Integrin β3 prevents apoptosis of HL-1 cardiomyocytes under conditions of oxidative stressThis article is one of a selection of papers published in a Special Issue on Oxidative Stress in Health and Disease.

2010 ◽  
Vol 88 (3) ◽  
pp. 324-330 ◽  
Author(s):  
Lee J. Shewchuk ◽  
Sean Bryan ◽  
Marina Ulanova ◽  
Neelam Khaper
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Flow Cytometry ◽  
Gene Silencing ◽  
Cardiac Remodeling ◽  
Caspase 3 ◽  
Annexin V ◽  
Fold Increase ◽  
Cardiomyocyte Apoptosis ◽  
Active Caspase

Integrin receptors are essential in the regulation of vital cardiac functions, and impaired integrin activity has been associated with cardiac remodeling. Oxidative stress is known to be involved in apoptosis and cardiac remodeling and thus may profoundly influence cardiac function via integrin modulation. The aim of this study was to determine the expression pattern and functional role of integrins in HL-1 cardiomyocytes under conditions of oxidative stress. Gene expression was studied by end-point and real-time PCR; surface protein expression was studied by flow cytometry; integrin knockdown was accomplished by siRNA gene silencing; and apoptosis was studied by annexin V staining and active caspase-3/7 using flow cytometry. Among the various subunits under study (αv, α5, α6, and β1, β3, β4, and β5), the expression of β3 integrin was significantly increased at both the mRNA and protein levels in cardiomyocytes exposed to 100 µmol/L hydrogen peroxide for 3 h. Gene silencing of β3 integrin by using siRNA resulted in a 2-fold increase in cardiomyocyte apoptosis upon treatment with hydrogen peroxide. This increase in apoptosis, as measured by annexin V staining, correlated with an increase in active caspase-3/7. Integrin β3 plays a vital role in preventing cardiomyocyte apoptosis under conditions of oxidative stress.

Effects of Arsenic Trioxide on Megakaryocytic Cell Lines: Apoptosis, Cell Cycle Dysfunction and Signaling Pathways.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4459-4459
Author(s):  
Hubert K.B. Lam ◽  
Karen K.H. Li ◽  
Ki Wai Chik ◽  
Mo Yang ◽  
Carmen K.Y. Chuen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Annexin V ◽  
Caspase 8 ◽  
Mrna Levels ◽  
Active Caspase

Abstract Despite progress made in the elucidation of the actions of arsenic trioxide (ATO) in acute promyelocytic leukemia, the molecular mechanisms leading to apoptosis in other malignancies remain unclear. In particular, the effects of ATO on the megakaryocytic (MK) lineage have not been well characterized. In this study, we focused on two MK cell lines CHRF-288-11 (CHRF) and MEG-01, which were derived from an infant and adult acute megakaryocytic leukemia (AMKL), respectively. Our data showed that these cells underwent apoptosis within 24 – 48 h post-ATO (6 μM) treatment, as demonstrated by the Annexin V assay (Table 1). By flow cytometry, significant activation of caspase-3 was detected in the MK cells at 24 h, and was preceded by the loss of mitochondrial membrane potential (8 h) as determined by the fluorescent dye JC-1. Western blotting experiments showed that ATO induced Bax expression and down-regulated Bcl-2, which led to an increase in Bax/Bcl-2 ratio. ATO exerted immediate and significant interference on the cell cycle by delaying S-phase progression and the subsequent accumulation of cells in the G2/M phase (43.2% vs 13.6%, p < 0.01). By multivariate analysis (BrdU and 7-AAD), active caspase-3 was detected in all phases of the cell cycle. The responses of CHRF and MEG-01 cells to ATO were similar, except that the latter appeared more resistant, in terms of the dosage of ATO and the slight delayed onset of apoptosis. We screened the expression levels of 96 genes involved in apoptosis using the GEArray Q Series Human Apoptosis Gene Array at 0, 4, 8 and 16 h (each n = 2) post-ATO treatment. We identified the up-regulation of mRNA of two extrinsic components of apoptosis. Fas was progressively increased in both cell lines (up to 6.14-fold) and caspase-8 was elevated in MEG-01 (3.58-fold). The protein expressions of Fas and activated caspase-8 were demonstrated in both cell lines by flow cytometry. Increased mRNA expressions of caspase-1 (2.30-fold) and CD137 (2.33-fold) were also noted, but their significance in apoptosis of our system remained to be investigated. To demonstrate the direct effect of ATO on gene expressions in AMKL cells, a more comprehensive microarray (Human 19K Array, Ontario Cancer Institute Microarray Centre) was used. Treatment with ATO for 4 h (n = 3) prompted an elevation in the mRNA levels of stress-associated proteins, such as metallothioneins (MT1G: 6.31-fold; MT2A: 3.64-fold), Hsp72 (5.81-fold), Hsp73 (3.77-fold), Hsp90 (2.11-fold), ferritin (2.02-fold) and ubiquitin (2.76-fold). Interestingly, WT1, a cell cycle regulatory gene elevated in many types of leukemia, was induced by ATO (2.44-fold). In conclusion, our results suggested that apoptosis in AMKL cells mediated by ATO involved a switch from pro-survival in the early phase to the activation of multiple death machineries, consisting of the intrinsic (mitochondrial, Bax, Bcl-2) and the extrinsic (Fas, caspase-8) compartments. Table 1: Signals regulated by ATO in CHRF cells 0 h 24 h 48 h Mean ± SEM; * p < 0.05 compared to 0 h; # n = 2, others n = 3–5. Annexin V +/PI − (%) 4.56±0.28 8.28±0.53* 9.83±0.73* Active caspase-3 (%) 2.28±0.13 4.58±0.87* 14.7±1.16* JC-1 greenhi/redlo (%) 4.18±0.52 8.05±0.60* 20.76±8.69* Bax/Bcl-2 (Fold)# 0.63±0.08 2.65±0.68 - Fas (Fold) 1 1.73±0.17* 1.96±0.20* CD137 (Fold) 1 1.55±0.08* 1.76±0.03*

scholarly journals Vitamin C Inhibits Aggravated Eryptosis by Hydrogen Peroxide in Glucose-6-Phosphated Dehydrogenase Deficiency

2016 ◽  
Vol 39 (4) ◽  
pp. 1453-1462 ◽  
Author(s):  
Feng Shan ◽  
Rui Yang ◽  
Tiemei Ji ◽  
Fengjun Jiao
Keyword(s):  
Hydrogen Peroxide ◽  
Vitamin C ◽  
G6pd Deficiency ◽  
Caspase 3 ◽  
Annexin V ◽  
Normal Activity ◽  
Protective Effects ◽  
Blank Group ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Active Caspase

Background/Aims: The study was aimed to investigate if vitamin C could exert protective effects on development of eryptosis caused by glucose-6-phosphate dehydrogenase (G6PD) deficiency and hydrogen peroxide. Methods: Isolated erythrocytes with different G6PD activity (normal or deficient) were divided into various groups treated by either Vitamin C or H2O2. Phosphatidylserine (PS) extroversion rate was detected by Annexin V binding. The intracellular Ca2+ concentration was detected by Fluo3-fluorescence, and western blot was used to detect the expression of apoptosis factor caspase 3. Results: Compared with the blank group, the PS extroversion rate (P < 0.001), intracellular Ca2+ concentration (P < 0.001) and active caspase 3 expression level (P < 0.05) of erythrocytes significantly increased after treatment of 0.05% H2O2. Then the index of eryptosis significantly decreased after erythrocytes were treated with Vitamin C (1 mg/ml) for 30 min (all P < 0.05). The decline in erythrocytes with G6PD normal activity was more significant than those with G6PD deficiency. Conclusion: Vitamin C could effectively inhibit the eryptosis contributed by H2O2 oxidative stress, and the suppression on eryptosis with G6PD normal activity was more effective than that with G6PD deficiency.

Combination of Histone Deacetylase Inhibitor with Cu(II) 5,5- diethylbarbiturate Complex Induces Apoptosis in Breast Cancer Stem Cells: A Promising Novel Approach

2020 ◽  
Vol 21 ◽  
Author(s):  
Merve Erkisa ◽  
Nazlihan Aztopal ◽  
Elif Erturk ◽  
Engin Ulukaya ◽  
Veysel T. Yilmaz ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Oxidative Stress ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Histone Deacetylases ◽  
Caspase 3 ◽  
Cancer Cell Line ◽  
Annexin V ◽  
Tumor Formation ◽  
Dependent Manner

Background: Cancer stem cells (CSC) are subpopulation within the tumor that acts a part in the initiation, progression, recurrence, resistance to drugs and metastasis of cancer. It is well known that epigenetic changes lead to tumor formation in cancer stem cells and show drug resistance. Epigenetic modulators and /or their combination with different agents have been used in cancer therapy. Objective: In our study we scope out the effects of combination of a histone deacetylases inhibitor, valproic acid (VPA), and Cu(II) complex [Cu(barb-κN)(barb-κ2N,O)(phen-κN,N’)]·H2O] on cytotoxicity/apoptosis in a stem-cell enriched population (MCF-7s) obtained from parental breast cancer cell line (MCF-7). Methods: Viability of the cells was measured by the ATP assay. Apoptosis was elucidated via the assessment of caspase-cleaved cytokeratin 18 (M30 ELISA) and a group of flow cytometry analysis (caspase 3/7 activity, phosphatidylserine translocation by annexin V-FITC assay, DNA damage and oxidative stress) and 2ˈ,7ˈ–dichlorofluorescein diacetate staining. Results: The VPA combined with Cu(II) complex showed anti proliferative activity on MCF-7s cells in a dose- and time-dependently. Treatment with combination of 2.5 mM VPA and 3.12 μM Cu(II) complex induces oxidative stress in a time-dependent manner, as well as apoptosis that is evidenced by the increase in caspase 3/7 activity, positive annexin-V-FITC, and increase in M30 levels. Conclusion: The results suggest that the combination therapy induces apoptosis following increased oxidative stress, thereby making it a possible promising therapeutic strategy that further analysis is required.

scholarly journals Selected Kefir Water from Malaysia Attenuates Hydrogen Peroxide-Induced Oxidative Stress by Upregulating Endogenous Antioxidant Levels in SH-SY5Y Neuroblastoma Cells

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 940
Author(s):  
Muganti Rajah Kumar ◽  
Swee Keong Yeap ◽  
Han Chung Lee ◽  
Nurul Elyani Mohamad ◽  
Muhammad Nazirul Mubin Aziz ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Neuroblastoma Cells ◽  
Annexin V ◽  
Morphological Features ◽  
Lower Percentage ◽  
Total Phenolic ◽  
Neuroprotective Effects ◽  
Antioxidant Power ◽  
Pre Treatment

Kefir, a fermented probiotic drink was tested for its potential anti-oxidative, anti-apoptotic, and neuroprotective effects to attenuate cellular oxidative stress on human SH-SY5Y neuroblastoma cells. Here, the antioxidant potentials of the six different kefir water samples were analysed by total phenolic content (TPC), total flavonoid content (TFC), ferric reducing antioxidant power (FRAP), and 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH) assays, whereas the anti-apoptotic activity on hydrogen peroxide (H2O2) induced SH-SY5Y cells was examined using MTT, AO/PI double staining, and PI/Annexin V-FITC assays. The surface and internal morphological features of SH-SY5Y cells were studied using scanning and transmission electron microscopy. The results indicate that Kefir B showed the higher TPC (1.96 ± 0.54 µg GAE/µL), TFC (1.09 ± 0.02 µg CAT eq/µL), FRAP (19.68 ± 0.11 mM FRAP eq/50 µL), and DPPH (0.45 ± 0.06 mg/mL) activities compared to the other kefir samples. The MTT and PI/Annexin V-FITC assays showed that Kefir B pre-treatment at 10 mg/mL for 48 h resulted in greater cytoprotection (97.04%), and a significantly lower percentage of necrotic cells (7.79%), respectively. The Kefir B pre-treatment also resulted in greater protection to cytoplasmic and cytoskeleton inclusion, along with the conservation of the surface morphological features and the overall integrity of SH-SY5Y cells. Our findings indicate that the anti-oxidative, anti-apoptosis, and neuroprotective effects of kefir were mediated via the upregulation of SOD and catalase, as well as the modulation of apoptotic genes (Tp73, Bax, and Bcl-2).

miR-187 modulates cardiomyocyte apoptosis and oxidative stress in myocardial infarction mice via negatively regulating DYRK2

2021 ◽  
Keyword(s):  
Oxidative Stress ◽  
Myocardial Infarction ◽  
Protective Effect ◽  
Myocardial Infarct ◽  
Annexin V ◽  
Cardiomyocyte Apoptosis ◽  
Ros Generation ◽  
Downstream Target

Myocardial infarction is a serious representation of cardiovescular disease, MicroRNAs play a role in modifying I/R injury and myocardial infarct remodeling. The present study therefore examined the potential role of miR-187 in cardiac I/R injury and its underlying mechanisms. miR-187 was inhibited or overexpressed in cardiomyocytes H/R models by pretreatment with miR-187 mimic or inhibitor to confirm the function of miR-187 in H/R. DYRK2 was inhibited or overexpressed in cardiomyocytes H/R models by pretreatment with DYRK2 inhibitor. A myocardium I/R mouse model was established. Circulating levels of miR-187 or DYRK2 was detected by quantitative realtime PCR and protein expression was detected by western blotting. The cell viability in all groups was determined by MTT assay and the apoptosis ratio was detected by flow cytometry after staining with Annexin V-FITC. The effect of miR-187 on cellular ROS generation was examined by DCFH-DA. The level of lipid peroxidation and SOD expression were determined by MDA and SOD assay. The findings indicated that miR-187 may be a possible regulator in the protective effect of H/R-induced cardiomyocyte apoptosis, cellular oxidative stress and leaded to DYRK2 suppression at a posttranscriptional level. Moreover, the improvement of miR-187 on H/R-induced cardiomyocyte injury contributed to the obstruction of DYRK2 expression. In addition, these results identified DYRK2 as the functional downstream target of miR-187 regulated myocardial infarction and oxidative stress.These present work provided the first insight into the function of miR-187 in successfully protect cardiomyocyte both in vivo and in vitro, and such a protective effect were mediated through the regulation of DYRK2 expression.

scholarly journals EXPERIMENTAL MODELING OF APOPTOSIS UNDER CONDITIONS OF STABLE STRONTIUM EXPOSURE

2019 ◽  
Vol 21 (1) ◽  
pp. 137-140
Author(s):  
O. V. Dolgikh ◽  
N. V. Zaitseva ◽  
D. G. Dianova ◽  
A. V. Krivtsov ◽  
K. D. Starkova ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Annexin V ◽  
Fold Increase ◽  
Apoptosis Markers ◽  
Stable Strontium ◽  
Cell Death Mechanisms ◽  
Apoptotic Pathways ◽  
And Control

Apoptosis is defined as a highly regulated form of programmed cell death with typical morphological and biochemical features. A variety of factors, including heavy metals, may influence the intensity of programmed cell death. The aim of the work was to simulate apoptosis in an in vitrosystem under the conditions of stable strontium exposure. The children’s population consuming drinking water with high strontium (Sr2+) content (n = 49) was observed. The level of lymphocyte apoptosis was determined with flow cytometry technique, by means of labeled annexin V-FITC conjugate (AnnV-FITC) and propidium iodide (PI) staining. AnnV-FITC+PI- cells were regarded as early apoptotic forms, whereas late apoptotic and/or necrotic cells were AnnV-FITC+PI+. The isolated leukocytes were incubated with Sr2+ at a concentration of 7.0 mg/l, the maximal permitted concentration (MPC) for water of aqueous objects, for 4 hours at 37 ºC. Expression of CD95 and p53 apoptosis markers was performed by flow cytometry using labeled monoclonal antibodies.In vitroexposure to strontium was associated with significantly decreased expression of apoptosisregulating factors, i.e., membrane marker CD95 and intracellular transcription protein p53, 1.56- and 1.68-fold, respectively. Meanwhile, we revealed a significantly (4.68-fold) decreased amounts of AnnV-FITC+PI--cells, as well as a statistically significant (1.35-fold) increase of the AnnV-FITC+PI+-cells. Moreover, the amounts of AnnV-FITC+ PI--lymphocytes in all samples were below the physiological ranges and control values. The number of samples with higher contents of AnnV-FITC+PI+-lymphocyte exceeding the established standards and control values, was 30.8%. Thus, it has been experimentally proven that strontium, at a concentration corresponding to MPC for water objects may significantly inhibit cell death along apoptotic pathways, with switching to necrotic cell death mechanisms, according to phosphatidylserine contents, as detected by annexin V binding test. The data have revealed an ability of strontium to have a significant effect upon the parameters of regulation and maintenance of cellular homeostasis, by influencing the apoptosis intensity, due to shifting a balance towards necrosis and reducing expression of apoptosis-regulating factors. The results of this study may be used in order to identify some marker indexes of immune disorders potentially induced by external influence of strontium upon human health under specific environmental factors.

Epicatechin and its in vivo metabolite, 3′-O-methyl epicatechin, protect human fibroblasts from oxidative-stress-induced cell death involving caspase-3 activation

2001 ◽  
Vol 354 (3) ◽  
pp. 493-500 ◽  
Author(s):  
Jeremy P. E. SPENCER ◽  
Hagen SCHROETER ◽  
Gunter KUHNLE ◽  
S. Kaila S. SRAI ◽  
Rex M. TYRRELL ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Cell Death ◽  
Caspase 3 ◽  
Cell Damage ◽  
Protective Action ◽  
Human Fibroblasts ◽  
Membrane Damage ◽  
Antioxidant Properties

There is considerable current interest in the cytoprotective effects of natural antioxidants against oxidative stress. In particular, epicatechin, a major member of the flavanol family of polyphenols with powerful antioxidant properties in vitro, has been investigated to determine its ability to attenuate oxidative-stress-induced cell damage and to understand the mechanism of its protective action. We have induced oxidative stress in cultured human fibroblasts using hydrogen peroxide and examined the cellular responses in the form of mitochondrial function, cell-membrane damage, annexin-V binding and caspase-3 activation. Since one of the major metabolites of epicatechin in vivo is 3′-O-methyl epicatechin, we have compared its protective effects with that of epicatechin. The results provide the first evidence that 3′-O-methyl epicatechin inhibits cell death induced by hydrogen peroxide and that the mechanism involves suppression of caspase-3 activity as a marker for apoptosis. Furthermore, the protection elicited by 3′-O-methyl epicatechin is not significantly different from that of epicatechin, suggesting that hydrogen-donating antioxidant activity is not the primary mechanism of protection.

scholarly journals Thimerosal-Derived Ethylmercury Is a Mitochondrial Toxin in Human Astrocytes: Possible Role of Fenton Chemistry in the Oxidation and Breakage of mtDNA

2012 ◽  
Vol 2012 ◽  
pp. 1-12 ◽  
Author(s):  
Martyn A. Sharpe ◽  
Andrew D. Livingston ◽  
David S. Baskin
Keyword(s):  
Hydrogen Peroxide ◽  
Caspase 3 ◽  
Permeability Transition ◽  
Fold Increase ◽  
Hydrogen Peroxide Production ◽  
Fenton Chemistry ◽  
Human Astrocytes ◽  
Mitochondrial Toxin ◽  
Normal Human

Thimerosal generates ethylmercury in aqueous solution and is widely used as preservative. We have investigated the toxicology of Thimerosal in normal human astrocytes, paying particular attention to mitochondrial function and the generation of specific oxidants. We find that ethylmercury not only inhibits mitochondrial respiration leading to a drop in the steady state membrane potential, but also concurrent with these phenomena increases the formation of superoxide, hydrogen peroxide, and Fenton/Haber-Weiss generated hydroxyl radical. These oxidants increase the levels of cellular aldehyde/ketones. Additionally, we find a five-fold increase in the levels of oxidant damaged mitochondrial DNA bases and increases in the levels of mtDNA nicks and blunt-ended breaks. Highly damaged mitochondria are characterized by having very low membrane potentials, increased superoxide/hydrogen peroxide production, and extensively damaged mtDNA and proteins. These mitochondria appear to have undergone a permeability transition, an observation supported by the five-fold increase in Caspase-3 activity observed after Thimerosal treatment.

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