Biochemical Markers Of Apoptosis Research Articles

Mitsugumin 53 mitigation of ischemia–reperfusion injury in a mouse model

ObjectivePrimary graft dysfunction is often attributed to ischemia–reperfusion injury, and prevention would be a therapeutic approach to mitigate injury. Mitsugumin 53, a myokine, is a component of the endogenous cell membrane repair machinery. Previously, exogenous administration of recombinant human (recombinant human mitsugumin 53) protein has been shown to mitigate acute lung injury. In this study, we aimed to quantify a therapeutic benefit of recombinant human mitsugumin 53 to mitigate a transplant-relevant model of ischemia–reperfusion injury. MethodsC57BL/6J mice were subjected to 1 hour of ischemia (via left lung hilar clamp), followed by 24 hours of reperfusion. mg53−/− mice were administered exogenous recombinant human mitsugumin 53 or saline before reperfusion. Tissue, bronchoalveolar lavage, and blood samples were collected at death and used to quantify the extent of lung injury via histology and biochemical assays. ResultsAdministration of recombinant human mitsugumin 53 showed a significant decrease in an established biometric profile of lung injury as measured by lactate dehydrogenase and endothelin-1 in the bronchoalveolar lavage and plasma. Biochemical markers of apoptosis and pyroptosis (interleukin-1β and tumor necrosis factor-α) were also significantly mitigated, overall demonstrating recombinant human mitsugumin 53's ability to decrease the inflammatory response of ischemia–reperfusion injury. Exogenous recombinant human mitsugumin 53 administration showed a trend toward decreasing overall cellular infiltrate and neutrophil response. Fluorescent colocalization imaging revealed recombinant human mitsugumin 53 was effectively delivered to the endothelium. ConclusionsThese data demonstrate that recombinant human mitsugumin 53 has the potential to prevent or reverse ischemia–reperfusion injury–mediated lung damage. Although additional studies are needed in wild-type mice to demonstrate efficacy, this work serves as proof-of-concept to indicate the potential therapeutic benefit of mitsugumin 53 administration to mitigate ischemia–reperfusion injury.

Gaillardin, a potent sesquiterpene lactone induces apoptosis via down-regulation of NF‐κβ in gastric cancer cells, AGS and MKN45

Ethnopharmacological relevanceThe genus Inula has been traditionally used as folk medicine in treating different illnesses such as kidney stones, urethra infection, jaundice, bronchitis, respiratory diseases and cancer. Aim of the studyGaillardin, a sesquiterpene lactone isolated from Inula oculus-christi, seems to have great potential as an anti-cancer agent. This study was carried out to evaluate the anti-cancer properties of Gaillardin in gastric cancer cells and also its possible underlying mechanism. MethodsThe colorimetric MTT assay was used to assess metabolic activity of cells as an indicator of viability and cytotoxicity. Flow cytometry using Annexin V-FITC/PI was applied to detect and quantify the level of apoptosis. Detection of activated caspase-3, as a biochemical marker of apoptosis, was done using caspase-3 assay kit. Activation of NF-κB pathway was determined by western blotting. The mRNA expression levels of NF-κB target genes were measured using quantitative RT-PCR. Moreover, intracellular reactive oxygen species (ROS) production was evaluated. ResultsGaillardin significantly reduced cell viability in a time and dose-dependent manner. The inhibitory effect of Gaillardin was attributed to induction of apoptosis. Investigation about the underlying mechanism revealed that Gaillardin exerts its action through inhibition of NF-κB activation and subsequently down-regulation of genes (COX-2, MMP-9, TWIST-1, and BCl-2) regulated by NF-κB. Moreover, Gaillardin caused remarkable elevation in ROS production in AGS and MKN45 cells. ConclusionWe provided evidences about the role of NF-κB pathway in the induction of apoptosis by the sesquiterpene lactone Gaillardin in AGS and MKN45 cells for the first time, which suggest Gaillardin could be considered as a promising natural agent for further investigations to identify new potent anticancer drugs in the future.

Olea europaea L. Flowers as a new promising anticancer natural product: phenolic composition, antiproliferative activity and apoptosis induction

The aim of this study was to characterise phenolic compounds of olive flower obtained from Olive tree cultivar Chemlali and to investigate their anticancer effect on MCF-7 cells. Phenolic characterisation was determined using LC/MS-MS. Cytotoxicity of the extract was determined using MTT. Biochemical markers of apoptosis were evaluated by immunoblotting. Our results showed that olive flower contained significant amounts of phenolic compounds mainly flavonoids, secoiridoids and simple phenols. Furthermore, the phenolic extract exerted a significant reduction in MCF-7 cell viability (EC50 values equal to 220.8 μg/ml). Western blot analysis revealed the presence of the cleaved forms of Parp-1. The DAPI staining analysis demonstrated a significant reduction in the number of cells and a considerable change in the morphology of the treated cells. In conclusion, Olea europaea. L flower contained great amounts of different bio-phenols able to reduce the proliferative activity of breast cancer MCF-7 cells by the induction of apoptosis.

Effect of oleuropein on morphine-induced hippocampus neurotoxicity and memory impairments in rats.

Oleuropein, as an olive leaf extract antioxidant polyphenol, has been reported to be a free radical scavenger. This study was done to investigate the effects of oleuropein, against morphine-induced hippocampus neurotoxicity and memory impairment in rats. The Morris water maze (MWM) test was used to assess the effect of oleuropein (5, 15, and 30mg/kg, i.p., co-administrated with morphine) on spatial learning and memory of male Wistar rats which were treated with morphine sulfate (45mg/kg, s.c., 4weeks). In order to evaluate the cleaved caspase-3, Bax, and Bcl2 protein expression (as biochemical markers of apoptosis) in CA1 area of hippocampus tissue, the western blot test was used. Also, to evaluate the oxidative stress status of hippocampus CA1 area tissue, the malondialdehyde (MDA) level, superoxide dismutase (SOD) activity, and glutathione peroxidase (GPx) activity were assessed. The data showed that oleuropein treatment (15 and 30mg/kg) improves the spatial learning and memory impairments in morphine-treated animals. Also, oleuropein treatment decreased the apoptosis and oxidative stress levels in the hippocampus CA1 area of morphine-treated rats. Oleuropein can prevent the spatial learning and memory impairments in morphine-treated rats. Molecular mechanisms underlying the observed effects could be at least partially related to the inhibition of neuronal apoptosis and oxidative stress in the hippocampus CA1 area of morphine-treated rats.

Phytohormone Abscisic Acid Protects Human Neuroblastoma SH-SY5Y Cells Against 6-Hydroxydopamine-Induced Neurotoxicity Through Its Antioxidant and Antiapoptotic Properties.

Parkinson's disease (PD) is a destroying and prevalent neurodegenerative disease that is characterized by a progressive death of midbrain dopaminergic neurons. It is important to understand the possible neuroprotective effects of reagents that rescue the neurons from death and apoptosis. In this study, we investigated the effects of abscisic acid (ABA) on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in human dopaminergic neuroblastoma SH-SY5Y cell line as an in vitro model of PD. Cell damage was induced by 150 μM 6-OHDA and the cell viability was examined by 2-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) assay. Reactive oxygen species (ROS) and mitochondrial membrane potential were assessed by fluorescence probe methods. Biochemical markers of apoptosis were also determined by immunoblotting. The data showed that 6-OHDA caused a significant loss of cell viability and mitochondrial membrane potential. In addition, intracellular ROS, cleaved caspase-3, Bax:Bcl-2 ratio, and cytochrome c release were significantly increased in 6-OHDA-incubated cells. ABA (100 μM) elicited a significant protective effect and reduced biochemical markers of cell damage and death. Blockage of peroxisome proliferator-activated receptor (PPAR)γ completely prevented the effect of ABA on 6-OHDA-induced cell toxicity. The results suggest that ABA has neuroprotective property against 6-OHDA-induced neurotoxicity, which is performed through PPARγ signaling. However, ABA antioxidant and antiapoptotic properties are involved, at least in part, in such protection.

Induction of cross-tolerance between protective effect of morphine and nicotine in 6-hydroxydopamine-induce neurotoxicity in SH-SY5Y human dopaminergic neuroblastoma cells

Purpose: Parkinson’s disease is a progressive neurodegenerative disease characterized by progressive and selective death of dopaminergic neurons. It has been reported that nicotine and morphine have protective roles during neuronal damage in Parkinson’s disease. In addition, the induction of cross-tolerance between their biological effects has been shown in numerous reports.Methods: Here, we investigated the effects of nicotine and morphine on 6-OHDA-induced neurotoxicity in human neuroblastoma SH-SY5Y cell line as an in vitro model of Parkinson’s disease. Cell damage was induced by 150 μM 6-OHDA and the cells viability was examined by MTT assay. Intracellular reactive oxygen species, calcium level, and mitochondrial membrane potential were determined by fluorescence spectrophotometer method. Biochemical markers of apoptosis were also evaluated by immunoblotting.Result: The data showed that morphine and nicotine prevent 6-OHDA- induced cell damage and apoptosis. However, the protective effects of nicotine were not observed in chronic morphine-pretreated cells. Morphine had no protective effects in chronic nicotine-incubated cells.Conclusion: A cross-tolerance between protective effects of morphine and nicotine was occurred in 6-OHDA-induced SH-SY5Y cell toxicity.

In-Vitro Anti-Proliferative and Pro-Apoptotic Properties of Sutureja Khuzestanica on Human Breast Cancer Cell Line (MCF-7) and Its Synergic Effects with Anticancer Drug Vincristine.

Satureja khuzestanica Jamzad (Marzeh Khuzestani in Persian) is an endemic plant that is widely distributed in the southern part of Iran. Despite the number of papers published on this plant, no one has focused on its anticancer effects. Therefore, the present study was designed to investigate the selective cytotoxic and anti-proliferative properties of satureja khuzestanica total extract (SKE). MCF-7 human breast cancer cells were used in this study. Cytotoxicity of the extract was determined using MTT and neutral red assaysafter 24 h treatment period. Biochemical markers of apoptosis (caspase-3, Bax, Bcl-2) and cell proliferation (cyclin D1) were evaluated by immunoblotting. Vincristine was used to evaluate the synergic effect of extract with an anticancer drug. The data showed that treatment of cells with SKE (150 and 200 µg/mL for 24 h) significantly reduced cell viability, activated caspase 3 and increased Bax/Bcl2 ratio. In addition, cyclin D1 expression was significantly decreased in the SKE-treated cells. In addition, concomitant treatment of the MCF-7 cells with SKE and vincristine produced a potent anti-proliferative and pro-apoptotic effect compared to extract or drug alone. In conclusion, satureja extract has a potential anti-cancer property against human breast cancer cells and its combination with chemotherapeutic agent vincristine may induce cell death effectively and be a potent modality to treat this type of cancer.

The BH3 mimetic drug ABT-737 induces apoptosis and acts synergistically with chemotherapeutic drugs in thyroid carcinoma cells.

BackgroundPatients with dedifferentiated and anaplastic thyroid carcinomas that do not take up radioiodine are resistant to chemotherapeutic treatment and external irradiation and thus are difficult to treat. Direct induction of apoptosis is a promising approach in these apoptosis-resistant tumor cells. The BH3 mimetic ABT-737 belongs to a new class of drugs that target anti-apoptotic proteins of the BCL-2 family and facilitate cell death. The purpose of this study was to investigate the effect of ABT-737 alone or in combination with chemotherapeutic drugs on thyroid carcinoma cell lines.MethodsA total of 16 cell lines derived from follicular, papillary, and anaplastic thyroid carcinomas were treated with ABT-737. Cell viability was measured with MTT assay. Cell death was determined by cell cycle phase distribution and subG1 peak analyses, determination of caspase 3/7 activity and caspase cleavage products, lactate dehydrogenase (LDH) liberation assays and LC3 analysis by western blot.ResultsThe number of viable cells was decreased in all cell lines examined after ABT-737 treatment, with IC50 values ranging from 0.73 to 15.6 μM. Biochemical markers of apoptosis like caspase activities, caspase cleavage products and DNA fragmentation determined as SubG1 peak were elevated after ABT-737 treatment, but no LC3 cleavage was induced by ABT-737 indicating no autophagic processes. In combination with doxorubicin and gemcitabine, ABT-737 showed synergistic effects on cell viability.ConclusionsWith these experiments we demonstrated the efficacy of the BH3 mimetic drug ABT-737 against dedifferentiated thyroid carcinoma cells of various histological origins and showed synergistic effects with chemotherapeutic drugs. ABT-737-treated cells underwent an apoptotic cell death. ABT-737 and related BH3 mimetic drugs, alone or in combination, may thus be of value as a new therapeutic option for dedifferentiated thyroid carcinomas.

Ouabain Enhances ADPKD Cell Apoptosis via the Intrinsic Pathway.

Progression of autosomal dominant polycystic kidney disease (ADPKD) is highly influenced by factors circulating in blood. We have shown that the hormone ouabain enhances several characteristics of the ADPKD cystic phenotype, including the rate of cell proliferation, fluid secretion and the capacity of the cells to form cysts. In this work, we found that physiological levels of ouabain (3 nM) also promote programmed cell death of renal epithelial cells obtained from kidney cysts of patients with ADPKD (ADPKD cells). This was determined by Alexa Fluor 488 labeled-Annexin-V staining and TUNEL assay, both biochemical markers of apoptosis. Ouabain-induced apoptosis also takes place when ADPKD cell growth is blocked; suggesting that the effect is not secondary to the stimulatory actions of ouabain on cell proliferation. Ouabain alters the expression of BCL family of proteins, reducing BCL-2 and increasing BAX expression levels, anti- and pro-apoptotic mediators respectively. In addition, ouabain caused the release of cytochrome c from mitochondria. Moreover, ouabain activates caspase-3, a key “executioner” caspase in the cell apoptotic pathway, but did not affect caspase-8. This suggests that ouabain triggers ADPKD cell apoptosis by stimulating the intrinsic, but not the extrinsic pathway of programmed cell death. The apoptotic effects of ouabain are specific for ADPKD cells and do not occur in normal human kidney cells (NHK cells). Taken together with our previous observations, these results show that ouabain causes an imbalance in cell growth/death, to favor growth of the cystic cells. This event, characteristic of ADPKD, further suggests the importance of ouabain as a circulating factor that promotes ADPKD progression.

Morphine Protects SH-SY5Y Human Neuroblastoma Cells Against 6-Hydroxydopamine–Induced Cell Damage: Involvement of Anti-Oxidant, Calcium Blocking, and Anti-Apoptotic Properties

Parkinson's disease is a neurodegenerative disorder characterized by progressive and selective death of dopaminergic neurons. Understanding the neuroprotective effects of chemical reagents has attracted increasing attention. The μ opioid agonist morphine exerts both toxic and protective effects. However, until recently, the neuroprotective role of morphine against 6-hydroxydopamine (6-OHDA)-induced cell death has not been studied. Here, we investigated the effects of morphine on 6-OHDA-induced neurotoxicity in human neuroblastoma SH-SY5Y cell line as an in vitro model of Parkinson's disease. Cell damage was induced by 150 μM 6-OHDA, and the cells' viability was examined by MTT assay. Intracellular calcium, reactive oxygen species (ROS), and mitochondrial membrane potential were determined by the fluorescence spectrophotometry method. Fragmented DNA and biochemical markers of apoptosis were also determined by gel electrophoresis and immunoblotting, respectively. The data showed that 6-OHDA caused a loss of cell viability and mitochondrial membrane potential. In addition, intracellular ROS and calcium levels, activated caspase-3, Bax:Bcl-2 ratio, cytochrome c release, as well as DNA fragmentation were significantly increased in 6-OHDA-treated cells. Incubation of SH-SY5Y cells with morphine (100 μM) elicited a protective effect and reduced biochemical markers of cell damage and death. These results suggest that morphine has neuroprotective effects against 6-OHDA-induced neurotoxicity, and such effects are accompanied by its anti-oxidant, calcium blocking, and anti-apoptotic properties.

Induction of intervertebral disc cell apoptosis and degeneration by chronic unpredictable stress

The purpose of this study was to evaluate the effects of chronic unpredictable stress on the intervertebral discs of rats. The cellular events involved in injury- and stress-induced disc degeneration were investigated in male Wistar rats. Disc degeneration and apoptosis were evaluated using microscopic (light and electron) and molecular (immunoblotting and immunohistochemistry) methods. Corticosterone levels were used as markers of stress and measured by radioimmunoassay. The data gathered in this study showed that chronic unpredictable stress can significantly increase corticosterone levels. Furthermore, biochemical markers of apoptosis (that is, increases in the Bax/Bcl2 ratio and TUNEL reactivity [p < 0.05]) were observed in the stressed animals. Electron and light microscopy also showed disc degeneration and apoptotic cells in the experimental groups. Taken together, these data demonstrated that chronic stress is most likely to be a risk factor for creating intervertebral disc degeneration and that programmed cell death may be one of the mechanisms of stress-induced disc degeneration.

Proapoptotic and Antiproliferative Effects of Thymus caramanicus on Human Breast Cancer Cell Line (MCF-7) and Its Interaction with Anticancer Drug Vincristine.

Thymus caramanicus Jalas is one of the species of thymus that grows in the wild in different regions of Iran. Traditionally, leaves of this plant are used in the treatment of diabetes, arthritis, and cancerous situation. Therefore, the present study was designed to investigate the selective cytotoxic and antiproliferative properties of Thymus caramanicus extract (TCE). MCF-7 human breast cancer cells were used in this study. Cytotoxicity of the extract was determined using MTT and neutral red assays. Biochemical markers of apoptosis (caspase 3, Bax, and Bcl-2) and cell proliferation (cyclin D1) were evaluated by immunoblotting. Vincristine was used as anticancer control drug in extract combination therapy. The data showed that incubation of cells with TCE (200 and 250 μg/mL) significantly increased cell damage, activated caspase 3 and Bax/Bcl2 ratio. In addition, cyclin D1 was significantly decreased in TCE-treated cells. Furthermore, concomitant treatment of cells with extract and anticancer drug produced a significant cytotoxic effect as compared to extract or drugs alone. In conclusion, thymus extract has a potential proapoptotic/antiproliferative property against human breast cancer cells and its combination with chemotherapeutic agent vincristine may induce cell death effectively and be a potent modality to treat this type of cancer.

Rebuttal from Sergei N. Orlov, Michael M. Model and Ryszard Grygorczyk

We agree with the title of the counter-argument article by Florian Lang and Else Hoffmann, Cell volume changes are essential step in the cell death machinery (Lang & Hoffmann, 2013), albeit with one important proviso: this statement is true for selected rather than for all cell types and cell death stimuli. Indeed, as shown in the major section of our paper as well as in our recent review (Orlov et al. 2013), cell volume perturbations do not contribute to the triggering or progression of the cell death machinery in several types of cells, including serum-deprived vascular smooth muscle cells (VSMC) or ouabain-treated renal epithelial cells, possessing biochemical markers of apoptosis and necrosis, respectively (Orlov et al. 1999a; Platonova et al. 2011). The proapoptotic action of ouabain documented in Fas-ligand treated Jurkat cells (Panayiotidis et al. 2010) contradicts its antiapoptotic action in rat VSMC (Orlov et al. 1999b). We also would like to point out methodological limitations of several studies cited in the counter argument article. Thus, in demonstrating that pro-caspase-3 activity (Bortner et al. 1997) and DNA degradation (Ajiro et al. 2008) is inhibited by high-K+ medium, the authors did not employ high-Na+ medium as a negative control. This comment seems to be important because in vascular smooth muscle cells, the pro-caspase-3 activity is equally inhibited by elevation of [KCl] and [NaCl], thus showing that augmented ionic strength rather than elevated [Na+]i/[K+]i ratio occurring in dying cells is responsible for the effect. The death of U937 and HeLa cells documented in Cl−-depleted medium (Maeno et al. 2006) might be caused by modulation of the activity of diverse [Cl−]i-sensitive enzymes (Orlov & Hamet, 2006) rather than by inhibition of inwardly directed Na+–K+–2Cl− cotransporter NKCC1 and cell shrinkage. Indeed, to the best of our knowledge, there are no reports of cell death triggered by sustained exposure to bumetanide or other potent NKCC1 inhibitors.

Chronic Unpredictable Stress Induces Intervertebral Disk Cell Apoptosis and Degeneration

Introduction Animal model was used to study the effects of chronic unpredictable stress (CUS) on the intervertebral disk (IVD). Stress is a predictor for low back disability in persons with earlier low back pain (LBP) and LBP is most common in the early stages of IVD degeneration. Stress can increase proinflammatory cytokines. Elevated levels of molecular mediators of inflammation have been described in the pathology of IVD degeneration; however, the effect of chronic stress on the induction of disk degeneration has not yet been clarified. Materials and Methods Cellular events involved in injury-and stress-induced disk degeneration in male Wistar rats were investigated. Disk degeneration and apoptosis was evaluated by microscopic (light and electron) and molecular (immunoblotting and immunohistochemistry) methods. Corticosterone levels were measured by radioimmunoassay as a marker of stress condition. Results The data showed that chronic stress could significantly increase corticosterone levels. Furthermore, biochemical markers of apoptosis (increased Bax to Bcl2 ratio ( p &lt; 0.05) and TUNEL reactivity) were observed in animals that had chronic stress. Electron and light microscopy also showed disk degeneration and apoptotic cells in over experimental group. Conclusion Taken together, the data demonstrated that chronic stress is probably a risk factor for creating IVD degeneration and that programmed cell death is one of the mechanisms of stress-induced disk degeneration, however, the effects of stress and its impact on the escalation of disk degeneration and LBP need to be clarified by further investigations. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Carnosine pretreatment protects against hypoxia–ischemia brain damage in the neonatal rat model

Perinatal hypoxia–ischemia brain injury is a major cause of mortality and morbidity in neonates and lacks an effective treatment thus far. Carnosine has been demonstrated to play a neuroprotective role in the adult brain injuries. However, there is no information available concerning its neuroprotective role in the immature brains after hypoxia–ischemia insults. Therefore, we investigated whether carnosine could also confer neuroprotective effects in a neonatal rat hypoxia–ischemia model. Hypoxia–ischemia was induced in rats on postnatal day 7 (P7). Carnosine (250 mg/kg) was administered intraperitoneally, 30 min prior to hypoxia–ischemia induction. Morphological brain injury and biochemical markers of apoptosis and oxidative stress were evaluated 24 h after hypoxia–ischemia induction. Cognitive performance was evaluated by the Morris Water Maze test on P28–P33. We found that pretreatment with carnosine significantly reduced the infarct volume and the number of terminal-deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells in the hypoxia–ischemia brain. Carnosine also inhibited mRNA expression of apoptosis-inducing factor(AIF) and caspase-3, which was accompanied by an increase in superoxide dismutase(SOD)activity and a decrease in the malondialdehyde(MDA)level in carnosine-treated rats. Furthermore, carnosine also improved the spatial learning and memory abilities of rats declined due to hypoxia–ischemia. These results demonstrate that carnosine can protect rats against hypoxia–ischemia-induced brain damage by antioxidation.

Temporal transcriptomic profiling reveals cellular targets that govern survival in HOCl-mediated neuronal apoptosis

Aims With the identification of hypochlorous acid (HOCl) as a biomarker in diseased brains and endogenous detection of its modified proteins, HOCl might be implicated in the development of neurodegenerative disorders. However, its effect on neuronal cell death has not yet been investigated at gene expression level. Main methods Therefore, DNA microarray was performed for screening of HOCl-responsive genes in primary mouse cortical neurons. Neurotoxicity caused by physiological relevant HOCl (250 μM) exhibited several biochemical markers of apoptosis. Key findings The biological processes affected during HOCl-mediated apoptosis included cell death, response to stress, cellular metabolism, and cell cycle. Among them, mRNAs level of cell death and stress response genes were up-regulated while expression of metabolism and cell cycle genes were down-regulated. Significance Our results showed, for the first time, that HOCl induces apoptosis in cortical neurons by upregulating apoptotic genes and gene expression of stress response such as heat shock proteins and antioxidant proteins were enhanced to provide protection. These data form a foundation for the development of screening platforms and define targets for intervention in HOCl neuropathologies where HOCl-mediated injury is causative.

ANGIOTENSIN II INDUCED APOPTOSIS IN ADULT CARDIOMYOCYTE IS SUPPRESSED BY ALL TRANS‐RETINOIC ACID

Circulatory levels of Angiotensin II (Ang II) and oxidative stress are increased in heart failure and both of these conditions are known to induce apoptosis. Vitamin A (retinol) is an important antioxidant and its uptake in the heart from plasma is increased in heart failure. Since vitamin A is metabolized to different bioactive molecules, most notably all trans‐retinoic acid (RA), we examined significance of RA in Ang II induced apoptosis in isolated adult cardiac myocytes. Ang II (10−9 M) caused a significant increase in myocytes apoptosis and pre‐treatment with RA (10−7 M) attenuated the morphological and biochemical markers of apoptosis. An increase in Bax / Bcl‐xl ratio by Ang II was prevented by RA. Ang II induced expression of phosphorylated ERK and JNK proteins was also modulated by RA. There was a moderate reduction in mRNA expression of c‐jun and c‐fos by RA treatment. Ang II treatment increased the levels of RXRs and pre‐treatment with RA lead to a reduction in the RXR levels and up‐ regulation of RAR receptors. RARs work in conjunction with RXRs and the present study indicates that low concentration of RA may activate only RAR receptors in cardiac myocytes in the presence of Ang II. The inhibitory effect of RA on the Ang II induced apoptosis in the cardiac myocytes is, thus, through a RAR‐dependent pathway. The results suggest that the interaction between RA and Ang II appears to be more complex than simple suppression of Ang II signaling (Supported by CIHR).

LEI/L-DNase II: interplay between caspase-dependent and independent pathways

Caspase activation has been seen, for several years, as the biochemical marker of apoptosis. However, in 2005 the Nomenclature Committee on Cell Death (NCCD) established that the 'official' classification of cell death had to rely on morphological criteria owing to the absence of a clear-cut equivalence between structural alterations and biochemical pathways. Actually, the controlled destruction of the cell is coordinated by a proteolytic system involving caspases but also other proteases like cathepsins, calpains and serine proteases. These enzymes participate in an activation cascade that culminates in cleavage of a set of proteins resulting in disassembly of the cell. This disassembling also includes the activation of endonucleases that will destroy a potentially harmful DNA. A caspase-activated DNase performs DNA degradation in caspase-dependent apoptosis, but other endonucleases like L-DNase II or GAAD are activated in caspase-independent apoptosis, allowing the complete dismantling of the cell.

Blood micromolar concentrations of kaempferol afford protection against ischemia/reperfusion-induced damage in rat brain

The slow time course of neurodegeneration after brain ischemia/reperfusion opened a realistic time window for the application of protective therapies to prevent spreading of brain damage. In this work, we studied the ability of micromolar concentrations of this flavonoid in the blood to protect against brain damage induced by transient focal cerebral ischemia in rats. Transient focal cerebral ischemia was induced by middle cerebral artery occlusion in adult rats and brain damage has been monitored by 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (H-E) staining, 'in situ' terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL), 'in situ' metalloproteinase activity using DQ-gelatin and loss of anti-laminin staining. Intravenous injections of kaempferol, at a dose of 10-15 mumol/L of blood 30 min before the induction of a 60 min ischemia-episode and just after reperfusion, led to >90% and 70-80% (TTC, H-E, TUNEL) decrease of brain damage in the temporal-frontal areas of neocortex and striatum, respectively, but only 40-50% decrease of brain damage was observed in the hippocampus and vicinal caudal areas of the striatum. This treatment with kaempferol also produced a similar reduction of metalloproteinase activation and loss of anti-laminin staining in cortical and striatum infarct areas. Kaempferol treatment efficiently protected against nitrosative-oxidative stress after ischemia/reperfusion, as shown by nearly complete protection against the increase of protein nitrotyrosines, and also afforded strong protection against the increase of apoptotic cell death (TUNEL) and biochemical markers of apoptosis, such as caspase-9 activity and poly-(ADP-ribose) polymerase degradation. On these grounds, a potential new therapeutic role of kaempferol to acute treatment of ischemic stroke is suggested.

The pro-inflammatory oxidant hypochlorous acid induces Bax-dependent mitochondrial permeabilisation and cell death through AIF-/EndoG-dependent pathways

At sites of chronic inflammation, such as in the inflamed rheumatoid joint, activated neutrophils release hydrogen peroxide (H 2O 2) and the enzyme myeloperoxidase to catalyse the formation of hypochlorous acid (HOCl). 3-chlorotyrosine, a marker of HOCl in vivo, has been observed in synovial fluid proteins from rheumatoid arthritis patients. However the mechanisms of HOCl-induced cytotxicity are unknown. We determined the molecular mechanisms by which HOCl induced cell death in human mesenchymal progenitor cells (MPCs) differentiated into a chondrocytic phenotype as a model of human cartilage cells and show that HOCl induced rapid Bax conformational change, mitochondrial permeability and release of intra-mitochondrial pro-apoptotic proteins which resulted in nuclear translocation of AIF and EndoG. siRNA-mediated knockdown of Bax substantially prevented mitochondrial permeability, release of intra-mitochondrial pro-apoptotic proteins. Cell death was inhibited by siRNA-mediated knockdown of Bax, AIF or EndoG. Although we observed several biochemical markers of apoptosis, caspase activation was not detected either by western blotting, fluorescence activity assays or by using caspase inhibitors to inhibit cell death. This was further supported by findings that (1) in vitro exposure of recombinant human caspases to HOCl caused significant inhibition of caspase activity and (2) the addition of HOCl to staurosporine-treated MPCs inhibited the activity of cellular caspases. Our results show for the first time that HOCl induced Bax-dependent mitochondrial permeability which led to cell death without caspase activity by processes involving AIF/EndoG-dependent pathways. Our study provides a novel insight into the potential mechanisms of cell death in the inflamed human joint.