fragmentation, and cytoplasmic and nuclear shrinkage, which are consistent with apoptosis. In contrast, H2O2-treated OPN−/−cardiac fibroblasts exhibited cell and nuclear swelling and membrane disruption that are indicative of cell necrosis. Treatment of OPN−/−and WT cells with a cell-permeable caspase-3 inhibitor reduced the percentage of TUNEL staining by more than fourfold in WT cells but decreased staining in OPN−/−cells by ∼30%. Although the percentage of PI-permeable WT cells was reduced threefold, the percent of PI-permeable OPN−/−cells was not altered. Restoration of OPN expression in OPN−/−fibroblasts reduced the percentage of PI-permeable cells but not TUNEL staining after H2O2treatment. Thus H2O2-induced cell death in OPN-deficient cardiac fibroblasts is mediated by a caspase-3-independent, necrotic pathway. We suggest that the increased expression of OPN in the myocardium after reperfusion may promote fibrosis by protecting cardiac fibroblasts from cell death.

scholarly journals Apoptosis as a Mechanism for Keratinocyte Death in Canine Toxic Epidermal Necrolysis

2016 ◽  
Vol 54 (2) ◽  
pp. 249-253 ◽  
Author(s):  
F. Banovic ◽  
S. Dunston ◽  
K. E. Linder ◽  
P. Rakich ◽  
T. Olivry
Keyword(s):  
Cell Death ◽  
Toxic Epidermal Necrolysis ◽  
Caspase 3 ◽  
Skin Lesions ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Epidermal Keratinocytes ◽  
Biopsy Specimens ◽  
Significant Difference ◽  
Keratinocyte Cell ◽  
Life Threatening

In humans and dogs, toxic epidermal necrolysis (TEN) is a life-threatening dermatosis characterized by sudden epidermal death resulting in extensive skin detachment. There is little information on the pathogenesis of keratinocyte cell death in canine TEN. We studied the occurrence of apoptosis in skin lesions of dogs with TEN to determine if apoptosis contributes to the pathogenesis of this disease. Immunostaining with antibodies to activated caspase-3 and the terminal deoxynucleotidyl-transferase (TdT)–mediated deoxyuridine triphosphate (dUTP) nick-end labeling technique revealed positive apoptotic keratinocytes in basal and suprabasal epidermal compartments in 17 biopsy specimens collected from 3 dogs with TEN and 16 from 3 dogs with erythema multiforme (EM). There was no significant difference in the number of positively stained epidermal cells between TEN and EM. These results suggest that apoptosis of epidermal keratinocytes and lymphocytic satellitosis represent one of the early steps in the pathogenesis of canine TEN, as in the human disease counterpart.

Extracellular ATP causes apoptosis and necrosis of cultured mesangial cells via P2Z/P2X7receptors

1998 ◽  
Vol 275 (6) ◽  
pp. F962-F971 ◽  
Author(s):  
Eckhard Schulze-Lohoff ◽  
Christian Hugo ◽  
Sylvia Rost ◽  
Susanne Arnold ◽  
Angela Gruber ◽  
...  
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Mesangial Cells ◽  
Lucifer Yellow ◽  
Fold Increase ◽  
Extracellular Atp ◽  
Glomerular Disease ◽  
Terminal Deoxynucleotidyl Transferase ◽  
P53 Expression ◽  
Apoptosis And Necrosis

Mesangial cells undergo cell death both by apoptosis and necrosis during glomerular disease. Since nucleotides are released from injured and destroyed cells in the glomerulus, we examined whether extracellular ATP and its receptors may regulate cell death of cultured mesangial cells. Addition of extracellular ATP (300 μM to 5 mM) to cultured rat mesangial cells for 90 min caused a 5.8-fold increase in DNA fragmentation (terminal deoxynucleotidyl transferase assay) and a 4.2-fold increase in protein levels of the tumor suppressor p53, which is thought to regulate apoptosis. Apoptotic DNA fragmentation was confirmed by the diphenylamine assay and by staining with the DNA-specific fluorochrome Hoechst 33258. The necrotic markers, release of lactate dehydrogenase and uptake of trypan blue, were not positive before 3 h of ATP addition. The effects of ATP on DNA fragmentation and p53 expression were reproduced by the purinergic P2Z/P2X7 receptor agonist, 3′- O-(4-benzoylbenzoyl)-ATP, and inhibited by the P2Z/P2X7 receptor blocker, oxidized ATP. Transcripts encoding the P2Z/P2X7 receptor were expressed by cultured mesangial cells as determined by Northern blot analysis. P2Z/P2X7 receptor-associated pore formation in the plasma membrane was demonstrated by the Lucifer yellow assay. We conclude that activation of P2Z/P2X7 receptors by extracellular ATP causes apoptosis and necrosis of cultured mesangial cells. Activation of purinergic P2Z/P2X7 receptors may play a role in causing death of mesangial cells during glomerular disease.

scholarly journals Embryo Buoyancy and Cell Death Gene Expression During Embryogenesis of Yellow-Tail Kingfish Seriola lalandi

2021 ◽  
Vol 9 ◽  
Author(s):  
Jaime Palomino ◽  
Camila Gómez ◽  
María Teresa Otarola ◽  
Phillip Dettleff ◽  
Daniel Patiño-García ◽  
...  
Keyword(s):  
Cell Death ◽  
Developmental Stages ◽  
Pelagic Fish ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Fish Embryo ◽  
Developmental Potential ◽  
Protein Levels ◽  
Apoptosis Pathways ◽  
Apoptosis Process ◽  
Seriola Lalandi

In pelagic fish, embryo buoyancy is a noteworthy aspect of the reproductive strategy, and is associated with overall quality, survival, and further developmental success. In captivity, the loss of buoyancy of early embryos correlates with high mortality that might be related to massive cell death. Therefore, the aim of this study was to evaluate under captivity conditions the expression of genes related to the apoptosis process during the early embryonic development of the pelagic fish Seriola lalandi, and its relationship to the buoyancy of embryos. The relative expression of bcl2, bax-like, casp9, casp8, and casp3 was evaluated by RT-qPCR and FasL/Fas protein levels by western blot in five development stages of embryos sorted as floating or low-floating. All genes examined were expressed in both floating and low-floating embryos up to 24 h of development. Expression of the pro-apoptotic factors bax, casp9, casp8, and casp3 was higher in low-floating as compared with floating embryos in a developmental stage-specific manner. In contrast, there was no difference in expression of bcl2 between floating and low-floating embryos. Fas protein was detected as a single band in floating embryos without changes in expression throughout development; however, in low-floating embryos, three higher intensity reactive bands were detected in the 24-h embryos. Interestingly, FasL was only detected at 24-h in floating embryos, whereas in low-floating samples this ligand was present at all stages, with a sharp increase as development progressed. Cell death, as evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, was highly increased in low-floating embryos as compared to floating embryos throughout all developmental stages, with the highest levels observed during the gastrula stage and at 24 h. The results of this study suggest that an increase in cell death, probably associated with the intrinsic and extrinsic apoptosis pathways, is present in low-floating embryos that might explain their lower developmental potential under captivity conditions.

scholarly journals Inhibiting autophagy targets human leukemic stem cells and hypoxic AML blasts by disrupting mitochondrial homeostasis

2021 ◽  
Vol 5 (8) ◽  
pp. 2087-2100
Author(s):  
Kaitlyn M. Dykstra ◽  
Hannah R. S. Fay ◽  
Ashish C. Massey ◽  
Neng Yang ◽  
Matthew Johnson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Mitochondrial Respiration ◽  
Prolonged Exposure ◽  
Tumor Burden ◽  
Therapeutic Interventions ◽  
Autophagic Flux ◽  
Mitochondrial Homeostasis ◽  
Nsg Mice

Abstract Leukemia stem cells (LSCs) and therapy-resistant acute myeloid leukemia (AML) blasts contribute to the reinitiation of leukemia after remission, necessitating therapeutic interventions that target these populations. Autophagy is a prosurvival process that allows for cells to adapt to a variety of stressors. Blocking autophagy pharmacologically by using mechanistically distinct inhibitors induced apoptosis and prevented colony formation in primary human AML cells. The most effective inhibitor, bafilomycin A1 (Baf A1), also prevented the in vivo maintenance of AML LSCs in NSG mice. To understand why Baf A1 exerted the most dramatic effects on LSC survival, we evaluated mitochondrial function. Baf A1 reduced mitochondrial respiration and stabilized PTEN-induced kinase-1 (PINK-1), which initiates autophagy of mitochondria (mitophagy). Interestingly, with the autophagy inhibitor chloroquine, levels of enhanced cell death and reduced mitochondrial respiration phenocopied the effects of Baf A1 only when cultured in hypoxic conditions that mimic the marrow microenvironment (1% O2). This indicates that increased efficacy of autophagy inhibitors in inducing AML cell death can be achieved by concurrently inducing mitochondrial damage and mitophagy (pharmacologically or by hypoxic induction) and blocking mitochondrial degradation. In addition, prolonged exposure of AML cells to hypoxia induced autophagic flux and reduced chemosensitivity to cytarabine (Ara-C), which was reversed by autophagy inhibition. The combination of Ara-C with Baf A1 also decreased tumor burden in vivo. These findings demonstrate that autophagy is critical for mitochondrial homeostasis and survival of AML cells in hypoxia and support the development of autophagy inhibitors as novel therapeutic agents for AML.

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