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17-β estradiol protects ARPE-19 cells from oxidative stress through estrogen receptor-β.
Giddabasappa A, Bauler M, Yepuru M, Chaum E, Dalton JT, Eswaraka J
(2010) Invest Ophthalmol Vis Sci 51: 5278-87
MeSH Terms: Adenosine Triphosphate, Apoptosis, Blotting, Western, Cells, Cultured, Cytoprotection, Enzyme-Linked Immunosorbent Assay, Estradiol, Estrogen Receptor beta, Estrogens, Humans, Hydrogen Peroxide, Membrane Potential, Mitochondrial, Microscopy, Confocal, Mitochondria, Oxidative Stress, RNA, Small Interfering, Reactive Oxygen Species, Retinal Pigment Epithelium, Reverse Transcriptase Polymerase Chain Reaction, Superoxide Dismutase, tert-Butylhydroperoxide
Show Abstract · Added June 11, 2018
PURPOSE - To elucidate the mechanism of 17-β estradiol (17β-E(2))-mediated protection of retinal pigment epithelium (RPE) from oxidative stress.
METHODS - Cultured ARPE-19 cells were subjected to oxidative stress with t-butyl hydroxide or hydrogen peroxide in the presence or absence of 17β-E(2). Reactive oxygen species (ROS) were measured using H(2)DCFDA fluorescence. Apoptosis was evaluated by cell-death ELISA kit and Hoechst-3486 staining. Mitochondrial membrane potential was measured using the JC-1 assay. Cellular localization of estrogen receptor (ER) was evaluated by confocal microscopy. Gene expression and protein expression was quantified using qRT-PCR and western blotting. Superoxide dismutase and ATP levels were measured using commercial kits.
RESULTS - ARPE-19 cells expressed significant amounts of ERα and ERβ. Pretreatment with 17β-E2 protected ARPE-19 cells from oxidative stress and apoptosis. 17β-E(2) reduced the ROS levels and mitochondrial depolarization. The 17β-E(2)-mediated cytoprotection was inhibited by ER antagonists ICI (ERα and ERβ) and THC (ERβ) but not by tamoxifen (ERα). Knockdown of ERβ expression by siRNA abolished the protective effects of 17β-E(2). Further, qRT-PCR analysis revealed that 17β-E(2) pretreatment upregulated the expression of ERβ and phase II cellular antioxidant genes.
CONCLUSIONS - These results indicate that 17β-E(2) protects ARPE-19 cells from oxidative stress through an ERβ-dependent mechanism. 17β-E(2)-mediated cytoprotection occurred through the preservation of mitochondrial function, reduction of ROS production, and induction of cellular antioxidant genes.
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MeSH Terms
Intracellular delivery of a proapoptotic peptide via conjugation to a RAFT synthesized endosomolytic polymer.
Duvall CL, Convertine AJ, Benoit DS, Hoffman AS, Stayton PS
(2010) Mol Pharm 7: 468-76
MeSH Terms: Antineoplastic Agents, Cell Proliferation, Flow Cytometry, HeLa Cells, Hemolysis, Humans, Hydrogen-Ion Concentration, Membrane Potential, Mitochondrial, Models, Theoretical, Peptides, Polymers
Show Abstract · Added March 14, 2018
Peptides derived from the third B-cell lymphoma 2 (Bcl-2) homology domain (BH3) can heterodimerize with antiapoptotic Bcl-2 family members to block their activity and trigger apoptosis. Use of these peptides presents a viable anticancer approach, but delivery barriers limit the broad application of intracellular-acting peptides as clinical therapeutics. Here, a novel diblock copolymer carrier is described that confers desirable pharmaceutical properties to intracellular-acting therapeutic peptides through site-specific molecular conjugation. This polymer was prepared using reversible addition-fragmentation chain transfer (RAFT) to form a pyridyl disulfide end-functionalized, modular diblock copolymer with precisely controlled molecular weight (M(n)) and low polydispersity (PDI). The diblock polymer (M(n) 19,000 g/mol, PDI 1.27) was composed of an N-(2-hydroxypropyl) methacrylamide (HPMA) first block (M(n) 13,800 g/mol, PDI 1.13) intended to enhance water solubility and circulation time. The second polymer block was a pH-responsive composition designed to enhance endosomal escape and consisted of equimolar quantities of dimethylaminoethyl methacrylate (DMAEMA), propylacrylic acid (PAA), and butyl methacrylate (BMA). A hemolysis assay indicated that the diblock polymer undergoes a physiologically relevant pH-dependent switch from a membrane inert (1% hemolysis, pH 7.4) to a membrane disruptive (61% hemolysis, pH 5.8) conformation. Thiol-disulfide exchange reactions were found to efficiently produce reversible polymer conjugates (75 mol % peptide reactivity with polymer) with a cell-internalized proapoptotic peptide. Microscopy studies showed that peptide delivered via polymer conjugates effectively escaped endosomes and achieved diffusion into the cytosol. Peptide-polymer conjugates also produced significantly increased apoptotic activity over peptide alone in HeLa cervical carcinoma cells as found using flow cytometric measurements of mitochondrial membrane depolarization (2.5-fold increase) and cell viability tests that showed 50% cytotoxicity after 6 h of treatment with 10 muM peptide conjugate. These results indicate that this multifunctional carrier shows significant promise for proapoptotic peptide cancer therapeutics and also as a general platform for delivery of peptide drugs with intracellular targets.
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11 MeSH Terms
Long-term exposure to AZT, but not d4T, increases endothelial cell oxidative stress and mitochondrial dysfunction.
Kline ER, Bassit L, Hernandez-Santiago BI, Detorio MA, Liang B, Kleinhenz DJ, Walp ER, Dikalov S, Jones DP, Schinazi RF, Sutliff RL
(2009) Cardiovasc Toxicol 9: 1-12
MeSH Terms: Biomarkers, Cell Death, Cell Line, Tumor, Cells, Cultured, DNA, Mitochondrial, Endothelial Cells, Glutathione, Humans, Lactic Acid, Membrane Potential, Mitochondrial, Mitochondria, Mitochondria, Liver, Oxidative Stress, Phosphorylation, Reverse Transcriptase Inhibitors, Stavudine, Superoxides, Time Factors, Zidovudine
Show Abstract · Added February 17, 2016
Nucleoside reverse transcriptase inhibitors (NRTIs), such as zidovudine (AZT) and stavudine (d4T), cause toxicities to numerous tissues, including the liver and vasculature. While much is known about hepatic NRTI toxicity, the mechanism of toxicity in endothelial cells is incompletely understood. Human aortic endothelial and HepG2 liver cells were exposed to 1 muM AZT or d4T for up to 5 weeks. Markers of oxidative stress, mitochondrial function, NRTI phosphorylation, mitochondrial DNA (mtDNA) levels, and cytotoxicity were monitored over time. In endothelial cells, AZT significantly oxidized glutathione redox potential, increased total cellular and mitochondrial-specific superoxide, decreased mitochondrial membrane potential, increased lactate release, and caused cell death from weeks 3 through 5. Toxicity occurred in the absence of di- and tri-phosphorylated AZT and mtDNA depletion. These data show that oxidative stress and mitochondrial dysfunction in endothelial cells occur with a physiologically relevant concentration of AZT, and require long-term exposure to develop. In contrast, d4T did not induce endothelial oxidative stress, mitochondrial dysfunction, or cytotoxicity despite the presence of d4T-triphosphate. Both drugs depleted mtDNA in HepG2 cells without causing cell death. Endothelial cells are more susceptible to AZT-induced toxicity than HepG2 cells, and AZT caused greater endothelial dysfunction than d4T because of its pro-oxidative effects.
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19 MeSH Terms
Lower respiratory capacity in extraocular muscle mitochondria: evidence for intrinsic differences in mitochondrial composition and function.
Patel SP, Gamboa JL, McMullen CA, Rabchevsky A, Andrade FH
(2009) Invest Ophthalmol Vis Sci 50: 180-6
MeSH Terms: Abdominal Muscles, Animals, Blotting, Western, Electron Transport, Electron Transport Complex I, Electron Transport Complex II, Electron Transport Complex III, Electron Transport Complex IV, Energy Metabolism, Male, Membrane Potential, Mitochondrial, Mitochondria, Muscle, Mitochondrial Membranes, Oculomotor Muscles, Oxygen Consumption, Rats, Rats, Sprague-Dawley
Show Abstract · Added April 25, 2016
PURPOSE - The constant activity of the extraocular muscles is supported by abundant mitochondria. These organelles may enhance energy production by increasing the content of respiratory complexes. The authors tested the hypothesis that extraocular muscle mitochondria respire faster than do mitochondria from limb muscles because of the higher content of respiratory complexes.
METHODS - Inner mitochondrial membrane density was determined by stereological analysis of triceps surae (a limb muscle) and extraocular muscles of adult male Sprague-Dawley rats. The authors measured respiration rates of isolated mitochondria using a Clark-type electrode. The activity of respiratory complexes I, II, and IV was determined by spectrophotometry. The content of respiratory complexes was estimated by Western blot.
RESULTS - States 3, 4, and 5 respiration rates in extraocular muscle mitochondria were 40% to 60% lower than in limb muscle mitochondria. Extraocular muscle inner mitochondrial membrane density was similar to that of other skeletal muscles. Activity of complexes I and IV was lower in extraocular muscle mitochondria (approximately 50% the activity in triceps), but their content was approximately 15% to 30% higher. There was no difference in complex II content or activity or complex III content. Finally, complex V was less abundant in extraocular muscle mitochondria.
CONCLUSIONS - The results demonstrate that extraocular muscle mitochondria respire at slower rates than mitochondria from limb muscles, despite similar mitochondrial ultrastructure. Instead, differences were found in the activity (I, IV) and content (I, IV, V) of electron transport chain complexes. The discrepancy between activity and content of some complexes is suggestive of alternative subunit isoform expression in the extraocular muscles compared with limb muscles.
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17 MeSH Terms
t-Darpp promotes cancer cell survival by up-regulation of Bcl2 through Akt-dependent mechanism.
Belkhiri A, Dar AA, Zaika A, Kelley M, El-Rifai W
(2008) Cancer Res 68: 395-403
MeSH Terms: Activating Transcription Factor 1, Carcinoma, Caspase 3, Caspase 9, Cell Survival, Cyclic AMP Response Element-Binding Protein, Cytochromes c, DNA-Binding Proteins, Dopamine and cAMP-Regulated Phosphoprotein 32, Gene Expression Regulation, Neoplastic, Genes, bcl-2, Humans, Membrane Potential, Mitochondrial, Nuclear Proteins, Oncogene Protein v-akt, Phosphorylation, Protein Isoforms, RNA, Messenger, Regulatory Factor X Transcription Factors, Signal Transduction, Stomach Neoplasms, Transcription Factors, Tumor Cells, Cultured, Up-Regulation
Show Abstract · Added March 5, 2014
t-Darpp is a cancer-related truncated isoform of Darpp-32 (dopamine and cyclic-AMP-regulated phosphoprotein of M(r) 32,000). We detected overexpression of t-Darpp mRNA in two thirds of gastric cancers compared with normal samples (P = 0.004). Using 20 micromol/L ceramide treatment as a model for induction of apoptosis in AGS cancer cells, we found that expression of t-Darpp led to an increase in Bcl2 protein levels and blocked the activation of caspase-3 and caspase-9. The MitoCapture mitochondrial apoptosis and cytochrome c release assays indicated that t-Darpp expression enforces the mitochondrial transmembrane potential and protects against ceramide-induced apoptosis. Interestingly, the expression of t-Darpp in AGS cells led to >or=2-fold increase in Akt kinase activity with an increase in protein levels of p-Ser(473) Akt and p-Ser(9) GSK3 beta. These findings were further confirmed using tetracycline-inducible AGS cells stably expressing t-Darpp. We also showed transcriptional up-regulation of Bcl2 using the luciferase assay with Bcl2 reporter containing P1 full promoter, quantitative reverse transcription-PCR, and t-Darpp small interfering RNA. The Bcl2 promoter contains binding sites for cyclic AMP-responsive element binding protein CREB/ATF1 transcription factors and using the electrophoretic mobility shift assay with a CREB response element, we detected a stronger binding in t-Darpp-expressing cells. The t-Darpp expression led to an increase in expression and phosphorylation of CREB and ATF-1 transcription factors that were required for up-regulating Bcl2 levels. Indeed, knockdown of Akt, CREB, or ATF1 in t-Darpp-expressing cells reduced Bcl2 protein levels. In conclusion, the t-Darpp/Akt axis underscores a novel oncogenic potential of t-Darpp in gastric carcinogenesis and resistance to drug-induced apoptosis.
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24 MeSH Terms
RNA suppression of ERK2 leads to collapse of mitochondrial membrane potential with acute oxidative stress in human lens epithelial cells.
Flynn JM, Lannigan DA, Clark DE, Garner MH, Cammarata PR
(2008) Am J Physiol Endocrinol Metab 294: E589-99
MeSH Terms: Cell Line, Cyclic AMP-Dependent Protein Kinases, Enzyme Activation, Epithelial Cells, Estradiol, Humans, Lens, Crystalline, Membrane Potential, Mitochondrial, Mitogen-Activated Protein Kinase 1, Oxidative Stress, Phosphorylation, RNA, Small Interfering, Serine, bcl-Associated Death Protein
Show Abstract · Added January 20, 2015
17beta-Estradiol (E(2)) reduces oxidative stress-induced depolarization of mitochondrial membrane potential (MMP) in cultured human lens epithelial cells (HLE-B3). The mechanism by which the nongenomic effects of E(2) contributed to the protection against mitochondrial membrane depolarization was investigated. Mitochondrial membrane integrity is regulated by phosphorylation of BAD, and it is known that phosphorylation of Ser(112) inactivates BAD and prevents its participation in the mitochondrial death pathway. We found that E(2) rapidly increased both the phosphorylation of ERK2 and Ser(112) in BAD. Ser(112) is phosphorylated by p90 ribosomal S6 kinase (RSK), a Ser/Thr kinase, which is a downstream effector of ERK1/2. Inhibition of RSK by the RSK-specific inhibitor SL0101 did not reduce the level of E(2)-induced phosphorylation of Ser(112). Silencing BAD using small interfering RNA did not alter mitochondrial membrane depolarization elicited by peroxide insult. However, under the same conditions, silencing ERK2 dramatically increased membrane depolarization compared with the control small interfering RNA. Therefore, ERK2, functioning through a BAD-independent mechanism regulates MMP in humans lens epithelial cells. We propose that estrogen-induced activation of ERK2 acts to protect cells from acute oxidative stress. Moreover, despite the fact that ERK2 plays a regulatory role in mitochondrial membrane potential, estrogen was found to block mitochondrial membrane depolarization via an ERK-independent mechanism.
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14 MeSH Terms
Molecular mechanisms of angiotensin II-mediated mitochondrial dysfunction: linking mitochondrial oxidative damage and vascular endothelial dysfunction.
Doughan AK, Harrison DG, Dikalov SI
(2008) Circ Res 102: 488-96
MeSH Terms: Angiotensin II, Animals, Aorta, Cattle, Cell Respiration, Cells, Cultured, Endothelial Cells, Glutathione, Hydrogen Peroxide, Membrane Potential, Mitochondrial, Mitochondria, Mitochondrial Diseases, Nitric Oxide, Oxidative Stress, Superoxides, Vascular Diseases, Vasoconstrictor Agents
Show Abstract · Added February 17, 2016
Mitochondrial dysfunction is a prominent feature of most cardiovascular diseases. Angiotensin (Ang) II is an important stimulus for atherogenesis and hypertension; however, its effects on mitochondrial function remain unknown. We hypothesized that Ang II could induce mitochondrial oxidative damage that in turn might decrease endothelial nitric oxide (NO.) bioavailability and promote vascular oxidative stress. The effect of Ang II on mitochondrial ROS, mitochondrial respiration, membrane potential, glutathione, and endothelial NO. was studied in isolated mitochondria and intact bovine aortic endothelial cells using electron spin resonance, dihydroethidium high-performance liquid chromatography -based assay, Amplex Red and cationic dye fluorescence. Ang II significantly increased mitochondrial H2O2 production. This increase was blocked by preincubation of intact cells with apocynin (NADPH oxidase inhibitor), uric acid (scavenger of peroxynitrite), chelerythrine (protein kinase C inhibitor), N(G)-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor), 5-hydroxydecanoate (mitochondrial ATP-sensitive potassium channels inhibitor), or glibenclamide. Depletion of p22(phox) subunit of NADPH oxidase with small interfering RNA also inhibited Ang II-mediated mitochondrial ROS production. Ang II depleted mitochondrial glutathione, increased state 4 and decreased state 3 respirations, and diminished mitochondrial respiratory control ratio. These responses were attenuated by apocynin, 5-hydroxydecanoate, and glibenclamide. In addition, 5-hydroxydecanoate prevented the Ang II-induced decrease in endothelial NO. and mitochondrial membrane potential. Therefore, Ang II induces mitochondrial dysfunction via a protein kinase C-dependent pathway by activating the endothelial cell NADPH oxidase and formation of peroxynitrite. Furthermore, mitochondrial dysfunction in response to Ang II modulates endothelial NO. and generation, which in turn has ramifications for development of endothelial dysfunction.
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17 MeSH Terms
Species- and tissue-specific relationships between mitochondrial permeability transition and generation of ROS in brain and liver mitochondria of rats and mice.
Panov A, Dikalov S, Shalbuyeva N, Hemendinger R, Greenamyre JT, Rosenfeld J
(2007) Am J Physiol Cell Physiol 292: C708-18
MeSH Terms: Animals, Brain, Calcium, Hydrogen, Male, Membrane Potential, Mitochondrial, Mice, Mice, Inbred C57BL, Mitochondria, Mitochondria, Liver, Mitochondrial Membranes, Organ Specificity, Permeability, Phosphates, Rats, Rats, Sprague-Dawley, Reactive Oxygen Species, Species Specificity
Show Abstract · Added February 17, 2016
In animal models of neurodegenerative diseases pathological changes vary with the type of organ and species of the animals. We studied differences in the mitochondrial permeability transition (mPT) and reactive oxygen species (ROS) generation in the liver (LM) and brain (BM) of Sprague-Dawley rats and C57Bl mice. In the presence of ADP mouse LM and rat LM required three times less Ca(2+) to initiate mPT than the corresponding BM. Mouse LM and BM sequestered 70% and 50% more Ca(2+) phosphate than the rat LM and BM. MBM generated 50% more ROS with glutamate than the RBM, but not with succinate. With the NAD substrates, generation of ROS do not depend on the energy state of the BM. Organization of the respiratory complexes into the respirasome is a possible mechanism to prevent ROS generation in the BM. With BM oxidizing succinate, 80% of ROS generation was energy dependent. Induction of mPT does not affect ROS generation with NAD substrates and inhibit with succinate as a substrate. The relative insensitivity of the liver to systemic insults is associated with its high regenerative capacity. Neuronal cells with low regenerative capacity and a long life span protect themselves by minimizing ROS generation and by the ability to withstand very large Ca(2+) insults. We suggest that additional factors, such as oxidative stress, are required to initiate neurodegeneration. Thus the observed differences in the Ca(2+)-induced mPT and ROS generation may underlie both the organ-specific and species-specific variability in the animal models of neurodegenerative diseases.
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18 MeSH Terms