Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent reports have linked neuronal cell death by necrosis to poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation. It is believed that under stress, the activity of this enzyme is up-regulated, resulting in extensive poly(ADP-ribosyl)ation of nuclear proteins, using NAD(+) as its substrate, which, in turn, leads to the depletion of NAD(+). In efforts to restore the level of NAD(+), depletion of ATP occurs, resulting in the shutdown of ATP-dependent ionic pumps. This results in cell swelling and eventual loss of membrane selectivity, hallmarks of necrosis. Reports from in vitro and in vivo studies in the brain have shown that NMDA receptor activation stimulates PARP activity and that blockade of the enzyme provides substantial neuroprotection. The present study was undertaken to determine whether PARP activity is regulated by NMDA in the rat retina, and whether blockade of PARP activity provides protection against toxic effects of NMDA. Rat retinas exposed to intravitreal injections containing NMDA, with or without the PARP inhibitor N-(6-oxo-5, 6-dihydrophenanthridin-2-yl)-(N,-dimethylamino) acetamide hydrochloride (PJ-34), were assessed for changes in PARP-1 activity as evidenced by poly(ADP-ribosyl)ation (PAR), loss of membrane integrity, morphological indicators of apoptosis and necrosis, and ganglion cell loss. Results showed that: NMDA increased PAR formation in a concentration-dependent manner and caused a decline in retinal ATP levels; PJ-34 blockade attenuated the NMDA-induced formation of PAR and decline in ATP; NMDA induced the loss of membrane selectivity to ethidium bromide (EtBr) in inner retinal neurons, but loss of membrane selectivity was not prevented by blocking PARP activity; cells stained with EtBr, or reacted for TUNEL-labeling, displayed features characteristic of both apoptosis and necrosis. In the presence of PJ-34, greater numbers of cells exhibited apoptotic features; PJ-34 provided partial neuroprotection against NMDA-induced ganglion cell loss. These findings suggest that although blockade of PARP activity fully attenuates NMDA-induced PAR formation and loss of retinal ATP content, and improves the survival of select populations of ganglion cells, this approach does not provide full neuroprotection. In contrast, blockade of PARP activity promotes apoptotic-like cell death in the majority of cells undergoing cell death. Furthermore, these studies show that the loss of membrane selectivity is not dependent upon PAR formation or the resulting decline of ATP, and suggests that an alternative pathway, other than PARP activation, exists to mediate this event.
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PMID:Blockade of PARP activity attenuates poly(ADP-ribosyl)ation but offers only partial neuroprotection against NMDA-induced cell death in the rat retina. 1690 75

Preconditioning-induced ischemic tolerance is well documented in the brain, but cell-specific responses and mechanisms require further elucidation. The aim of this study was to develop an in vitro model of ischemic tolerance in human brain microvascular endothelial cells (HBMECs) and to examine the roles of phosphatidylinositol 3-kinase (PI3-kinase)/Akt and the inhibitor-of- apoptosis protein, survivin, in the ability of hypoxic preconditioning (HP) to protect endothelium from apoptotic cell death. Cultured HBMECs were subjected to HP, followed 16 h later by complete oxygen and glucose deprivation (OGD) for 8 h; cell viability was quantified at 20 h of reoxygenation (RO) by the 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide assay. HBMECs were examined at various times after HP or OGD/RO using immunoblotting and confocal laser scanning immunofluorescence microscopy for appearance of apoptotic markers and expression of phosphorylated (p)-Akt and p-survivin. Causal evidence for the participation of the PI3-kinase/Akt pathway in HP-induced protection and p-survivin upregulation was assessed by the PI3-kinase inhibitor LY-294002. HP significantly reduced OGD/RO-induced injury by 50% and also significantly reduced the OGD-induced translocation of apoptosis-inducing factor (AIF) from mitochondria to nucleus and the concomitant cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). PI3-kinase inhibition blocked HP-induced increases in Akt phosphorylation, reversed the effects of HP on OGD-induced AIF translocation and PARP-1 cleavage, blocked HP-induced survivin phosphorylation, and ultimately attenuated HP-induced protection of HBMECs from OGD. Thus HP promotes an antiapoptotic phenotype in HBMECs, in part by activating survivin via the PI3-kinase/Akt pathway. Survivin and other phosphorylation products of p-Akt may be therapeutic targets to protect cerebrovascular endothelium from apoptotic injury following cerebral ischemia.
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PMID:Hypoxic preconditioning protects human brain endothelium from ischemic apoptosis by Akt-dependent survivin activation. 1740 Jul 25

We recently identified a novel type III secretion system (T3SS) effector, AexU, from a diarrheal isolate SSU of Aeromonas hydrophila, and demonstrated that mice infected with the DeltaaexU mutant were significantly protected from mortality. Although the NH(2)-terminal domain of this toxin exhibits homology to AexT of A. salmonicida, a fish pathogen, and ExoT/S of Pseudomonas aeruginosa, the COOH-terminal domain of AexU is unique, with no homology to any known proteins in the NCBI database. In this study, we purified the full-length AexU and its NH(2)-terminal (amino acid residues 1-231) and COOH-terminal (amino acid residues 232-512) domains after expression of their corresponding genes in Escherichia coli as histidine-tag fusion proteins using the bacteriophage T7 RNA polymerase/promoter-based pET-30a vector system. The full-length and NH(2)- and COOH-terminal domains of AexU exhibited ADP-ribosyltransferase activity, with the former two exhibiting much higher activity than the latter. These different forms of AexU were also successfully expressed and produced in the HeLa Tet-Off cell system using a pBI-EGFP vector, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and intracellular staining of the toxin using flow cytometric analysis. Production of AexU in HeLa cells resulted in possible actin reorganization and cell rounding, as determined by phalloidin staining and confocal microscopy. Based on electron microscopy, the toxin also caused chromatin condensation, which is indicative of apoptosis. Apoptosis of HeLa cells expressing and producing AexU was confirmed by 7-amino actinomycin D (7-AAD) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] assays, by detection of cytoplasmic histone-associated DNA fragments, and by activation of caspases 3 and 9. These effects were much more pronounced in host cells that expressed and produced the full-length or NH(2)-terminal domain of AexU, compared to those that expressed and produced the COOH-terminal domain or the vector alone. This study represents the first characterization of this novel T3SS effector.
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PMID:Biological characterization of a new type III secretion system effector from a clinical isolate of Aeromonas hydrophila-part II. 1758 31

Among 13 different cell lines, gossypol (GOS) showed the most potent cytotoxic effect against human colorectal carcinoma cells including HT29, COLO205, COLO320HSR and COLO320DM cells according to an MTT assay. The cytotoxic effect of GOS was mediated by its induction of apoptosis as characterized by the occurrence of DNA ladders, apoptotic bodies and chromosome condensation in both COLO205 and HT29 cells. Activation of caspase 3, 6, 8 and 9, but not caspase 1, accompanied by the appearance of cleaved fragments of PARP (85 kDa), and caspase 3 (p17/p15), was identified in GOS-treated cells. Decreases in Bcl-xL and phosphorylated Bad proteins were found in GOS-treated cells. GOS induction of ROS production was detected by in vitro plasmid digestion, and an increase in the intracellular peroxide level was observed in GOS-treated COLO205 cells by the DCHF-DA assay. Antioxidants including N-acetyl-L-cysteine (NAC), catalase (CAT), tempol (TEM) and melatonin (MEL), but not allopurinol (ALL), pyrrolidine dithiocarbamate (PDTC) or diphenylene iodonium (DPI), significantly inhibited GOS-induced Reactive oxygen species (ROS) production through blocking the occurrence of apoptosis. GOS induced mitochondrial dysfunction characterized by a loss of the mitochondria membrane potential via DiOC6 staining, and the release of cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) from mitochondria to the cytoplasm was observed. Removing mitochondria by ethidium bromide (EtBr) treatment significantly reduced the apoptotic effect of GOS in COLO205 cells. Furthermore, an intraperitoneal injection of GOS or gossypol acetic acid (GAA) significantly reduced the growth of colorectal carcinoma induced by a subcutaneous injection of COLO205 cells in nude mice. Results of the present study provide the first evidences demonstrating the in vitro and in vivo antitumor effects of GOS via an ROS-dependent mitochondrial apoptosis in colorectal carcinoma.
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PMID:Gossypol reduction of tumor growth through ROS-dependent mitochondria pathway in human colorectal carcinoma cells. 1759 9

Homoharringtonine (HHT) is a plant alkaloid with antileukemic activity which is currently being used for treatment of acute and chronic leukemias. The present studies have evaluated the effect of HHT on proliferation and apoptosis in human myeloma cells. Myeloma cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptotic cells and cell cycle were evaluated by flow cytometry. Level of caspase-8, caspase-9, caspase-3, and DNA repair enzyme poly (ADP-ribose) polymerase (PARP), were investigated using Western blot analysis. We found that HHT significantly inhibited the proliferation of human multiple myeloma (MM) cell lines and tumor cells from patients with relapsed refractory MM in a dose-dependent manner. HHT also induced apoptosis in myeloma cells as evidenced by flow cytometric detection of annexin V binding assay. This apoptotic process was associated with the activation of caspase-8, caspase-9, caspase-3 and PARP. The results also demonstrate that HHT potentiates dexamethasone-induced killing of MM cells. These findings indicate that HHT may be effective in the treatment of MM.
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PMID:Homoharringtonine induces apoptosis and growth arrest in human myeloma cells. 1761 69

Myocyte injury due to myocardial reperfusion injury plays a crucial role in the pathogenesis of acute myocardial infarction even after successful coronary revascularization. Identification of compounds that reduce reperfusion-associated myocyte death is important. Therefore, we developed an in vitro model of myocardial reperfusion injury in H9c2 rat cardiomyocytes and applied a cell-based high-throughput approach to screen a standard library of pharmacologically active compounds (LOPAC) in order to identify drugs with cardioprotective effects. Oxidative stress was induced with hydrogen peroxide (H2O2) treatment, which resulted in approximately 50% reduction in cell viability. Test compounds were added at a 3-microM final concentration as a pretreatment or in a delayed fashion (30 min after the peroxide challenge in order to imitate pharmacological treatment following angioplasty). Cells were cultured for 3 or 24 h. Viability was quantitated with the methylthiazolyldiphenyl-tetrazolium bromide method. Cytotoxicity and cytoprotection were also evaluated by measuring the lactate dehydrogenase activity in the cell culture supernatant. The screening identified a number of compounds with cytoprotective action, including molecules that are known to interfere with components of DNA repair and cell cycle progression, e.g. poly(ADP-ribose) polymerase (PARP) inhibitors, topoisomerase inhibitors, and cyclin dependent kinase inhibitors, or reduce energy consumption by interfering with cardiac myofilament function. A number of dopamine D1 receptor agonists also provided significant cytoprotection at 3 h, but only three of them showed a similar effect at 24 h: chloro- and bromo-APB and chloro-PB hydrobromide. Chloro-APB hydrobromide significantly reduced peroxide-induced PARP activation in the myocytes independently of its action on dopamine D1 receptors, but lacked PARP inhibitor capacity in a cell-free PARP assay system. In conclusion, the pattern of cytoprotective drugs identified in the current assay supports the overall validity of our model system. The findings demonstrate that cytoprotective agents, including novel indirect inhibitors of cellular PARP activation can be identified with the method, chloro-APB hydrobromide being one such compound. The current experimental setting can be employed for cell-based high-throughput screening of various compound libraries.
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PMID:Oxidant-induced cardiomyocyte injury: identification of the cytoprotective effect of a dopamine 1 receptor agonist using a cell-based high-throughput assay. 1791 70

Parkinson's disease is characterized by the progressive degeneration of midbrain dopaminergic neurons. Buddleia lindleyana is a traditional Chinese herb, commonly called Zui Yu Cao. The purification and identification of pedicularioside A and other phenylethanoid glycosides from this plant have been reported. However, their neuroprotective effects on the 1-methyl-4-phenylpyridinium ion (MPP(+))-induced death of rat mesencephalic neuron primary cultures and the precise mechanism of this protection remains unclear. We used the 3-(4, 5-dimethylthiozol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay for cellular growth to examine the effects of five phenylethanoid glycosides isolated from B. lindleyana, including pedicularioside A, leucosceptoside A, isoacteoside, acteoside, and arenariside, on the viability of mesencephalic neurons treated with MPP(+). Of the compounds tested, pedicularioside A exhibited the greatest degree of protection from MPP(+)-induced cell death. We also observed a marked increase in the number of tyrosine hydroxylase immunoreactive neurons. Pedicularioside A inhibited expression of the caspase-3 gene and cleavage of poly (ADP-ribose) polymerase (PARP) in cultures exposed to MPP(+). Our results suggest that pedicularioside A has a neuroprotective effect to improve the survival of mesencephalic neurons (dopaminergic neurons and non-dopaminergic neurons). The mode of action appears to be the inhibition of caspase-3 gene expression, thereby protecting mesencephalic neurons from MPP(+)-induced cell death.
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PMID:Pedicularioside A from Buddleia lindleyana inhibits cell death induced by 1-methyl-4-phenylpyridinium ions (MPP+) in primary cultures of rat mesencephalic neurons. 1803 49

Fig fruit latex (FFL) contains significant amounts of polyphenolic compounds and can serve as a source of antioxidants after human consumption. The purpose of this study is to confirm anticancer activity of FFL against human cancer cells and to further elucidate its mechanism of activity. Human glioblastoma, hepatocellular carcinoma, and normal liver cells were used for in vitro tests of FFL effects. Those tests included cytotoxicity, colony formation inhibition, Brdu incorporation, acridine orange/ethidium bromide (AO/EB) staining for apoptotic cells, cell cycle distribution through flow cytometry (FCM), and ADP-ribosyltransferase (NAD+; poly(ADP-ribose) polymerase)-like 1 (ADPERL1) mRNA expression through RT-PCR in response to FFL treatment. After FFL treatment, the proliferation, colony formation, and Brdu labeling indices of cancer cells decreased (P<0.05), while the AO/EB stained apoptotic cells increased (P<0.05). By FCM analysis, an increase of G(0)/G(1) phase cell population and decrease of S and G(2)/M phase cells were observed (P<0.01), while both ADPRTL1 mRNA expression and apoptotic indices increased (P<0.01). The findings in these studies suggested that FFL exhibited potent cytotoxicity in some human cancer cells with little effect in normal cells at certain concentration. The mechanism for such effects might be associated with the inhibition of DNA synthesis, induction of apoptosis, and cell cycle arrest of cancer cells.
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PMID:Cytotoxicity of fig fruit latex against human cancer cells. 1807 3

The neurotoxicity of l-3,4-dihydroxyphenylalanine (L-DOPA), used for the treatment of Parkinson's disease, remains controversial. Although there are many reports suggesting that long-term treatment of L-DOPA causes neuronal death, an increasing body of recent evidence has proposed that L-DOPA might be neuroprotective rather than neurotoxic. We investigated the effect of L-DOPA on neuronally differentiated PC12 (nPC12) cells by treating cells with various concentrations of L-DOPA for 24h. We also studied whether glycogen synthase kinase (GSK)-3 activation is related to L-DOPA-induced neurotoxicity by simultaneously treating cells with several concentrations of L-DOPA and a GSK-3 inhibitor for 24h. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, trypan blue staining, cell counting kit-8, and DAPI staining all showed that L-DOPA decreased nPC12 cell viability at high concentrations. In addition, 100 microM L-DOPA treatment significantly increased the activity of GSK-3 and death signals including cytochrome c, activated caspase-3 and cleaved PARP, and decreased survival signals including heat shock transcription factor-1 in a concentration-dependent manner. Treatment with GSK-3 inhibitor VIII or lithium chloride prevented L-DOPA-induced cell death. Together, these results suggest that L-DOPA induces neuronal cell death at high concentrations and that the neurotoxic effect of L-DOPA might be mediated in part by GSK-3 activation.
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PMID:Inhibition of glycogen synthase kinase-3 reduces L-DOPA-induced neurotoxicity. 1838 27

Mitochondrial dysfunction resulting from mitochondrial DNA (mtDNA) mutations and/or depletion has been correlated with cancer progression and drug resistance. To investigate the role of mtDNA in prostate cancer progression, we used LNCaP and PC-3 prostate carcinoma cells as experimental model. Compared to minimally invasive androgen-dependent LNCaP cells, highly invasive androgen-independent PC-3 cells, as well as androgen-independent DU145 and C4-2 cells, exhibited significantly reduced mtDNA content. In PC-3 cells, reduction of mtDNA was accompanied by decreased mitochondrial membrane potential (DeltaPsi(m)), increased migration onto the basement membrane protein laminin-1, reduced chemosensitivity to paclitaxel (IC(50)=110 nM vs. 22 nM) and decreased expression of poly(ADP-ribose) polymerase (PARP)-1. To investigate the relationship between mtDNA depletion and these phenotypic characteristics, we established mtDNA-depleted LNCaP cells [Rho(-)] by long-term exposure to ethidium bromide or treated wild-type LNCaP cells with a mitochondrial ionophore, carbonyl cyanide m-chlorophenylhydrazone. Both manipulations resulted in DeltaPsi(m) loss, acquisition of invasive cytology, increased motility onto laminin-1, reduced sensitivity to paclitaxel (IC(50)= approximately 100 nM) and approximately 75% reduction in PARP-1 protein levels, resembling PC-3 cells. Overall, these results provide novel evidence demonstrating that mtDNA depletion in early prostate carcinoma may contribute to the acquisition of a more invasive phenotype that is less sensitive to paclitaxel-induced apoptosis.
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PMID:Mitochondrial DNA depletion reduces PARP-1 levels and promotes progression of the neoplastic phenotype in prostate carcinoma. 1860 66


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