Abstract 146: Biphasic Effect Of Cyclic Amp Axis On Cardiomyocyte Survival

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Morihiko Aoyama ◽  
Yasuko K Bando ◽  
Haruya Kawase ◽  
Akio Monji ◽  
Toyoaki Murohara
Keyword(s):  
Cell Death ◽  
Cyclic Amp ◽  
Pressure Overload ◽  
Myocardial Cell ◽  
Threshold Concentration ◽  
High Concentration ◽  
Neonatal Cardiomyocytes ◽  
Myocardial Apoptosis ◽  
Vitro Analysis

Background: Persistent cardiac hypertrophy in response to pathological stimuli is results in maladaptive myocardial remodeling and cell death. Clinical evidence revealed that one of the most significantly beneficial medications for targeting heart failure with pathologic myocardial hypertrophy is beta1-adrenergic receptor (β1R) blockers. The molecular pathway of β1R is mediated by the second messenger cyclic AMP (cAMP). However, there are some debate regarding the role of cAMP in myocardial survival. We hypothesized whether there may be threshold concentration of cAMP in cell susceptibility to cardiomyocyte cell death. Methods: Male 14-week-old C57BL6 mice were subjected to the surgery of thoracic aortic constriction (TAC) to induce pressure overload. Changes in apoptosis were evaluated in each heart section and in vitro culture of neonatal cardiomyocytes using TUNEL. To elucidate the concentration-dependent distinct effect of cAMP on myocardial cell death, we tested the different concentration of cell-permeable cAMP (8-br-cAMP) at low (60 μM) and high concentration (6 mM), and receptor-mediated cAMP-stimulators (Ex4; exendin-4, ISO; isoproterenol). Results: In vitro analysis revealed that the high-cAMP and ISO exhibited marked increase in TUNEL-positivity (15.46%±3.09% for high-cAMP versus 6.71%±0.33% for ISO), which was reversed by Rp-cAMP (1.80%±0.17% and 2.05%±0.25%, respectively). Unexpectedly, the 8-p-Methoxyphenylthon-2-O-methyl-cAMP (pMe-cAMP, 50 μM), the specific activator of another cAMP-sensitive target Epac, reversed the high-cAMP-induced cell death even at a less extent compared to that observed by PKA-inhibitor Rp-cAMP (3.73%±0.70%). Serum depletion induced 3.22±0.24% of TUNEL-positive cell count of NRVM, which was reversed by pMe-cAMP, (50 μM) and Ex4 (1.74±0.18%, n=6, P<0.01), which was insensitive to PKA inhibition by Rp-cAMP (100 μM). TAC increased myocardial apoptosis. TAC-CON heart exhibited 1.66-fold decrease in cardiac cAMP concentration compared to sham-CON. Ex4 ameliorated the TAC-induced cardiac dysfunction and apoptosis by increase in cAMP. Conclusions: The cAMP-related cell death was mediated by PKA activation, which were reversed by Epac activation.

Abstract 145: Glucose Depletion Is Essential For The Calorie-restriction-mediated Cardiac Angiogenesis Via Pka/ampk-dependent Autophagy.

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Akio Monji ◽  
Yasuko K Bando ◽  
Morihiko Aoyama ◽  
Toko Mitsui ◽  
Haruya Kawase ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cyclic Amp ◽  
Culture Media ◽  
Pressure Overload ◽  
Cardiovascular Effects ◽  
Tube Formation ◽  
Simultaneous Increase ◽  
Glucose Depletion ◽  
Vitro Analysis

Background: Caloric restriction (CR) promotes beneficial cardiovascular effects; however, its effects on cardiac angiogenesis remain unclear. We thus examined whether CR may modulate cardiac angiogenesis via activation of autophagy and seeked for the essential nutrient factor(s) that may be responsible for the CR-mediated angiogenesis. Methods: To confirm whether CR-induced angiogenic effects may be universally observed, we allocated 2 distinct heart failure models; #1 pressure overload (4 weeks) generated by transaortic constriction (TAC) and #2 diet-induced obesity (DIO) mice, as a model of diabetic cardiomyopathy. Male mice (14 w/o) were randomly allocated to a 4-week CR (TAC-CR and DIO-CR) and ad libitum (TAC-AL and DIO-AL). Cultured endothelial cells (ECs) were exposed to culture media of 1) ordinary composition, 2) glucose-depleted, 3) amino acid-depleted, and 4) serum-depleted condition. To visualize cardiac autophagic changes, GFP-LC3 mice were allocated to DIO and TAC treatment. Analyses for the changes in activities of autophagy (LC3-turnover assay and p62 level) and angiogenesis (tube formation and Akt/AMPK/eNOS activity) were evaluated. Results: Immunohistochemical analyses revealed that CR enhanced cardiac angiogenesis both in TAC and DIO mice. CR promoted increased in myocardial cyclic AMP concentration with concomitant PKA/AMPK/eNOS activation and the simultaneous increase in autophagic activity both in TAC and DIO hearts. Of note, cardiac Akt activity remains unchanged by CR. In vitro analysis revealed that glucose depletion, as well as forskolin (10 μM) and 8-bromo-cyclic AMP (1 mM), activated PKA/AMPK axis thereby facilitated angiogenesis and autophagy in ECs. The enhanced in vitro angiogenesis induced by glucose depletion and PKA enhancers was abrogated by autophagy inhibitor 3-MA treatment. PKA inhibitors (H89 and RP-cAMP) abrogated the increase in AMPK/eNOS phosphorylation levels induced by glucose depletion. Neither amino acid- nor serum-depletion had no effect on angiogenesis and autophagy. Conclusions: Glucose depletion is essential for the CR-mediated activation of cardiac angiogenesis and autophagy in a cAMP/PKA/AMPK-dependent fashion.

scholarly journals Effects of acetylsalicylic acid and acetic acid solutions in VX2 carcinoma cells: In vitro analysis

2006 ◽  
Vol 21 (3) ◽  
pp. 151-154 ◽  
Author(s):  
Rogério Saad-Hossne ◽  
René Gamberini Prado ◽  
William Saad Hossne
Keyword(s):  
Cell Death ◽  
Acetic Acid ◽  
Cell Viability ◽  
Acetylsalicylic Acid ◽  
Neoplastic Cell ◽  
Carcinoma Cells ◽  
Acid Solutions ◽  
Vx2 Carcinoma ◽  
Vitro Analysis

PURPOSE: To analyze, in vitro, the effects of acetylsalicylic acid (aspirin) and acetic acid solutions on VX2 carcinoma cells in suspension and to examine the correlation between these effects and neoplastic cell death. METHODS: The VX2 tumor cells (10(7) cells/ml) were incubated in solutions containing differing concentrations (2.5% and 5%) of either acetylsalicylic acid or acetic acid, or in saline solution (controls). Every five minutes, cell viability was tested (using the trypan blue test) and analyzed under light microscopy. RESULTS: Tumor cell viability (in %) decreased progressively and, by 30 minutes, neoplastic cell death had occurred in all solutions. CONCLUSION: Based on this experimental model and the methodology employed, we conclude that these solutions cause neoplastic cell death in vitro.

Abstract 346: Lipophilic Statins Attenuate Human Atrial Fibroblast Viability In Vitro

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Kiranjit K Sran ◽  
Yun Li ◽  
Saeid Ghavami ◽  
Melanie Ngo ◽  
Rakesh C Arora ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Heart Surgery ◽  
Cardiac Fibrosis ◽  
Therapeutic Option ◽  
Open Heart Surgery ◽  
Myocardial Cell ◽  
Dependent Manner ◽  
Vitro Effect

Cardiovascular diseases (CVD) leading to heart failure are associated with myocardial cell loss and cardiac fibrosis. Hydroxymethylglutaryl-Coenzyme-A Reductase (HMGR) inhibitors ("statins") are widely used to limit cardiovascular events in patients with hypercholesterolemia and CVD by altering their lipid profile. HMGR inhibition reduces cholesterol precursor L-mevalonate production, whose depletion induces autophagy, apoptosis, and endoplasmic reticulum stress in various cell types. However it is unclear if this is a class effect or a phenomenon specific to various compounds. We examined the in vitro effect of HMGR inhibition on human atrial fibroblast (hATF) viability with particular reference to hydrophilic vs lipophilic compounds. Hypothesis- Lipophilic statins induce cell death in primary hATF via mevalonate depletion; whereas hydrophilic statins do not have any effect on hATF viability. IRB approval was obtained for collection of hATF from consenting patients undergoing open heart surgery. Cells were treated with atorvastatin, simvastatin or pravastatin (0.1, 1.0 or 10 λM) for 24, 48, 72 or 96 hours. Expression of proteins involved in the regulation of apoptosis and autophagy was assessed using immunoblotting. Cell viability was assessed using MTT assay. Treatment of hATF with 0.1 - 10 λM atorvastatin or simvastatin (lipophilic statins) resulted in progressively reduced cell viability in time and dose-dependent manner. Viability could be rescued by coincubation with mevalonate. Expression of key apoptotic cascade proteins -Bcl2, Bax and cleaved Caspase3 showed a clear induction of apoptosis. Also, there was an increase in Atg5-12 expression at 24h indicating induction of early autophagic response. Pravastatin (hydrophilic statin) did not affect cell viability or autophagy and apoptosis. We conclude that statin-induced cell death is mediated by mevalonate depletion, which activates intrinsic apoptotic pathways in hATF. Lipophilic statins impair the viability of hATFs in vitro, whereas hydrophilic statins have no effect on cell growth and cell viability of hATFs. This may represent an additional pleiotropic effect of statins, and may represent a novel therapeutic option for the prevention and treatment of cardiac fibrosis.

Oncogene-induced mitotic catastrophe and apoptosis in glioblastoma cells in vitro

2016 ◽  
Vol 4 ◽  
pp. 63-78
Author(s):  
Jolanta Zięba ◽  
Ewelina Stoczyńska-Fidelus ◽  
Piotr Rieske
Keyword(s):  
Cell Death ◽  
Cell Division ◽  
Mitotic Catastrophe ◽  
Glioblastoma Cells ◽  
Genetic Changes ◽  
Primary Brain Tumour ◽  
Problematic Issue ◽  
Vitro Analysis ◽  
In Vitro Analysis

Glioblastoma (GB) is one of the most prevalent and aggressive primary brain tumour. The median survival of GB patients who underwent standard therapy (neurosurgical resection, radiotherapy and chemotherapy) is 14 months. There for, GB treatment remains a great challenge. Furthermore, in vitro culturing of glioblastoma cells constitutes problematic issue. The reason for these difficulties might be found in 3 processes that were observed during in vitro cell culturing: apoptosis, mitotic catastrophe and senescence. The spontaneous occurrence of these phenomena leads to inhibition of cell division and / or cell death. Only a small percentage of glioblastoma cases present a genetic predisposition to be cultured in vitro. Analysis of available date indicate that 50% of GB cell lines are characterised by the coexistence of TP53 mutations and CDKN2A deletions. Comparing this percentage to 5% of coexistence of above-mentioned genetic changes in samples collected from patients (presenting) we can presume that the presence of those alterations has impact on stabilization of glioblastoma cell cultures. Importantly, proteins p16, p14 (encoded by CDKN2A) and TP53 protein are involved in the mentioned processes. In vitro dominates, senescence, apoptosis and mitotic catastrophe of glioblastoma cells. Identification of factors, responsible for spontaneous inhibition of cell division and / or cell death of glioblastoma cells in vitro, may provide a ground for a new GB therapeutic approach.

scholarly journals TAXOL INDUCES CELL DEATH WITH CYTOPLASM VACUOLIZATION IN PARAPTOSIS-LIKE BUT NOT ONCOSIS FASHION IN ASTC-a-1 CELLS

2013 ◽  
Vol 06 (04) ◽  
pp. 1350046
Author(s):  
YING-YAO QUAN ◽  
CHAOYANG WANG ◽  
XIAO-PING WANG ◽  
TONG-SHENG CHEN
Keyword(s):  
Cell Death ◽  
Annexin V ◽  
Typical Characteristic ◽  
High Concentration ◽  
Human Lung Carcinoma ◽  
Apoptosis Detection ◽  
Fcm Analysis ◽  
Cytoplasm Vacuolization

Recently, we found that high concentration of taxol (70 μM) induced cell death with cytoplasm vacuolization, the typical characteristic of both paraptosis and oncosis, in human lung carcinoma (ASTC-a-1) cells. This report was designed to further determine the form of taxol-induced cell death with cytoplasm vacuolization. It is generally considered that the cytoplasm vacuolization in oncosis due to the swelling of endoplasmic reticulum (ER), mitochondria, lysosomes and nuclei occurs after the loss of mitochondrial membrane potential (ΔΨm). However, flow cytometry (FCM) analysis showed that taxol-induced cytoplasm vacuolization preceded the loss of ΔΨm. Moreover, taxol treatment did not induce the collapse of microtubule, the typical characteristic of oncosis. These data demonstrated that taxol-induced cell death with cytoplasm vacuolization is not oncosis. FCM analysis by Annexin V-FITC/PI apoptosis detection kit further demonstrated that taxol-induced cell death with cytoplasm vacuolization is not apoptosis. In conclusion, in combination with our recent in vitro and in vivo data, this report further demonstrates that high concentration of taxol induces cell death with cytoplasm vacuolization in paraptosis-like but not oncosis fashion.

OSU03012, a Novel Celecoxib Derivative, Induces Apoptosis in Multiple Myeloma Cells Independently of Caspase Activation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5075-5075
Author(s):  
Shuhong Zhang ◽  
Valerie L. White ◽  
Amy Johnson ◽  
Ching-Shih Chen ◽  
Sherif S. Farag
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Plasma Cells ◽  
Parp Cleavage ◽  
Caspase Inhibitor ◽  
High Concentration ◽  
Down Regulation ◽  
Vitro Effect

Abstract Multiple myeloma (MM) is a clonal disorder affecting terminally differentiated B cells, with the accumulation of plasma cells in the bone marrow. Previous studies showed that OSU03012 is a novel celecoxib derivative lacking cyclooxygenase-2 inhibitory activity that induces apoptosis in various types of cancer cells and is being developed as an anti-cancer therapy in the NCI Rapid Access to Intervention Therapy (RAID). Here, we examined the in vitro effect of OSU03012 in MM cell lines (U266, ARH-77, IM-9 and RPMI8226). Cytotoxicity data indicated that mean LC50 (lethal concentration 50%) of OSU03012 was 6.25±0.86 μM at 24 hours and 4.23±0.87 μM at 72 hours in these four cell lines. Using annexin V/PI (propidium iodide) flow cytometry assay, OSU03012 was shown to induce apoptosis in MM cells. OSU03012 activated caspases-8, -9, and -3, induced PARP (POLY ADP-RIBOSE Polymerase) cleavage, and reduced survivin and XIAP expression after 6 and 24 hour exposure. Although the caspase inhibitor Q-VD-OPH treatment strongly blocked OSU03012-induced PARP cleavage, it did not inhibit OSU03012-induced apoptosis of MM cells. The pan-caspase inhibitor z-VAD-fmk did not prevent OSU03012 mediated cell death. Cell death with OSU03012 treatment was associated with significant down-regulation of phospho-Akt. Several substrates of AKT, including phospho-GSK-3 beta (Ser9), phospho-FoxO1a (Ser256) and phospho-MDM2 (Ser166) were also down-regulated by OSU03012 drug. OSU03012 triggered both early (6h) and late (24h) down-regulation of cyclin D1 expression, but cyclin A and B1 expression was down-regulated only at 24h. There was no induction of p21 or p27 protein levels by OSU03012. After 24-hour exposure, low concentration (1–5 μM) OSU03012 arrested MM cell lines in the G1 phase of the cell cycle while high concentration (10 μM) OSU03012 induced G2 phase arrested. OSU03012 decreased both phospho-Stat3 (Ser727) and Stat3 expression. OSU03012 has on effect on phosphorylated MAP kinase kinase1/2 (pMEK1/2) but it decreased MEK1/2 expression at 24h. The expression levels of Bcl-2 family proteins, Bcl-2, Mcl-1, BAX, and BIM did not alter with OSU03012 treatment suggesting that Bcl-2 members may not play direct or significant roles in inducing cell death. Taken together, we conclude that OSU03012 is potently active against MM cells by predominantly caspase-independent mechanisms, and may involve downstream pathways consequent to phopho-Akt down-regulation. These studies provide preclinical rationale for investigating OSU03012 in the treatment of MM.

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.

scholarly journals Calcium mediated functional interplay between myocardial cells upon laser-induced single-cell injury: an in vitro study of cardiac cell death signaling mechanisms

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Krishna Chander Sridhar ◽  
Nils Hersch ◽  
Georg Dreissen ◽  
Rudolf Merkel ◽  
Bernd Hoffmann
Keyword(s):  
Cell Death ◽  
Single Cell ◽  
Cell Injury ◽  
Specific Effect ◽  
Myocardial Cell ◽  
Myocardial Tissue ◽  
Cell Clusters ◽  
Signaling Events ◽  
Intracellular Ca

Abstract Background The electromechanical function of myocardial tissue depends on the intercellular communication between cardiomyocytes (CMs) as well as their crosstalk with other cell types. Cell injury, and subsequent death trigger inflammation as in myocardial infarction (MI) resulting in myocardial remodeling. Although mechanisms underlying myocardial cell death have been studied so far, the signaling events following single cell death and spontaneous response of connected cells in the myocardial tissue is still barely understood. Methods Here, we investigated the effect of laser-induced single cell death on Calcium (Ca2+) concentrations and transport in myocardial cell clusters in vitro. Spatial and temporal changes in intracellular Ca2+ concentrations [Ca2+]i were studied using a fluorescent calcium indicator, Fluo-4AM. Spontaneous signaling events following cell death were studied in rat embryonic cardiomyocytes and non-myocytes using separate cell culture systems. Results Cell death triggered spontaneous increase in intracellular Ca2+ levels ([Ca2+]i) of surrounding cells. The spread of the observed propagating Ca2+ signal was slow and sustained in myocytes while it was rapid and transient in fibroblasts (Fbs). Further, sustained high Ca2+ levels temporarily impaired the contractility in CMs. The cell-type specific effect of ablation was confirmed using separate cultures of CMs and Fbs. Comparing Ca2+ propagation speed in myocytes and fibroblasts, we argue for a diffusion-driven Ca2+ propagation in myocytes, but not in fibroblasts. Radial and sequential Ca2+ diffusion across the CMs through cell–cell contacts and presence of Cx43-based intercellular junctions indicated a gap junction flow of Ca2+. Conclusions These findings illustrate the spontaneous Ca2+-mediated functional interplay in myocardial cell clusters upon mechanical injury and, further, the difference in Ca2+ signaling in cardiomyocytes and fibroblasts.

Adaphostin-induced apoptosis in CLL B cells is associated with induction of oxidative stress and exhibits synergy with fludarabine

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 2099-2106 ◽  
Author(s):  
Tait D. Shanafelt ◽  
Yean K. Lee ◽  
Nancy D. Bone ◽  
Ann K. Strege ◽  
Ven L. Narayanan ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cells ◽  
Annexin V ◽  
Adenosine Diphosphate ◽  
Lymphocytic Leukemia ◽  
Parp Cleavage ◽  
Ros Generation ◽  
Preclinical Development ◽  
Vitro Analysis

AbstractB-cell chronic lymphocytic leukemia (CLL) is characterized by accumulation of clonal lymphocytes resistant to apoptosis. We evaluated the ability of the investigational antileukemic agent adaphostin to induce apoptosis in CLL B cells and synergize with fludarabine in vitro. Analysis by annexin V/propidium iodide (PI) staining revealed that the concentration of adaphostin required to induce 50% cell death (IC50) at 24 hours was 4.2 μM (range, 1.10-11.25 μM; median, 4.25 μM; n = 29) for CLL isolates and more than 10 μM for B and T cells from healthy donors. Immunoblots demonstrated adaphostin induced poly(adenosine diphosphate-ribose) polymerase (PARP) cleavage and cleavage of caspase-3 substrates, suggesting that adaphostin induces apoptosis. Adaphostin increased the level of reactive oxygen species (ROS) within CLL B cells, and the antioxidant N-acetylcysteine blocked both adaphostin-induced ROS generation and apoptosis. Adaphostin also caused a decrease in the level of the antiapoptotic protein Bcl-2. When adaphostin was combined with fludarabine (F-ARA-AMP), a synergistic effect on cell death was observed in all 10 CLL samples. These findings not only indicate that adaphostin induces apoptosis selectively in CLL B cells through a mechanism that involves ROS generation but also demonstrate its ability to augment the effects of fludarabine. Further preclinical development of adaphostin as a novel agent for the treatment of CLL appears warranted.

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