scholarly journals Differential Profile of Nix Upregulation and Translocation during Hypoxia/Ischaemia in Vivo Versus in Vitro

2005 ◽  
Vol 25 (10) ◽  
pp. 1356-1365 ◽  
Author(s):  
Jui-Lee A Birse-Archbold ◽  
Lorraine E Kerr ◽  
Paul A Jones ◽  
James McCulloch ◽  
John Sharkey
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Mrna Level ◽  
Hypoxia Inducible Factor ◽  
Neuronal Loss ◽  
Promoter Sequence ◽  
Ischaemic Brain

Nix, a hypoxia-sensitive member of the Bcl-2 family, is upregulated at the mRNA level during hypoxia through induction of a hypoxia-inducible factor-1α (HIF-1α) response element in its promoter sequence. However, the mechanism(s) regulating Nix protein activation remain unclear. The present studies examine Nix protein expression and subcellular distribution in response to hypoxic stimuli in vivo and in culture and to two disparate apoptotic stimuli in vitro. Upregulation and translocation of Nix (by day 5) in hypoxic/serum-deprived CHO-K1 cells, was preceded by Bax activation (by day 4) and caspase-3 processing (by day 2), suggesting that initiation of cell death in vitro is a Nix-independent event. In contrast, an early Nix response (upregulation and translocation to the mitochondria) was observed after 6 h of middle cerebral artery occlusion in the rat. Nix translocation was observed in the ipsilateral cortex and striatum before other histological (infarct development, neuronal loss, apoptotic body formation) or biochemical (Bax activation or caspase-3 cleavage) markers of damage were detected. While fundamental differences between hypoxia/ischaemia in culture and in vivo likely explain the different temporal profiles of Nix, Bax, and caspase-3 activation observed, these studies show that like Bax, mitochondrial accumulation is a common event during Nix activation. These are the first studies to show upregulation and translocation of Nix in the ischaemic brain and suggest Nix to be a novel therapeutic target in ischaemic research. Moreover, Nix upregulation in staurosporine-treated SH-SY5Y cells and dexamethasone-treated A1.1 cells supports a more generalized role for Nix in apoptotic cell death.

scholarly journals Protective Effect of Panaxynol Isolated from Panax vietnamensis against Cisplatin-Induced Renal Damage: In Vitro and In Vivo Studies

Biomolecules ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 890
Author(s):  
Dahae Lee ◽  
Jaemin Lee ◽  
Kim Long Vu-Huynh ◽  
Thi Hong Van Le ◽  
Thi Hong Tuoi Do ◽  
...  
Keyword(s):  
Cell Death ◽  
Protective Effect ◽  
Renal Damage ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Body Weight Loss ◽  
Apoptotic Cell Death ◽  
In Vivo Studies

Polyacetylenic compounds isolated from Panax species are comprised of non-polar C17 compounds, exhibiting anti-inflammatory, antitumor, and antifungal activities. Panaxynol represents the major component of the essential oils of ginseng. We investigated whether panaxynol isolated from Panax vietnamensis (Vietnamese ginseng, VG) could prevent cisplatin-induced renal damage induced in vitro and in vivo. Cisplatin-induced apoptotic cell death was observed by staining with annexin V conjugated with Alexa Fluor 488, and western blotting evaluated the molecular mechanism. Panaxynol at concentrations above 0.25 μM prevented cisplatin-induced LLC-PK1 porcine renal proximal tubular cell death. LLC-PK1 cells treated with cisplatin demonstrated an increase in apoptotic cell death, whereas pretreatment with 2 and 4 μM panaxynol decreased this effect. Cisplatin demonstrated a marked increase in the phosphorylation of c-Jun N-terminal kinase (JNK), P38, and cleaved caspase-3. However, pretreatment with 2 and 4 μM panaxynol reversed the upregulated phosphorylation of JNK, P38, and the expression of cleaved caspase-3. We confirmed that the protective effect of panaxynol isolated from P. vietnamensis in LLC-PK1 cells was at least partially mediated by reducing the cisplatin-induced apoptotic damage. In the animal study, panaxynol treatment ameliorated body weight loss and blood renal function markers and downregulated the mRNA expression of inflammatory mediators.

Anti-Angiongenic Effects of mTOR-Inhibitors Is Regulated by AKT Activity in Multiple Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3406-3406
Author(s):  
Patrick Frost ◽  
Bao Hoang ◽  
Yijiang Shi ◽  
Huajun Yan ◽  
Alan Lichtenstein
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Apoptotic Cell ◽  
Mammalian Target Of Rapamycin ◽  
Hypoxia Inducible Factor ◽  
Mtor Inhibitors ◽  
Underlying Mechanisms ◽  
Salvage Pathways

Abstract Inhibitors of the mammalian target of rapamycin (mTOR), such as rapamycin (RAPA) and CCI-779 (CCI), have potential as anti-tumor agents against multiple myeloma (MM). Since other tumor models have demonstrated that heightened AKT activity induces hypersensitivity to mTOR inhibitors, we stably transfected U266 human MM cells with a constitutively activated AKT allele (U266-AKT) or empty vector control (U266-EV) in order to further explore the underlying mechanisms of this phenomena. Analysis of cell death demonstrated that U266-AKT were significantly more sensitive to RAPA in vitro, with an ED50 of 0.01 nM versus an ED50 of >100 nM for U266-EV control cells. A similar alteration of sensitivity to CCI was demonstrated in U266 isogenic tumors grown in NOD/SCID mice and treated with CCI in vivo. Analysis of the excised tumor nodules demonstrated a >5 fold inclease in apoptotic nuclei in U266-AKT tumors treated with CCI compared to isogenic control tumors, despite previous reports that mTOR inhibitors do not induce apoptosis in MM cells in vitro. One potential explanation for this is that AKT regulates the ability of CCI to inhibit angiogenesis, which is only relevent in vivo, and thereby indirectly induces apoptotic cell death. In support of this hypothesis, we demonstrated that CCI significantly decreased angiogenesis (measued by in situ staining of excised tumor nodules with CD34, a marker for endothelial cells) by 80% in U266-AKT, and only by 67% in isogenic controls. Since previous studies demonstrated that AKT/mTOR regulates the expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1a (HIF1a), we hypothesized that MM cells with heightened AKT activity may be more sensitive to the CCI-mediated inhibition of these critical angiogenic factors. In vitro, RAPA was markedly more effective at inhibiting HIF-1a and VEGF expression from U266-AKT compared to U266-EV control cells. One possible explanation for the regulatory role of AKT in the RAPA/CCI response is that MM cells with hyperactive AKT function depend upon mTOR-mediated (i.e. cap-dependent) translational pathway to express critical proteins like VEGF and HIF-1a, while “low-AKT” MM cells may be able to utilize non-mTOR dependent (i.e. cap-independent) salvage pathways to express these critical proteins and are thereby resistant to mTOR inhibitors.

scholarly journals Differential Efficacy of Caspase Inhibitors on Apoptosis Markers during Sepsis in Rats and Implication for Fractional Inhibition Requirements for Therapeutics

2004 ◽  
Vol 199 (2) ◽  
pp. 199-207 ◽  
Author(s):  
Nathalie Méthot ◽  
JingQi Huang ◽  
Nathalie Coulombe ◽  
John P. Vaillancourt ◽  
Dita Rasper ◽  
...  
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Mitochondrial Pathway ◽  
Caspase Inhibitors ◽  
Caspase Inhibition ◽  
Caspase Activated Dnase

A rodent model of sepsis was used to establish the relationship between caspase inhibition and inhibition of apoptotic cell death in vivo. In this model, thymocyte cell death was blocked by Bcl-2 transgene, indicating that apoptosis was predominantly dependent on the mitochondrial pathway that culminates in caspase-3 activation. Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly different efficacy for different cell death markers. Inhibition of DNA fragmentation required substantially higher levels of caspase-3 attenuation than that required for blockade of other apoptotic events such as spectrin proteolysis and phosphatidylserine externalization. These data indicate a direct relationship between caspase inhibition and some apoptotic manifestations but that small quantities of uninhibited caspase-3 suffice to initiate genomic DNA breakdown, presumably through the escape of catalytic quantities of caspase-activated DNase. These findings suggest that putative caspase-independent apoptosis may be overestimated in some systems since blockade of spectrin proteolysis and other cell death markers does not accurately reflect the high degrees of caspase-3 inhibition needed to prevent DNA fragmentation. Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.

scholarly journals KUS121 attenuates the progression of monosodium iodoacetate-induced osteoarthritis in rats

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sachiko Iwai ◽  
Hanako O. Ikeda ◽  
Hisashi Mera ◽  
Kohei Nishitani ◽  
Motoo Saito ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Apoptotic Cell ◽  
Intraperitoneal Administration ◽  
Atp Depletion ◽  
Knee Joints ◽  
Cellular Protection ◽  
Monosodium Iodoacetate

AbstractCurrently there is no effective treatment available for osteoarthritis (OA). We have recently developed Kyoto University Substances (KUSs), ATPase inhibitors specific for valosin-containing protein (VCP), as a novel class of medicine for cellular protection. KUSs suppressed intracellular ATP depletion, endoplasmic reticulum (ER) stress, and cell death. In this study, we investigated the effects of KUS121 on chondrocyte cell death. In cultured chondrocytes differentiated from ATDC5 cells, KUS121 suppressed the decline in ATP levels and apoptotic cell death under stress conditions induced by TNFα. KUS121 ameliorated TNFα-induced reduction of gene expression in chondrocytes, such as Sox9 and Col2α. KUS121 also suppressed ER stress and cell death in chondrocytes under tunicamycin load. Furthermore, intraperitoneal administration of KUS121 in vivo suppressed chondrocyte loss and proteoglycan reduction in knee joints of a monosodium iodoacetate-induced OA rat model. Moreover, intra-articular administration of KUS121 more prominently reduced the apoptosis of the affected chondrocytes. These results demonstrate that KUS121 protects chondrocytes from stress-induced cell death in vitro and in vivo, and indicate that KUS121 is a promising novel therapeutic agent to prevent the progression of OA.

Biochemical Characterisation of an In Vitro Model of Hepatocellular Apoptotic Cell Death

2009 ◽  
Vol 37 (2) ◽  
pp. 209-218 ◽  
Author(s):  
Mathieu Vinken ◽  
Elke Decrock ◽  
Elke De Vuyst ◽  
Luc Leybaert ◽  
Tamara Vanhaecke ◽  
...  
Keyword(s):  
Cell Death ◽  
In Vitro Model ◽  
Caspase 3 ◽  
Fas Ligand ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Apoptotic Cell Death ◽  
Biochemical Characterisation ◽  
Vitro Model

This study was set up to critically evaluate a commonly-used in vitro model of hepatocellular apoptotic cell death, in which freshly isolated hepatocytes, cultured in a monolayer configuration, are exposed to a combination of Fas ligand and cycloheximide for six hours. A set of well-acknowledged cell death markers was addressed: a) cell morphology was studied by light microscopy; b) apoptotic and necrotic cell populations were quantified by in situ staining with Annexin-V, Hoechst 33342 and propidium iodide (PI); c) apoptotic and necrotic activities were monitored by probing caspase 3-like activity and measuring the extracellular leakage of lactate dehydrogenase (LDH), respectively; and d) the expression of apoptosis regulators was investigated by immunoblotting. The initiation of apoptosis was evidenced by the activation of caspase 8 and caspase 9, and increased Annexin-V reactivity. Progression through the apoptotic process was confirmed by the activation of caspase 3 and Bid, the enhanced expression of Bax, and the occurrence of nuclear fragmentation. Late transition to a necrotic appearance was demonstrated by an increased number of PI-positive cells and augmented extracellular release of LDH. Thus, the in vitro model allows the study of the entire course of Fas-mediated hepatocellular apoptotic cell death, which is not possible in vivo. This experimental system can serve a broad range of in vitro pharmaco-toxicological purposes, thereby directly assisting in the reduction of animal experimentation.

scholarly journals Iron toxicity, lipid peroxidation and ferroptosis after intracerebral haemorrhage

2019 ◽  
Vol 4 (2) ◽  
pp. 93-95 ◽  
Author(s):  
Jieru Wan ◽  
Honglei Ren ◽  
Jian Wang
Keyword(s):  
Lipid Peroxidation ◽  
Cell Death ◽  
Intracerebral Haemorrhage ◽  
Neuronal Death ◽  
Molecular Mechanisms ◽  
Apoptotic Cell ◽  
Iron Toxicity ◽  
Secondary Brain Injury

Intracerebral haemorrhage (ICH) is a devastating type of stroke with high mortality and morbidity. However, we have few options for ICH therapy and limited knowledge about post-ICH neuronal death and related mechanisms. In the aftermath of ICH, iron overload within the perihaematomal region can induce lethal reactive oxygen species (ROS) production and lipid peroxidation, which contribute to secondary brain injury. Indeed, iron chelation therapy has shown efficacy in preclinical ICH studies. Recently, an iron-dependent form of non-apoptotic cell death known as ferroptosis was identified. It is characterised by an accumulation of iron-induced lipid ROS, which leads to intracellular oxidative stress. The ROS cause damage to nucleic acids, proteins and lipid membranes, and eventually cell death. Recently, we and others discovered that ferroptosis does occur after haemorrhagic stroke in vitro and in vivo and contributes to neuronal death. Inhibition of ferroptosis is beneficial in several in vivo and in vitro ICH conditions. This minireview summarises current research on iron toxicity, lipid peroxidation and ferroptosis in the pathomechanisms of ICH, the underlying molecular mechanisms of ferroptosis and the potential for combined therapeutic strategies. Understanding the role of ferroptosis after ICH will provide a vital foundation for cell death-based ICH treatment and prevention.

scholarly journals Soluble Asialoglycoprotein Receptors Reflect the Apoptosis of Hepatocytes

2002 ◽  
Vol 11 (5) ◽  
pp. 407-415 ◽  
Author(s):  
Tetsuji Kakegawa ◽  
Hirohiko Ise ◽  
Nobuhiro Sugihara ◽  
Toshio Nikaido ◽  
Naoki Negishi ◽  
...  
Keyword(s):  
Cell Death ◽  
Liver Tissue ◽  
Liver Diseases ◽  
Culture Medium ◽  
Apoptotic Cell ◽  
Rabbit Serum ◽  
Mouse Serum ◽  
Apoptosis And Necrosis

Cell death is thought to take place through at least two distinct processes: apoptosis and necrosis. There is increasing evidence that dysregulation of the apoptotic program is involved in liver diseases. However, there is no method to simply evaluate apoptosis in the liver tissue at present. It has been reported that the expression of asialoglycoprotein receptors (AGPRs) increases with apoptosis, but there is no report until now that investigates the influence of soluble AGPRs on apoptosis of hepatocytes. Soluble AGPRs have been reported to be present in human serum under physiological conditions. In the present study, in order to investigate the correlation between apoptosis of hepatocytes and soluble AGPR, mouse soluble AGPRs were detected using SDS-PAGE and Western blot analysis was conducted using anti-extracellular mouse hepatic lectin-1 (Ex-MHL-1) antiserum (polyclonal rabbit serum). The mouse soluble AGPRs were present in culture medium and mouse serum when hepatocytes were damaged. The soluble AGPRs increased proportionately, as the number of dead hepatocytes increased. In addition, soluble AGPRs existed more when apoptotic cell death was observed in in vitro and in vivo than when necrotic cell death was observed. The extracellular moiety of MHL-1 exists in the culture medium and mouse serum as a soluble AGPR, but the detailed mechanism of releasing soluble AGPR from hepatocytes has not been revealed yet. We described the first evidence for the relation between quantity of soluble AGPRs with two kinds of cell death: necrosis and apoptosis. Based on the results of our study, soluble AGPRs might become a new marker of apoptosis in the liver tissue and be useful for clinical diagnosis and treatment for liver diseases.

scholarly journals Neuroprotective Activity ofSibjeondaebo-tangon AβPeptide-Induced Damages

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Hyeon Ju Yim ◽  
Jung Hwa Lim ◽  
Min Hee Kim ◽  
Uk Namgung ◽  
Sang Ryong Lee ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Cortical Neurons ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Immunofluorescence Staining ◽  
Neuroprotective Activity ◽  
Potential Therapeutic Target ◽  
Protein Levels

Background.Sibjeondaebo-tang(SJDBT) has been used to treat diverse disorders including neuropsychiatric disabilities in traditional Korean medicine.Objective. The present study aims to investigate the potential effects of SJDBT on neuroprotection against Aβ peptide-induced damage usingin vitroculture andin vivorat brain systems.Materials and Methods. PC12 cell viability was analyzed by MTT assay, and neurite arborizations and caspase 3 protein signals in cultured PC12 cells andin vivocortical neurons were analyzed by immunofluorescence staining. Phospho-Erk1/2 protein was analyzed by immunofluorescence staining and western blot analysis.Results. In PC12 cells, atrophied cell body and reduced neurite extension by Aβtreatment were recovered by SJDBT treatment. Caspase 3 protein signals were increased in Aβ-treated PC12 cells, but SJDBT treatment decreased apoptotic cell death. Caspase 3 activation in cortical neurons, which was induced similarly by Aβtreatment, was reduced by SJDBT treatment. Furthermore, phospho-Erk1/2 protein levels, which had been decreased by Aβtreatment, were elevated in the cortical neurons by SJDBT treatment.Conclusion. These data show that SJDBT may play a role in protecting from damages induced by Aβin neuronal tissue and further suggest that SJDBT can be explored as the potential therapeutic target for AD treatments in human.

Intracellular NAD+ Depletion Enhances Bortezomib-Induced Myeloma Cytotoxicity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 330-330
Author(s):  
Antonia Cagnetta ◽  
Michele Cea ◽  
Chirag Acharya ◽  
Teresa Calimeri ◽  
Yu-Tzu Tai ◽  
...  
Keyword(s):  
Cell Death ◽  
Synergistic Effect ◽  
Cell Lines ◽  
Caspase 3 ◽  
Statistical Significance ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Nicotinamide Phosphoribosyltransferase

Abstract Abstract 330 Background: Our previous study demonstrated that inhibition of nicotinamide phosphoribosyltransferase (Nampt) acts by severely depleting intracellular NAD+ content and thus eliciting mitochondrial dysfunction and autophagic MM cell death. The proteasome inhibitor Bortezomib induces anti-MM activity by affecting a variety of signaling pathways. However, as with other agents, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we demonstrate that combining Nampt inhibitor and bortezomb induces synergistic anti-MM cell death both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. Material and Methods: We utilized MM.1S, MM.1R, RPMI-8226, and U266 human MM cell lines, as well as purified tumor cells from patients relapsing after prior therapies. Cell viability and apoptosis assays were performed using Annexin V/PI staining. Intracellular NAD+ level and proteasome activity were quantified after 12, 24, and 48h exposure to single/combination drugs by specific assays. In vitro angiogenesis was assessed by Matrigel capillary-like tube structure formation assay. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, and tubulin. CB-17 SCID male mice (n = 28; 7 mice/EA group) were subcutaneously inoculated with 5.0 × 106 MM.1S cells in 100 microliters of serum free RPMI-1640 medium. When tumors were measurable (3 weeks after MM cell injection), mice were treated for three weeks with vehicle alone, FK866 (30mg/kg 4 days weekly), Bortezomib (0.5 mg/kg twice weekly), or FK866 (30 mg/kg) plus Bortezomib (0.5 mg/kg). Statistical significance of differences observed in FK866, Bortezomib or combination-treated mice was determined using a Student t test. Isobologram analysis was performed using “CalcuSyn” software program. A combination index < 1.0 indicates synergism. Results/Discussion: Combining FK866 and Bortezomib induces synergistic anti-MM activity in vitro against MM cell lines (P<0.005, CI < 1) or patient CD138-positive MM cells (P< 0.004). FK866 plus Bortezomib-induced synergistic effect is associated with: 1)activation of caspase-8, caspase-9, caspase-3, and PARP; 2) improved intracellular NAD+ dissipation; 3) suppression of chymotrypsin-like, caspase-like, and trypsin-like proteolytic activities; 4) inhibition of NF-kappa B signaling; and 5) inhibition of angiogenesis. Importantly, the ectopic overexpression of Nampt rescues this observed synergistic effect; conversely, Nampt knockdown by RNAi significantly enhances the anti-MM effect of bortezomib. In the murine xenograft MM model, low dose combination FK866 (30 mg/kg) and Bortezomib (0.5 mg/kg) is well tolerated, significantly inhibits tumor growth (P < 0.001), and prolongs host survival (2–2.5 months in mice receiving combined drugs, P = 0.001). These findings demonstrate that intracellular NAD+ levels represent a major determinant in the ability of bortezomib to induce apoptosis of MM cells, providing the rationale for clinical protocols evaluating FK866 together with Bortezomib to improve patient outcome in MM. Disclosures: Munshi: Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

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