The intrinsic apoptotic pathway lies upstream of oxidative stress in multiple organs.
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ABSTRACT:
SUBMITTER: Patel MY
PROVIDER: S-EPMC7484315 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Oxidative stress is a common feature of the aging process and of many neurodegenerative disorders, including Alzheimer's disease. Understanding the direct causative relationship between oxidative stress and amyloid pathology, and determining the underlying molecular mechanisms is crucial for the development of more effective therapeutics for the disease. By employing microdialysis technique, we report local increase in the amyloid-?42 levels and elevated amyloid-?42/40 ratio in the interstitial fluid within 6h of direct infusion of oxidizing agents into the hippocampus of living and awake wild type mice. The increase in the amyloid-?42/40 ratio correlated with the pathogenic conformational change of the amyloid precursor protein-cleaving enzyme, presenilin1/?-secretase. Furthermore, we found that the product of lipid peroxidation 4-hydroxynonenal, binds to both nicastrin and BACE, differentially affecting ?- and ?-secretase activity, respectively. The present study demonstrates a direct cause-and-effect correlation between oxidative stress and altered amyloid-? production, and provides a molecular mechanism by which naturally occurring product of lipid peroxidation may trigger generation of toxic amyloid-?42 species.
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Project description:Mutations in p150glued cause hereditary motor neuropathy with vocal cord paralysis (HMN7B) and Perry syndrome (PS). Here we show that both overexpression of p150glued mutants and knockdown of endogenous p150glued induce apoptosis. Overexpression of a p150glued plasmid containing either a HMN7B or PS mutation resulted in cytoplasmic p150glued-positive aggregates and was associated with cell death. Cells containing mutant p150glued aggregates underwent apoptosis that was characterized by an increase in cleaved caspase-3- or Annexin V-positive cells and was attenuated by both zVAD-fmk (a pan-caspase inhibitor) application and caspase-3 siRNA knockdown. In addition, overexpression of mutant p150glued decreased mitochondrial membrane potentials and increased levels of translocase of the mitochondrial outer membrane (Tom20) protein, indicating accumulation of damaged mitochondria. Importantly, siRNA knockdown of endogenous p150glued independently induced apoptosis via caspase-8 activation and was not associated with mitochondrial morphological changes. Simultaneous knockdown of endogenous p150glued and overexpression of mutant p150glued had additive apoptosis induction effects. These findings suggest that both p150glued gain-of-toxic-function and loss-of-physiological-function can cause apoptosis and may underlie the pathogenesis of p150glued-associated disorders.
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Project description:Apoptosis is a known regulator of morphogenetic events. In mammals, the critical role of oxidative stress-induced apoptosis has been well-studied; however, in insects the role of oxidative stress in apoptosis is not clear. In a previous study, we showed that apoptosis-related genes are present in the silkworm Bombyx mori, an important lepidopteran insect model. In this study, we evaluated the effect of H2O2-induced oxidative stress on apoptosis, reactive oxygen species (ROS) levels, mitochondrial response, cytochrome c release and apoptosis-related gene expression in the BmN-SWU1 cell line from B. mori ovaries. Our results showed that BmN-SWU1 cells exposed to H2O2 showed cell protuberances, cytoplasmic condensation, apoptotic bodies, DNA ladder formation and caspase activities indicating apoptosis. H2O2-induced apoptosis also increased intracellular ROS level, changed mitochondrial distribution, reduced mitochondrial membrane potential and increased the release of cytochrome c from mitochondria. Furthermore, western blot analysis revealed a significant increase in p53 and cytochrome c expression, and a decrease in Bcl-2 expression compared to the controls. Moreover, quantitative real-time PCR (qRT-PCR) showed an increase in the transcript levels of BmICE, Bmapaf-1 and BmEndoG by 439.5%, 423.9% and 42.2%, respectively, after treatment with 1 ?M H2O2 for 24 h. However, the transcript levels of Bmbuffy declined by 41.4% after 24 h of exposure to 1 ?M H2O2. These results show that H2O2 treatment induced apoptosis in BmN-SWU1 cells via the mitochondrial apoptotic pathway. Further, it appears that oxidative stress induced by H2O2 activates both caspase-dependent and caspase-independent mitochondrial apoptotic pathways in silkworm cells. Taken together, these findings improve our knowledge of apoptosis in silkworm and the apoptotic pathways in insects.
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Project description:The Prep1 homeodomain transcription factor is essential in embryonic development. Prep1 hypomorphic mutant mouse (Prep1(i/i)) embryos (embryonic day 9.5) display an increased terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling reaction compared to wild-type (WT) littermates. Prep1(i/i) mouse embryo fibroblasts (MEFs) show an increased basal level of annexin V binding activity, reduction of the mitochondrial-membrane potential, and increased caspase 9 and 3 activation, indicating increased apoptosis. Prep1(i/i) MEFs also respond faster than WT MEFs to genotoxic stress, indicating increased activation of the intrinsic apoptotic pathways. We did not observe an increase in p53 or an abnormal p53 response to apoptotic stimuli. However, hypomorphic MEFs have decreased endogenous levels of antiapoptotic Bcl-X(L) mRNA and protein, and Bcl-x overexpression rescues the defect of Prep1(i/i) MEFs. Using transient transfections and chromatin immunoprecipitation, we identified the Bcl-x promoter as a novel target of Prep1. Thus, Prep1 directly controls mitochondrial homeostasis (and the apoptotic potential) by modulating Bcl-x gene expression.
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Project description:High levels of reactive oxygen species (ROS) contribute to muscle cell death in aging and disuse. We have previously found that resveratrol can reduce oxidative stress in response to aging and hindlimb unloading in rodents in vivo, but it was not known if resveratrol would protect muscle stem cells during repair or regeneration when oxidative stress is high. To test the protective role of resveratrol on muscle stem cells directly, we treated the C2C12 mouse myoblast cell line with moderate (100 ?M) or very high (1 mM) levels of H2O2 in the presence or absence of resveratrol. The p21 promoter activity declined in myoblasts in response to high ROS, and this was accompanied a greater nuclear to cytoplasmic translocation of p21 in a dose-dependent matter in myoblasts as compared to myotubes. Apoptosis, as indicated by TdT-mediated dUTP nick-end labeling, was greater in C2C12 myoblasts as compared to myotubes (P<.05) after treatment with H2O2. Caspase-9, -8 and -3 activities were elevated significantly (P<.05) in myoblasts treated with H2O2. Myoblasts were more susceptible to ROS-induced oxidative stress than myotubes. We treated C2C12 myoblasts with 50 ?M of resveratrol for periods up to 48 h to determine if myoblasts could be rescued from high-ROS-induced apoptosis by resveratrol. Resveratrol reduced the apoptotic index and significantly reduced the ROS-induced caspase-9, -8 and -3 activity in myoblasts. Furthermore, Bcl-2 and the Bax/Bcl-2 ratio were partially rescued in myoblasts by resveratrol treatment. Similarly, muscle stem cells isolated from mouse skeletal muscles showed reduced Sirt1 protein abundance with H2O2 treatment, but this could be reversed by resveratrol. Reduced apoptotic susceptibility in myoblasts as compared to myotubes to ROS is regulated, at least in part, by enhanced p21 promoter activity and nuclear p21 location in myotubes. Resveratrol confers further protection against ROS by improving Sirt1 levels and increasing antioxidant production, which reduces mitochondrial associated apoptotic signaling, and cell death in myoblasts.
| S-EPMC5694367 | biostudies-literature
Project description:Two members of a recently discovered family of protein kinases {WNK1 and WNK4 [with no K (lysine) kinases-1 and -4]} are the cause of an inherited disease known as pseudohypoaldosteronism type II that features arterial hypertension. The family is known as WNK due to a lack of the invariant catalytic lysine in kinase subdomain II. The mechanisms by which WNKs regulate blood pressure are beginning to be understood at the physiological level from recent studies showing effects of WNK4 on several plasma membrane co-transporters and ion channels. However, little is known about the function of WNKs at the biochemical level. In this issue of the Biochemical Journal, Vitari et al. have shown that WNK1 and WNK4 interact with other kinases, SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress response kinase-1), which are involved in the regulation of ion transporters. WNK1 and WNK4 phosphorylate SPAK and OSR1, which in turn phosphorylate the N-terminal domain of the basolateral Na+-K+-2Cl- co-transporter, NKCCl. The phosphorylation site involved in SPAK or OSR1 activation is identified as a threonine residue within the T-loop.
| S-EPMC1237149 | biostudies-other
Project description:Estrogen receptor-? positive (ER?+) breast cancer accounts for approximately 70-80% of the nearly 25,0000 new cases of breast cancer diagnosed in the US each year. Endocrine-targeted therapies (those that block ER? activity) serve as the first line of treatment in most cases. Despite the proven benefit of endocrine therapies, however, ER?+ breast tumors can develop resistance to endocrine therapy, causing disease progression or relapse, particularly in the metastatic setting. Anti-apoptotic Bcl-2 family proteins enhance breast tumor cell survival, often promoting resistance to targeted therapies, including endocrine therapies. Herein, we investigated whether blockade of anti-apoptotic Bcl-2 family proteins could sensitize luminal breast cancers to anti-estrogen treatment. We used long-term estrogen deprivation (LTED) of human ER?+ breast cancer cell lines, an established model of sustained treatment with and acquired resistance to aromatase inhibitors (AIs), in combination with Bcl-2/Bcl-xL inhibition (ABT-263), finding that ABT-263 induced only limited tumor cell killing in LTED-selected cells in culture and in vivo. Interestingly, expression and activity of the Bcl-2-related factor Mcl-1 was increased in LTED cells. Genetic Mcl-1 ablation induced apoptosis in LTED-selected cells, and potently increased their sensitivity to ABT-263. Increased expression and activity of Mcl-1 was similarly seen in clinical breast tumor specimens treated with AI + the selective estrogen receptor downregulator fulvestrant. Delivery of Mcl-1 siRNA loaded into polymeric nanoparticles (MCL1 si-NPs) decreased Mcl-1 expression in LTED-selected and fulvestrant-treated cells, increasing tumor cell death and blocking tumor cell growth. These findings suggest that Mcl-1 upregulation in response to anti-estrogen treatment enhances tumor cell survival, decreasing response to therapeutic treatments. Therefore, strategies blocking Mcl-1 expression or activity used in combination with endocrine therapies would enhance tumor cell death.
| S-EPMC5833697 | biostudies-literature
Project description:Oxidative stress contributes to disease and can alter endothelial cell (EC) function. EC from different vascular beds are heterogeneous in structure and function, thus we assessed the apoptotic responses of EC from lung and heart to oxidative stress. Since protein kinase C? (PKC?) is activated by oxidative stress and is an important modulator of apoptosis, experiments assessed the level of apoptosis in fixed lung and heart sections of PKC? wild-type (PKC?(+/+)) and null (PKC?(-/-)) mice housed under normoxia (21% O(2)) or hyperoxia (~95% O(2)). We noted a significantly greater number of TUNEL-positive cells in lungs of hyperoxic PKC?(+/+) mice, compared to matched hearts or normoxic organs. We found that 33% of apoptotic cells identified in hyperoxic lungs of PKC?(+/+) mice were EC, compared to 7% EC in hyperoxic hearts. We further noted that EC apoptosis was significantly reduced in lungs of PKC?(-/-) hyperoxic mice, compared to lungs of PKC?(+/+) hyperoxic mice. In vitro, both hyperoxia and H(2)O(2) promoted apoptosis in EC isolated from microvasculature of lung (LMVEC), but not from the heart (HMVEC). H(2)O(2) treatment significantly increased p38 activity in LMVEC, but not in HMVEC. Inhibition of p38 attenuated H(2)O(2)-induced LMVEC apoptosis. Baseline expression of total PKC? protein, as well as the caspase-mediated, catalytically active PKC? cleavage fragment, was higher in LMVEC, compared to HMVEC. PKC? inhibition significantly attenuated H(2)O(2)-induced LMVEC p38 activation. Conversely, overexpression of wild-type PKC? or the catalytically active PKC? cleavage product greatly increased H(2)O(2)-induced HMVEC caspase and p38 activation. We propose that enhanced susceptibility of lung EC to oxidant-induced apoptosis is due to increased PKC??p38 signaling, and we describe a PKC?-centric pathway which dictates the differential response of EC from distinct vascular beds to oxidative stress.
| S-EPMC3533229 | biostudies-literature
Project description:Background and aimsDefective autophagy has been proposed as an important event in a growing number of autoimmune and inflammatory diseases such as rheumatoid arthritis and lupus. However, the precise role of mechanistic target of rapamycin (mTOR)-dependent autophagy and its underlying regulatory mechanisms in the intestinal epithelium in response to inflammation and oxidative stress remain poorly understood.MethodsThe levels of p-mTOR, LC3B, p62 and autophagy in mice and LPS-treated cells were examined by immunoblotting, immunohistochemistry, confocal microscopy and transmission electron microscopy (TEM). We evaluated the expression of IL-1?, IL-8, TNF-?, MDA, SOD and T-AOC by quantitative real time-polymerase chain reaction (qRT-PCR) and commercially available kits after silencing of mTOR and ATG5. In vivo modulation of mTOR and autophagy was achieved by using AZD8055, rapamycin and 3-methyladenine. Finally, to verify the involvement of TLR4 signalling and the NF-?B pathway in cells and active ulcerative colitis (UC) patients, immunofluorescence, qRT-PCR, immunoblotting and TEM were performed to determine TLR4 signalling relevance to autophagy and inflammation.ResultsThe mTOR-dependent autophagic flux impairment in a murine model of colitis, human intestinal epithelial cells and active UC patients is probably regulated by TLR4-MyD88-MAPK signalling and the NF-?B pathway. Silencing mTOR remarkably attenuated, whereas inhibiting ATG5 aggravated, LPS-induced inflammation and oxidative injury. Pharmacological administration of mTOR inhibitors and autophagy stimulators markedly ameliorated experimental colitis and oxidative stress in vivo.ConclusionsOur findings not only shed light on the regulatory mechanism of mTOR-dependent autophagy, but also provided potential therapeutic targets for intestinal inflammatory diseases such as refractory inflammatory bowel disease.
| S-EPMC6161481 | biostudies-literature
Project description:BACKGROUND AND PURPOSE: Ototoxicity is a known adverse effect of cisplatin (CDDP). Since apoptosis is involved in the development of some pathological conditions associated with the administration of anticancer drugs, we examined, using immunohistochemical and electrophysiological techniques, the apoptotic changes in the cochlea of Sprague-Dawley (SD) rats after an injection of CDDP (5 mgkg(-1) body weight). EXPERIMENTAL APPROACH: Luciferase assays were used to determine the different caspase activities and ATP levels in protein extracts of whole cochleae. The expression of several apoptotic-related proteins was measured by means of Western blotting. These analyses were performed 2, 7 and 30 days after the CDDP injection. The auditory brain stem response was obtained before and at the different times after the injection of CDDP, before the animals were killed. KEY RESULTS: CDDP significantly increased the levels of caspase-3/7 activity and active caspase-3 protein expression and caspase-3 immunofluorescence staining, caspase-9 activity, and Bax protein expression but decreased Bcl-2 protein expression within the rat cochleae. Threshold shifts were significantly elevated 2 days after CDDP treatment. CONCLUSIONS AND IMPLICATIONS: These findings support the hypothesis that cisplatin-related apoptosis evokes an intrinsic pathway of pro-apoptotic signalling within the rat cochleae. Thus, selective inhibition of the sequence of events involved in the intrinsic apoptotic pathway could provide a strategy to minimize cisplatin-induced ototoxicity.
| S-EPMC2095105 | biostudies-other