Activity-based assay for human mono-ADP-ribosyltransferases ARTD7/PARP15 and ARTD10/PARP10 aimed at screening and profiling inhibitors.

Science.gov (United States)

Venkannagari, Harikanth; Fallarero, Adyary; Feijs, Karla L H; Lüscher, Bernhard; Lehtiö, Lari

2013-05-13

Poly(ADP-ribose) polymerases (PARPs) or diphtheria toxin like ADP-ribosyl transferases (ARTDs) are enzymes that catalyze the covalent modification of proteins by attachment of ADP-ribose units to the target amino acid residues or to the growing chain of ADP-ribose. A subclass of the ARTD superfamily consists of mono-ADP-ribosyl transferases that are thought to modify themselves and other substrate proteins by covalently adding only a single ADP-ribose moiety to the target. Many of the ARTD enzymes are either established or potential drug targets and a functional activity assay for them will be a valuable tool to identify selective inhibitors for each enzyme. Existing assays are not directly applicable for screening of inhibitors due to the different nature of the reaction and different target molecules. We modified and applied a fluorescence-based assay previously described for PARP1/ARTD1 and tankyrase/ARTD5 for screening of PARP10/ARTD10 and PARP15/ARTD7 inhibitors. The assay measures the amount of NAD(+) present after chemically converting it to a fluorescent analog. We demonstrate that by using an excess of a recombinant acceptor protein the performance of the activity-based assay is excellent for screening of compound libraries. The assay is homogenous and cost effective, making it possible to test relatively large compound libraries. This method can be used to screen inhibitors of mono-ARTDs and profile inhibitors of the enzyme class. The assay was optimized for ARTD10 and ARTD7, but it can be directly applied to other mono-ARTDs of the ARTD superfamily. Profiling of known ARTD inhibitors against ARTD10 and ARTD7 in a validatory screening identified the best inhibitors with submicromolar potencies. Only few of the tested ARTD inhibitors were potent, implicating that there is a need to screen new compound scaffolds. This is needed to create small molecules that could serve as biological probes and potential starting points for drug discovery projects against

Evaluation of candidate biomarkers to predict cancer cell sensitivity or resistance to PARP-1 inhibitor treatment

DEFF Research Database (Denmark)

Oplustilova, L.; Wolanin, K.; Bartkova, J.

2012-01-01

combinations with camptothecin or ionizing radiation. Furthermore, monitoring pARsylation and Rad51 foci formation as surrogate markers for PARP activity and HR, respectively, supported their candidacy for biomarkers of PARP-1i responses. As to resistance mechanisms, we confrmed the role of the multidrug......(ADp-ribose) polymerase-1 (PARP-1), an enzyme critical for repair pathways alternative to HR. While promising, treatment with PARP-1 inhibitors (PARP-1i) faces some hurdles, including (1) acquired resistance, (2) search for other sensitizing, non-BRCA1/2 cancer defects and (3) lack of biomarkers to predict response...

A Gene Expression Profile of BRCAness That Predicts for Responsiveness to Platinum and PARP Inhibitors

Science.gov (United States)

2017-02-01

affecting the function of Fanconi Anemia (FA) genes ( FANCA /B/C/D2/E/F/G/I/J/L/M, PALB2) or DNA damage response genes involved in HR 5 (ATM, ATR...Award Number: W81XWH-10-1-0585 TITLE: A Gene Expression Profile of BRCAness That Predicts for Responsiveness to Platinum and PARP Inhibitors...To) 15 July 2010 – 2 Nov.2016 4. TITLE AND SUBTITLE A Gene Expression Profile of BRCAness That Predicts for Responsiveness to Platinum and PARP

Human mass balance study and metabolite profiling of 14C-niraparib, a novel poly(ADP-Ribose) polymerase (PARP)-1 and PARP-2 inhibitor, in patients with advanced cancer

NARCIS (Netherlands)

van Andel, Lotte; Zhang, Z; Lu, S.; Kansra, V; Agarwal, S.; Hughes, L.; Tibben, M.; Gebretensae, A.; Lucas, L.; Hillebrand, Michel J X; Rosing, H.; Schellens, J H M|info:eu-repo/dai/nl/073926272; Beijnen, J H|info:eu-repo/dai/nl/071919570

2017-01-01

Niraparib is an investigational oral, once daily, selective poly(ADP-Ribose) polymerase (PARP)-1 and PARP-2 inhibitor. In the pivotal Phase 3 NOVA/ENGOT/OV16 study, niraparib met its primary endpoint of improving progression-free survival (PFS) for adult patients with recurrent, platinum sensitive,

A phenanthrene derived PARP inhibitor is an extra-centrosomes de-clustering agent exclusively eradicating human cancer cells

Directory of Open Access Journals (Sweden)

Izraeli Shai

2011-09-01

Full Text Available Abstract Background Cells of most human cancers have supernumerary centrosomes. To enable an accurate chromosome segregation and cell division, these cells developed a yet unresolved molecular mechanism, clustering their extra centrosomes at two poles, thereby mimicking mitosis in normal cells. Failure of this bipolar centrosome clustering causes multipolar spindle structures and aberrant chromosomes segregation that prevent normal cell division and lead to 'mitotic catastrophe cell death'. Methods We used cell biology and biochemical methods, including flow cytometry, immunocytochemistry and live confocal imaging. Results We identified a phenanthrene derived PARP inhibitor, known for its activity in neuroprotection under stress conditions, which exclusively eradicated multi-centrosomal human cancer cells (mammary, colon, lung, pancreas, ovarian while acting as extra-centrosomes de-clustering agent in mitosis. Normal human proliferating cells (endothelial, epithelial and mesenchymal cells were not impaired. Despite acting as PARP inhibitor, the cytotoxic activity of this molecule in cancer cells was not attributed to PARP inhibition alone. Conclusion We identified a water soluble phenanthridine that exclusively targets the unique dependence of most human cancer cells on their supernumerary centrosomes bi-polar clustering for their survival. This paves the way for a new selective cancer-targeting therapy, efficient in a wide range of human cancers.

The PARP inhibitor PJ-34 sensitizes cells to UVA-induced phototoxicity by a PARP independent mechanism.

Science.gov (United States)

Lakatos, Petra; Hegedűs, Csaba; Salazar Ayestarán, Nerea; Juarranz, Ángeles; Kövér, Katalin E; Szabó, Éva; Virág, László

2016-08-01

A combination of a photosensitizer with light of matching wavelength is a common treatment modality in various diseases including psoriasis, atopic dermatitis and tumors. DNA damage and production of reactive oxygen intermediates may impact pathological cellular functions and viability. Here we set out to investigate the role of the nuclear DNA nick sensor enzyme poly(ADP-ribose) polymerase 1 in photochemical treatment (PCT)-induced tumor cell killing. We found that silencing PARP-1 or inhibition of its enzymatic activity with Veliparib had no significant effect on the viability of A431 cells exposed to 8-methoxypsoralen (8-MOP) and UVA (2.5J/cm(2)) indicating that PARP-1 is not likely to be a key player in either cell survival or cell death of PCT-exposed cells. Interestingly, however, another commonly used PARP inhibitor PJ-34 proved to be a photosensitizer with potency equal to 8-MOP. Irradiation of PJ-34 with UVA caused changes both in the UV absorption and in the 1H NMR spectra of the compound with the latter suggesting UVA-induced formation of tautomeric forms of the compound. Characterization of the photosensitizing effect revealed that PJ-34+UVA triggers overproduction of reactive oxygen species, induces DNA damage, activation of caspase 3 and caspase 8 and internucleosomal DNA fragmentation. Cell death in this model could not be prevented by antioxidants (ascorbic acid, trolox, glutathione, gallotannin or cell permeable superoxide dismutase or catalase) but could be suppressed by inhibitors of caspase-3 and -8. In conclusion, PJ-34 is a photosensitizer and PJ-34+UVA causes DNA damage and caspase-mediated cell death independently of PARP-1 inhibition. Copyright © 2016 Elsevier B.V. All rights reserved.

Suppression of Homologous Recombination by insulin-like growth factor-1 inhibition sensitizes cancer cells to PARP inhibitors

International Nuclear Information System (INIS)

Amin, Oreekha; Beauchamp, Marie-Claude; Nader, Paul Abou; Laskov, Ido; Iqbal, Sanaa; Philip, Charles-André; Yasmeen, Amber; Gotlieb, Walter H.

2015-01-01

Impairment of homologous recombination (HR) is found in close to 50 % of ovarian and breast cancer. Tumors with BRCA1 mutations show increased expression of the Insulin-like growth factor type 1 receptor (IGF-1R). We previously have shown that inhibition of IGF-1R results in growth inhibition and apoptosis of ovarian tumor cells. In the current study, we aimed to investigate the correlation between HR and sensitivity to IGF-1R inhibition. Further, we hypothesized that IGF-1R inhibition might sensitize HR proficient cancers to Poly ADP ribose polymerase (PARP) inhibitors. Using ovarian and breast cancer cellular models with known BRCA1 status, we evaluated their HR functionality by RAD51 foci formation assay. The 50 % lethal concentration (LC50) of Insulin-like growth factor type 1 receptor kinase inhibitor (IGF-1Rki) in these cells was assessed, and western immunoblotting was performed to determine the expression of proteins involved in the IGF-1R pathway. Moreover, IGF-1R inhibitors were added on HR proficient cell lines to assess mRNA and protein expression of RAD51 by qPCR and western blot. Also, we explored the interaction between RAD51 and Insulin receptor substance 1 (IRS-1) by immunoprecipitation. Next, combination effect of IGF-1R and PARP inhibitors was evaluated by clonogenic assay. Cells with mutated/methylated BRCA1 showed an impaired HR function, and had an overactivation of the IGF-1R pathway. These cells were more sensitive to IGF-1R inhibition compared to HR proficient cells. In addition, the IGF-IR inhibitor reduced RAD51 expression at mRNA and protein levels in HR proficient cells, and sensitized these cells to PARP inhibitor. Targeting IGF-1R might lead to improved personalized therapeutic approaches in cancer patients with HR deficiency. Targeting both PARP and IGF-1R might increase the clinical efficacy in HR deficient patients and increase the population of patients who may benefit from PARP inhibitors

New approaches of PARP-1 inhibitors in human lung cancer cells and cancer stem-like cells by some selected anthraquinone-derived small molecules.

Directory of Open Access Journals (Sweden)

Yu-Ru Lee

Full Text Available Poly (ADP-ribose polymerase-1 (PARP-1 and telomerase, as well as DNA damage response pathways are targets for anticancer drug development, and specific inhibitors are currently under clinical investigation. The purpose of this work is to evaluate anticancer activities of anthraquinone-derived tricyclic and tetracyclic small molecules and their structure-activity relationships with PARP-1 inhibition in non-small cell lung cancer (NSCLC and NSCLC-overexpressing Oct4 and Nanog clone, which show high-expression of PARP-1 and more resistance to anticancer drug. We applied our library selected compounds to NCI's 60 human cancer cell-lines (NCI-60 in order to generate systematic profiling data. Based on our analysis, it is hypothesized that these drugs might be, directly and indirectly, target components to induce mitochondrial permeability transition and the release of pro-apoptotic factors as potential anti-NSCLC or PARP inhibitor candidates. Altogether, the most active NSC747854 showed its cytotoxicity and dose-dependent PARP inhibitory manner, thus it emerges as a promising structure for anti-cancer therapy with no significant negative influence on normal cells. Our studies present evidence that telomere maintenance should be taken into consideration in efforts not only to overcome drug resistance, but also to optimize the use of telomere-based therapeutics. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and telomerase inhibitors, in particular. Together, the data presented here expand our insight into the PARP inhibitors and support the resource-demanding lead optimization of structurally related small molecules for human cancer therapy.

New Approaches of PARP-1 Inhibitors in Human Lung Cancer Cells and Cancer Stem-Like Cells by Some Selected Anthraquinone-Derived Small Molecules

Science.gov (United States)

Yu, Dah-Shyong; Huang, Kuo-Feng; Chou, Shih-Jie; Chen, Tsung-Chih; Lee, Chia-Chung; Chen, Chun-Liang; Chiou, Shih-Hwa; Huang, Hsu-Shan

2013-01-01

Poly (ADP-ribose) polymerase-1 (PARP-1) and telomerase, as well as DNA damage response pathways are targets for anticancer drug development, and specific inhibitors are currently under clinical investigation. The purpose of this work is to evaluate anticancer activities of anthraquinone-derived tricyclic and tetracyclic small molecules and their structure-activity relationships with PARP-1 inhibition in non-small cell lung cancer (NSCLC) and NSCLC-overexpressing Oct4 and Nanog clone, which show high-expression of PARP-1 and more resistance to anticancer drug. We applied our library selected compounds to NCI's 60 human cancer cell-lines (NCI-60) in order to generate systematic profiling data. Based on our analysis, it is hypothesized that these drugs might be, directly and indirectly, target components to induce mitochondrial permeability transition and the release of pro-apoptotic factors as potential anti-NSCLC or PARP inhibitor candidates. Altogether, the most active NSC747854 showed its cytotoxicity and dose-dependent PARP inhibitory manner, thus it emerges as a promising structure for anti-cancer therapy with no significant negative influence on normal cells. Our studies present evidence that telomere maintenance should be taken into consideration in efforts not only to overcome drug resistance, but also to optimize the use of telomere-based therapeutics. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and telomerase inhibitors, in particular. Together, the data presented here expand our insight into the PARP inhibitors and support the resource-demanding lead optimization of structurally related small molecules for human cancer therapy. PMID:23451039

IDH1/2 Mutations Sensitize Acute Myeloid Leukemia to PARP Inhibition and This Is Reversed by IDH1/2-Mutant Inhibitors.

Science.gov (United States)

Molenaar, Remco J; Radivoyevitch, Tomas; Nagata, Yasunobu; Khurshed, Mohammed; Przychodzen, Bartolomiej; Makishima, Hideki; Xu, Mingjiang; Bleeker, Fonnet E; Wilmink, Johanna W; Carraway, Hetty E; Mukherjee, Sudipto; Sekeres, Mikkael A; van Noorden, Cornelis J F; Maciejewski, Jaroslaw P

2018-04-01

Purpose: Somatic mutations in IDH1/2 occur in approximately 20% of patients with myeloid neoplasms, including acute myeloid leukemia (AML). IDH1/2 MUT enzymes produce D -2-hydroxyglutarate ( D 2HG), which associates with increased DNA damage and improved responses to chemo/radiotherapy and PARP inhibitors in solid tumor cells. Whether this also holds true for IDH1/2 MUT AML is not known. Experimental Design: Well-characterized primary IDH1 MUT , IDH2 MUT , and IDH1/2 WT AML cells were analyzed for DNA damage and responses to daunorubicin, ionizing radiation, and PARP inhibitors. Results: IDH1/2 MUT caused increased DNA damage and sensitization to daunorubicin, irradiation, and the PARP inhibitors olaparib and talazoparib in AML cells. IDH1/2 MUT inhibitors protected against these treatments. Combined treatment with a PARP inhibitor and daunorubicin had an additive effect on the killing of IDH1/2 MUT AML cells. We provide evidence that the therapy sensitivity of IDH1/2 MUT cells was caused by D 2HG-mediated downregulation of expression of the DNA damage response gene ATM and not by altered redox responses due to metabolic alterations in IDH1/2 MUT cells. Conclusions: IDH1/2 MUT AML cells are sensitive to PARP inhibitors as monotherapy but especially when combined with a DNA-damaging agent, such as daunorubicin, whereas concomitant administration of IDH1/2 MUT inhibitors during cytotoxic therapy decrease the efficacy of both agents in IDH1/2 MUT AML. These results advocate in favor of clinical trials of PARP inhibitors either or not in combination with daunorubicin in IDH1/2 MUT AML. Clin Cancer Res; 24(7); 1705-15. ©2018 AACR . ©2018 American Association for Cancer Research.

The irreversible ERBB1/2/4 inhibitor neratinib interacts with the PARP1 inhibitor niraparib to kill ovarian cancer cells.

Science.gov (United States)

Booth, Laurence; Roberts, Jane L; Samuel, Peter; Avogadri-Connors, Francesca; Cutler, Richard E; Lalani, Alshad S; Poklepovic, Andrew; Dent, Paul

2018-06-03

The irreversible ERBB1/2/4 inhibitor neratinib has been shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET, PDGFRα and mutant RAS proteins via autophagic degradation. Neratinib interacted in an additive to synergistic fashion with the approved PARP1 inhibitor niraparib to kill ovarian cancer cells. Neratinib and niraparib caused the ATM-dependent activation of AMPK which in turn was required to cause mTOR inactivation, ULK-1 activation and ATG13 phosphorylation. The drug combination initially increased autophagosome levels followed later by autolysosome levels. Preventing autophagosome formation by expressing activated mTOR or knocking down of Beclin1, or knock down of the autolysosome protein cathepsin B, reduced drug combination lethality. The drug combination caused an endoplasmic reticulum stress response as judged by enhanced eIF2α phosphorylation that was responsible for reducing MCL-1 and BCL-XL levels and increasing ATG5 and Beclin1 expression. Knock down of BIM, but not of BAX or BAK, reduced cell killing. Expression of activated MEK1 prevented the drug combination increasing BIM expression and reduced cell killing. Downstream of the mitochondrion, drug lethality was partially reduced by knock down of AIF, but expression of dominant negative caspase 9 was not protective. Our data demonstrate that neratinib and niraparib interact to kill ovarian cancer cells through convergent DNA damage and endoplasmic reticulum stress signaling. Cell killing required the induction of autophagy and was cathepsin B and AIF -dependent, and effector caspase independent.

Vasoactivity of rucaparib, a PARP-1 inhibitor, is a complex process that involves myosin light chain kinase, P2 receptors, and PARP itself.

Directory of Open Access Journals (Sweden)

Cian M McCrudden

Full Text Available Therapeutic inhibition of poly(ADP-ribose polymerase (PARP, as monotherapy or to supplement the potencies of other agents, is a promising strategy in cancer treatment. We previously reported that the first PARP inhibitor to enter clinical trial, rucaparib (AG014699, induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK 10-fold more potently than its commercially available inhibitor ML-9. Moreover, rucaparib produces additive relaxation above the maximal degree achievable with ML-9, suggesting that MLCK inhibition is not solely responsible for dilation. Inhibition of nitric oxide synthesis using L-NMMA also failed to impact rucaparib's activity. Rucaparib contains the nicotinamide pharmacophore, suggesting it may inhibit other NAD+-dependent processes. NAD+ exerts P2 purinergic receptor-dependent inhibition of smooth muscle contraction. Indiscriminate blockade of the P2 purinergic receptors with suramin abrogated rucaparib-induced vasodilation in rat arterial tissue without affecting ML-9-evoked dilation, although the specific receptor subtypes responsible have not been unequivocally identified. Furthermore, dorsal window chamber and real time tumor vessel perfusion analyses in PARP-1-/- mice indicate a potential role for PARP in dilation of tumor-recruited vessels. Finally, rucaparib provoked relaxation in 70% of patient-derived tumor-associated vessels. These data provide tantalising evidence of the complexity of the mechanism underlying rucaparib-mediated vasodilation.

Effect of 3-aminobenzamide, PARP inhibitor, on matrix metalloproteinase-9 level in plasma and brain of ischemic stroke model

International Nuclear Information System (INIS)

Koh, Seong-Ho; Chang, Dae-Il; Kim, Hee-Tae; Kim, Juhan; Kim, Myung-Ho; Kim, Kyung Suk; Bae, Inhee; Kim, Haekwon; Kim, Dong Won; Kim, Seung Hyun

2005-01-01

We investigated the effect of poly(ADP-ribose) polymerase (PARP) inhibitor on the levels of plasma and brain matrix metalloproteinase-9 (MMP-9) and the expression of nuclear factor kappa B (NF-κB) during experimental focal cerebral ischemia. The 3-aminobenzamide (3-AB), a PARP inhibitor, and saline were administered to 80 Sprague-Dawley rats [3-AB group; 5 rats for plasma sampling, 35 for brain sampling, and 40 for TTC staining] and to 85 rats (10, 35, and 40, respectively), respectively, 10 min before the occlusion of the left middle cerebral artery (MCAo) for 2 h. Infarct volume was measured by TTC staining, the serial levels of plasma and brain MMP-9 were measured by zymography just before and 2, 4, 8, 24, 48, and 72 h after MCAo, brain NF-κB activity was determined by Western blotting, and neutrophil infiltration was evaluated by assessing myeloperoxidase activity. Compared with control group, the levels of plasma and brain MMP-9, brain NF-κB, and MPO activities were significantly reduced in 3-AB group at each time point (p < 0.05). Plasma MMP-9 increased maximally at 4 h and then decreased rapidly, brain MMP-9 increased maximally at 24 h and persisted until 72 h, and NF-κB increased maximally at 24 h and then decreased slowly in both groups. Therefore, the PARP inhibitor reduces the expression of MMP-9 and NF-κB and the infiltration of neutrophils in ischemic stroke

PARP Inhibition Restores Extrinsic Apoptotic Sensitivity in Glioblastoma

Science.gov (United States)

Karpel-Massler, Georg; Pareja, Fresia; Aimé, Pascaline; Shu, Chang; Chau, Lily; Westhoff, Mike-Andrew; Halatsch, Marc-Eric; Crary, John F.; Canoll, Peter; Siegelin, Markus D.

2014-01-01

Background Resistance to apoptosis is a paramount issue in the treatment of Glioblastoma (GBM). We show that targeting PARP by the small molecule inhibitors, Olaparib (AZD-2281) or PJ34, reduces proliferation and lowers the apoptotic threshold of GBM cells in vitro and in vivo. Methods The sensitizing effects of PARP inhibition on TRAIL-mediated apoptosis and potential toxicity were analyzed using viability assays and flow cytometry in established GBM cell lines, low-passage neurospheres and astrocytes in vitro. Molecular analyses included western blots and gene silencing. In vivo, effects on tumor growth were examined in a murine subcutaneous xenograft model. Results The combination treatment of PARP inhibitors and TRAIL led to an increased cell death with activation of caspases and inhibition of formation of neurospheres when compared to single-agent treatment. Mechanistically, pharmacological PARP inhibition elicited a nuclear stress response with up-regulation of down-stream DNA-stress response proteins, e.g., CCAAT enhancer binding protein (C/EBP) homology protein (CHOP). Furthermore, Olaparib and PJ34 increased protein levels of DR5 in a concentration and time-dependent manner. In turn, siRNA-mediated suppression of DR5 mitigated the effects of TRAIL/PARP inhibitor-mediated apoptosis. In addition, suppression of PARP-1 levels enhanced TRAIL-mediated apoptosis in malignant glioma cells. Treatment of human astrocytes with the combination of TRAIL/PARP inhibitors did not cause toxicity. Finally, the combination treatment of TRAIL and PJ34 significantly reduced tumor growth in vivo when compared to treatment with each agent alone. Conclusions PARP inhibition represents a promising avenue to overcome apoptotic resistance in GBM. PMID:25531448

Synergetic Effects of PARP Inhibitor AZD2281 and Cisplatin in Oral Squamous Cell Carcinoma in Vitro and in Vivo

Directory of Open Access Journals (Sweden)

Masaaki Yasukawa

2016-02-01

Full Text Available Cisplatin is a commonly used chemotherapeutic drug for treatment of oral carcinoma, and combinatorial effects are expected to exert greater therapeutic efficacy compared with monotherapy. Poly(ADP-ribosylation is reported to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, and genomic stability. Based on these properties, poly(ADP-ribose polymerase (PARP inhibitors are used for treatment of cancers, such as BRCA1/2 mutated breast and ovarian cancers, or certain solid cancers in combination with anti-cancer drugs. However, the effects on oral cancer have not been fully evaluated. In this study, we examined the effects of PARP inhibitor on the survival of human oral cancer cells in vitro and xenografted tumors derived from human oral cancer cells in vivo. In vitro effects were assessed by microculture tetrazolium and survival assays. The PARP inhibitor AZD2281 (olaparib showed synergetic effects with cisplatin in a dose-dependent manner. Combinatorial treatment with cisplatin and AZD2281 significantly inhibited xenografted tumor growth compared with single treatment of cisplatin or AZD2281. Histopathological analysis revealed that cisplatin and AZD2281 increased TUNEL-positive cells and decreased Ki67- and CD31-positive cells. These results suggest that PARP inhibitors have the potential to improve therapeutic strategies for oral cancer.

Microglial activation induced by brain trauma is suppressed by post-injury treatment with a PARP inhibitor

Directory of Open Access Journals (Sweden)

d'Avila Joana C

2012-02-01

Full Text Available Abstract Background Traumatic brain injury (TBI induces activation of microglia. Activated microglia can in turn increase secondary injury and impair recovery. This innate immune response requires hours to days to become fully manifest, thus providing a clinically relevant window of opportunity for therapeutic intervention. Microglial activation is regulated in part by poly(ADP-ribose polymerase-1 (PARP-1. Inhibition of PARP-1 activity suppresses NF-kB-dependent gene transcription and thereby blocks several aspects of microglial activation. Here we evaluated the efficacy of a PARP inhibitor, INO-1001, in suppressing microglial activation after cortical impact in the rat. Methods Rats were subjected to controlled cortical impact and subsequently treated with 10 mg/kg of INO-1001 (or vehicle alone beginning 20 - 24 hours after the TBI. Brains were harvested at several time points for histological evaluation of inflammation and neuronal survival, using markers for microglial activation (morphology and CD11b expression, astrocyte activation (GFAP, and neuronal survival (NeuN. Rats were also evaluated at 8 weeks after TBI using measures of forelimb dexterity: the sticky tape test, cylinder test, and vermicelli test. Results Peak microglial and astrocyte activation was observed 5 to 7 days after this injury. INO-1001 significantly reduced microglial activation in the peri-lesion cortex and ipsilateral hippocampus. No rebound inflammation was observed in rats that were treated with INO-1001 or vehicle for 12 days followed by 4 days without drug. The reduced inflammation was associated with increased neuronal survival in the peri-lesion cortex and improved performance on tests of forelimb dexterity conducted 8 weeks after TBI. Conclusions Treatment with a PARP inhibitor for 12 days after TBI, with the first dose given as long as 20 hours after injury, can reduce inflammation and improve histological and functional outcomes.

PARP Inhibition Prevents Ethanol-Induced Neuroinflammatory Signaling and Neurodegeneration in Rat Adult-Age Brain Slice Cultures

Science.gov (United States)

Tajuddin, Nuzhath; Kim, Hee-Yong

2018-01-01

Using rat adult-age hippocampal-entorhinal cortical (HEC) slice cultures, we examined the role of poly [ADP-ribose] polymerase (PARP) in binge ethanol’s brain inflammatory and neurodegenerative mechanisms. Activated by DNA strand breaks, PARP (principally PARP1 in the brain) promotes DNA repair via poly [ADP-ribose] (PAR) products, but PARP overactivation triggers regulated neuronal necrosis (e.g., parthanatos). Previously, we found that brain PARP1 levels were upregulated by neurotoxic ethanol binges in adult rats and HEC slices, and PARP inhibitor PJ34 abrogated slice neurodegeneration. Binged HEC slices also exhibited increased Ca+2-dependent phospholipase A2 (PLA2) isoenzymes (cPLA2 IVA and sPLA2 IIA) that mobilize proinflammatory ω6 arachidonic acid (ARA). We now find in 4-day–binged HEC slice cultures (100 mM ethanol) that PARP1 elevations after two overnight binges precede PAR, cPLA2, and sPLA2 enhancements by 1 day and high-mobility group box-1 (HMGB1), an ethanol-responsive alarmin that augments proinflammatory cytokines via toll-like receptor-4 (TLR4), by 2 days. After verifying that PJ34 effectively blocks PARP activity (↑PAR), we demonstrated that, like PJ34, three other PARP inhibitors—olaparib, veliparib, and 4-aminobenzamide—provided neuroprotection from ethanol. Importantly, PJ34 and olaparib also prevented ethanol’s amplification of the PLA2 isoenzymes, and two PLA2 inhibitors were neuroprotective—thus coupling PARP to PLA2, with PLA2 activity promoting neurodegeneration. Also, PJ34 and olaparib blocked ethanol-induced HMGB1 elevations, linking brain PARP induction to TLR4 activation. The results provide evidence in adult brains that induction of PARP1 may mediate dual neuroinflammatory pathways (PLA2→phospholipid→ARA and HMGB1→TLR4→proinflammatory cytokines) that are complicit in binge ethanol-induced neurodegeneration. PMID:29339456

PARP-1 inhibition alleviates diabetic cardiac complications in experimental animals.

Science.gov (United States)

Zakaria, Esraa M; El-Bassossy, Hany M; El-Maraghy, Nabila N; Ahmed, Ahmed F; Ali, Abdelmoneim A

2016-11-15

Cardiovascular complications are the major causes of mortality among diabetic population. Poly(ADP-ribose) polymerase-1 enzyme (PARP-1) is activated by oxidative stress leading to cellular damage. We investigated the implication of PARP-1 in diabetic cardiac complications. Type 2 diabetes was induced in rats by high fructose-high fat diet and low streptozotocin dose. PARP inhibitor 4-aminobenzamide (4-AB) was administered daily for ten weeks after diabetes induction. At the end of study, surface ECG, blood pressure and vascular reactivity were studied. PARP-1 activity, reduced glutathione (GSH) and nitrite contents were assessed in heart muscle. Fasting glucose, fructosamine, insulin, and tumor necrosis factor alpha (TNF-α) levels were measured in serum. Finally, histological examination and collagen deposition detection in rat ventricular and aortic sections were carried out. Hearts isolated from diabetic animals showed increased PARP-1 enzyme activity compared to control animals while significantly reduced by 4-AB administration. PARP-1 inhibition by 4-AB alleviated cardiac ischemia in diabetic animals as indicated by ECG changes. PARP-1 inhibition also reduced cardiac inflammation in diabetic animals as evidenced by histopathological changes. In addition, 4-AB administration improved the elevated blood pressure and the associated exaggerated vascular contractility, endothelial destruction and vascular inflammation seen in diabetic animals. Moreover, PARP-1 inhibition decreased serum levels of TNF-α and cardiac nitrite but increased cardiac GSH contents in diabetic animals. However, PARP-1 inhibition did not significantly affect the developed hyperglycemia. Our findings prove that PARP-1 enzyme plays an important role in diabetic cardiac complications through combining inflammation, oxidative stress, and fibrosis mechanisms. Copyright © 2016. Published by Elsevier B.V.

Combination strategy of PARP inhibitor with antioxidant prevent bioenergetic deficits and inflammatory changes in CCI-induced neuropathy.

Science.gov (United States)

Komirishetty, Prashanth; Areti, Aparna; Gogoi, Ranadeep; Sistla, Ramakrishna; Kumar, Ashutosh

2017-02-01

Neuropathic pain, a debilitating pain condition and the underlying pathogenic mechanisms are complex and interwoven amongst each other and still there is scant information available regarding therapies which promise to treat the condition. Evidence indicate that oxidative/nitrosative stress induced poly (ADP-ribose) polymerase (PARP) overactivation initiate neuroinflammation and bioenergetic crisis culminating into neurodegenerative changes following nerve injury. Hence, we investigated the therapeutic effect of combining an antioxidant, quercetin and a PARP inhibitor, 4-amino 1, 8-naphthalimide (4-ANI) on the hallmark deficits induced by chronic constriction injury (CCI) of sciatic nerve in rats. Quercetin (25 mg/kg, p.o.) and 4-ANI (3 mg/kg, p.o.) were administered either alone or in combination for 14 days to examine sciatic functional index, allodynia and hyperalgesia using walking track analysis, Von Frey, acetone spray and hot plate tests respectively. Malondialdehyde, nitrite and glutathione levels were estimated to detect oxidative/nitrosative stress; mitochondrial membrane potential and cytochrome c oxidase activity to assess mitochondrial function; NAD & ATP levels to examine the bioenergetic status and levels of inflammatory markers were evaluated in ipsilateral sciatic nerve. Quercetin and 4-ANI alone improved the pain behaviour and biochemical alterations but the combination therapy demonstrated an appreciable reversal of CCI-induced changes. Nitrotyrosine and Poly ADP-Ribose (PAR) immunopositivity was decreased and nuclear factor erythroid 2-related factor (Nrf-2) levels were increased significantly in micro-sections of the sciatic nerve and dorsal root ganglion (DRG) of treatment group. These results suggest that simultaneous inhibition of oxidative stress-PARP activation cascade may potentially be useful strategies for management of trauma induced neuropathic pain. Copyright © 2016 Elsevier Ltd. All rights reserved.

The safety of antiangiogenic agents and PARP inhibitors in platinum-sensitive recurrent ovarian cancer.

Science.gov (United States)

Lorusso, Domenica; Fontanella, Caterina; Maltese, Giuseppa; Lepori, Stefano; Tripodi, Elisa; Bogani, Giorgio; Raspagliesi, Francesco

2017-06-01

Recurrence is a common event in endothelial ovarian cancer (EOC) patients, and the choice of the most appropriate treatment is driven by the platinum-free interval, molecular characteristics of the disease such as BRCA mutational status, previous treatments and toxicity. Areas covered: This review focuses on the main hematologic and non-hematologic toxicities correlated with the use of licensed antiangiogenic agents and PARP inhibitors in recurrent platinum-sensitive EOC, providing recommendations for their management. Expert opinion: The clinical research over the next years will be focused on a more precise characterization of molecular pathways underlying tumorigenesis of the five ovarian tumors, to improve the decision-making process in these rare diseases. For this purpose, new study designs and international collaborations will become mandatory. Immunotherapy, antiangiogenic agents and PARP inhibitors will be combined to build a treatment strategy algorithm which will allow patients to receive all the available treatment option, in the more appropriate sequence.

Increased oxidative stress mediates the antitumor effect of PARP inhibition in ovarian cancer

Directory of Open Access Journals (Sweden)

Dong Hou

2018-07-01

Full Text Available PARP inhibitors have been widely tested in clinical trials, especially for the treatment of breast cancer and ovarian cancer, and were shown to be highly successful. Because PARP primarily functions in sensing and repairing DNA strand breaks, the therapeutic effect of PARP inhibition is generally believed to be attributed to impaired DNA repair. We here report that oxidative stress is also increased by PARP inhibition and mediates the antitumor effect. We showed that PARP1 is highly expressed in specimens of high grade serous ovarian carcinoma and its activity is required for unperturbed proliferation of ovarian cancer cells. Inhibition or depletion of PARP leads to not only an increase in DNA damage, but also an elevation in the levels of reactive oxygen species (ROS. Importantly, antioxidant N-acetylcysteine (NAC significantly attenuated the induction of DNA damage and the perturbation of proliferation by PARP inhibition or depletion. We further showed that NADPH oxidases 1 and 4 were significantly upregulated by PARP inhibition and were partially responsible for the induction of oxidative stress. Depletion of NOX1 and NOX4 partially rescued the growth inhibition of PARP1-deficient tumor xenografts. Our findings suggest that in addition to compromising the repair of DNA damage, PARP inhibition or depletion may exert extra antitumor effect by elevating oxidative stress in ovarian cancer cells. Keywords: PARP1, Oxidative stress, NADPH oxidases, Ovarian cancer

Cediranib, a pan-VEGFR inhibitor, and olaparib, a PARP inhibitor, in combination therapy for high grade serous ovarian cancer.

Science.gov (United States)

Ivy, S Percy; Liu, Joyce F; Lee, Jung-Min; Matulonis, Ursula A; Kohn, Elise C

2016-01-01

An estimated 22,000 women are diagnosed annually with ovarian cancer in the United States. Initially chemo-sensitive, recurrent disease ultimately becomes chemoresistant and may kill ~14,000 women annually. Molecularly targeted therapy with cediranib (AZD2171), a vascular endothelial growth factor receptor (VEGFR)-1, 2, and 3 signaling blocker, and olaparib (AZD2281), a poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitor, administered orally in combination has shown anti-tumor activity in the treatment of high grade serous ovarian cancer (HGSOC). This combination has the potential to change the treatment of HGSOC. Preclinical and clinical studies of single agent cediranib and olaparib or their combination are reviewed. Data are presented from peer-reviewed published manuscripts, completed and ongoing early phase clinical trials registered in ClinicalTrials.gov, National Cancer Institute-sponsored clinical trials, and related recent abstracts. Advances in the treatment of HGSOC that improve progression-free and overall survival have proven elusive despite examination of molecularly targeted therapy. HGSOC patients with deleterious germline or somatic mutations in BRCA1 or BRCA2 (BRCAm) are most responsive to PARP inhibitors (PARPi). PARPi combined with angiogenesis inhibition improved anti-cancer response and duration in both BRCAm and BRCA wild type HGSOC patients, compared to olaparib single agent treatment, demonstrating therapeutic chemical and contextual synthetic lethality.

A Phase 1 trial of the PARP inhibitor olaparib (AZD2281) in combination with the anti-angiogenic cediranib (AZD2171) in recurrent epithelial ovarian or triple-negative breast cancer

Science.gov (United States)

Liu, Joyce F.; Tolaney, Sara M.; Birrer, Michael; Fleming, Gini F.; Buss, Mary K.; Dahlberg, Suzanne E.; Lee, Hang; Whalen, Christin; Tyburski, Karin; Winer, Eric; Ivy, Percy; Matulonis, Ursula A.

2014-01-01

Background PARP-inhibitors and anti-angiogenics have activity in recurrent ovarian and breast cancer; however, the effect of combined therapy against PARP and angiogenesis in this population has not been reported. We investigated the toxicities and recommended phase 2 dosing (RP2D) of the combination of cediranib, a multitargeted inhibitor of VEGFR-1/2/3, and olaparib, a PARP-inhibitor (NCT01116648). Methods Cediranib tablets once daily and olaparib capsules twice daily were administered orally in a standard 3+3 dose escalation design. Patients with recurrent ovarian or metastatic triple-negative breast cancer were eligible. Patients had measurable disease by RECIST 1.1 or met GCIG CA125 criteria. No prior PARP-inhibitors or anti-angiogenics in the recurrent setting were allowed. Results 28 patients (20 ovarian, 8 breast) enrolled to 4 dose levels. 2 DLTs (1 grade 4 neutropenia ≥4 days; 1 grade 4 thrombocytopenia) occurred at the highest dose level (cediranib 30mg daily; olaparib 400mg BID). The RP2D was cediranib 30mg daily and olaparib 200mg BID. Grade 3 or higher toxicities occurred in 75% of patients, and included grade 3 hypertension (25%) and grade 3 fatigue (18%). One grade 3 bowel obstruction occurred. The overall response rate (ORR) in the 18 RECIST-evaluable ovarian cancer patients was 44%, with a clinical benefit rate (ORR plus SD >24 weeks) of 61%. None of the 7 evaluable breast cancer patients achieved clinical response; 2 patients had stable disease for >24 weeks. Interpretation The combination of cediranib and olaparib has hematologic DLTs and anticipated class toxicities, with promising evidence of activity in ovarian cancer patients. PMID:23810467

PARP Inhibition Attenuates Histopathological Lesion in Ischemia/Reperfusion Renal Mouse Model after Cold Prolonged Ischemia

Directory of Open Access Journals (Sweden)

Raimundo M. G. del Moral

2013-01-01

Full Text Available We test the hypothesis that PARP inhibition can decrease acute tubular necrosis (ATN and other renal lesions related to prolonged cold ischemia/reperfusion (IR in kidneys preserved at 4°C in University of Wisconsin (UW solution. Material and Methods. We used 30 male Parp1+/+ wild-type and 15 male Parp10/0 knockout C57BL/6 mice. Fifteen of these wild-type mice were pretreated with 3,4-dihydro-5-[4-(1-piperidinylbutoxyl]-1(2H-isoquinolinone (DPQ at a concentration of 15 mg/kg body weight, used as PARP inhibitor. Subgroups of mice were established (A: IR 45 min/6 h; B: IR + 48 h in UW solution; and C: IR + 48 h in UW solution plus DPQ. We processed samples for morphological, immunohistochemical, ultrastructural, and western-blotting studies. Results. Prolonged cold ischemia time in UW solution increased PARP-1 expression and kidney injury. Preconditioning with PARP inhibitor DPQ plus DPQ supplementation in UW solution decreased PARP-1 nuclear expression in renal tubules and renal damage. Parp10/0 knockout mice were more resistant to IR-induced renal lesion. In conclusion, PARP inhibition attenuates ATN and other IR-related renal lesions in mouse kidneys under prolonged cold storage in UW solution. If confirmed, these data suggest that pharmacological manipulation of PARP activity may have salutary effects in cold-stored organs at transplantation.

Trial watch – inhibiting PARP enzymes for anticancer therapy

Science.gov (United States)

Sistigu, Antonella; Manic, Gwenola; Obrist, Florine; Vitale, Ilio

2016-01-01

ABSTRACT Poly(ADP-ribose) polymerases (PARPs) are a members of family of enzymes that catalyze poly(ADP-ribosyl)ation (PARylation) and/or mono(ADP-ribosyl)ation (MARylation), two post-translational protein modifications involved in crucial cellular processes including (but not limited to) the DNA damage response (DDR). PARP1, the most abundant family member, is a nuclear protein that is activated upon sensing distinct types of DNA damage and contributes to their resolution by PARylating multiple DDR players. Recent evidence suggests that, along with DDR, activated PARP1 mediates a series of prosurvival and proapoptotic processes aimed at preserving genomic stability. Despite this potential oncosuppressive role, upregulation and/or overactivation of PARP1 or other PARP enzymes has been reported in a variety of human neoplasms. Over the last few decades, several pharmacologic inhibitors of PARP1 and PARP2 have been assessed in preclinical and clinical studies showing potent antineoplastic activity, particularly against homologous recombination (HR)-deficient ovarian and breast cancers. In this Trial Watch, we describe the impact of PARP enzymes and PARylation in cancer, discuss the mechanism of cancer cell killing by PARP1 inactivation, and summarize the results of recent clinical studies aimed at evaluating the safety and therapeutic profile of PARP inhibitors in cancer patients. PMID:27308587

Risk of severe hematologic toxicities in cancer patients treated with PARP inhibitors: results of monotherapy and combination therapy trials

Directory of Open Access Journals (Sweden)

Alecu I

2018-02-01

Full Text Available Iulian Alecu, Tsveta Milenkova, Simon R Turner Research and Development, AstraZeneca UK Limited, Cambridge, UKThe tolerability profile of PARP inhibitors often includes hematologic toxicities, and the characterization of these adverse events is important to allow effective management by clinicians. Zhou et al1 recently carried out a meta-analysis of the incidence and relative risks of severe neutropenia, thrombocytopenia, and anemia events in 12 randomized controlled trials of PARP inhibitors, either as monotherapy or in combination with chemotherapy or radiotherapy. The authors concluded that olaparib resulted in a higher incidence of severe (common terminology criteria for adverse events [CTCAE] grade $3 neutropenia when compared with niraparib and veliparib; however, these conclusions are based on inappropriate and incomplete comparisons of hematologic toxicity with olaparib or veliparib in combination with myelotoxic chemotherapy versus niraparib monotherapy. While both monotherapy and combination therapy olaparib studies are discussed in the paper, the neutropenia analysis is based on olaparib data solely from studies in combination with paclitaxel or paclitaxel plus carboplatin. In order to inform the practicing clinician of the relative risk of hematologic toxicity associated with different PARP inhibitors, direct comparison needs to be conducted based on monotherapy, where applicable, as per the approved drug indication, otherwise the reader is given misleading information.View the original paper by Zhou et al.

Concurrent targeting of nitrosative stress-PARP pathway corrects functional, behavioral and biochemical deficits in experimental diabetic neuropathy

Energy Technology Data Exchange (ETDEWEB)

Negi, Geeta; Kumar, Ashutosh [Molecular Neuropharmacology Laboratory, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar, Punjab 160062 (India); Sharma, Shyam S., E-mail: sssharma@niper.ac.in [Molecular Neuropharmacology Laboratory, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar, Punjab 160062 (India)

2010-01-01

Peroxynitrite mediated nitrosative stress, an indisputable initiator of DNA damage and overactivation of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated after sensing DNA damage, are two crucial pathogenetic mechanisms in diabetic neuropathy. The intent of the present study was to investigate the effect of combination of a peroxynitrite decomposition catalyst (PDC), FeTMPyP and a PARP inhibitor, 4-ANI against diabetic peripheral neuropathy. The end points of evaluation of the study included motor nerve conduction velocity (MNCV) and nerve blood flow (NBF) for evaluating nerve functions; thermal hyperalgesia and mechanical allodynia for assessing nociceptive alterations, malondialdehyde and peroxynitrite levels to detect oxidative stress-nitrosative stress; NAD concentration in sciatic nerve to assess overactivation of PARP. Additionally immunohistochemical studies for nitrotyrosine and Poly(ADP-ribose) (PAR) was also performed. Treatment with the combination of FeTMPyP and 4-ANI led to significant improvement in nerve functions and pain parameters and also attenuated the oxidative-nitrosative stress markers. Further, the combination also reduced the overactivation of PARP as evident from increased NAD levels and decreased PAR immunopositivity in sciatic nerve microsections. Thus, it can be concluded that treatment with the combination of a PDC and PARP inhibitor attenuates alteration in peripheral nerves in diabetic neuropathy (DN).

PARP inhibition versus PARP-1 silencing: different outcomes in terms of single-strand break repair and radiation susceptibility

International Nuclear Information System (INIS)

Godon, C.; Cordelieres, F.P.; Giocanti, N.; Megnin-Chanet, F.; Hall, J.; Favaudon, V.; Godon, C.; Giocanti, N.; Megnin-Chanet, F.; Hall, J.; Favaudon, V.; Cordelieres, F.P.; Cordelieres, F.P.; Biard, D.

2008-01-01

The consequences of PARP-1 disruption or inhibition on DNA single-strand break repair (SSBR) and radio-induced lethality were determined in synchronized, iso-genic HeLa cells stably silenced or not for poly(ADP-ribose) polymerase-1 (PARP-1) (PARP-1(KD)) or XRCC1 (XRCC1(KD)). PARP-1 inhibition prevented XRCC1-YFP recruitment at sites of 405 nm laser micro irradiation, slowed SSBR 10-fold and triggered the accumulation of large persistent foci of GFP-PARP-1 and GFP-PCNA at photo damaged sites. These aggregates are presumed to hinder the recruitment of other effectors of the base excision repair (BER) pathway.PARP-1 silencing also prevented XRCC1-YFP recruitment but did not lengthen the lifetime of GFP-PCNA foci. Moreover, PARP-1(KD) and XRCC1(KD) cells in S phase completed SSBR as rapidly as controls, while SSBR was delayed in G1. Taken together, the data demonstrate that a PARP-1- and XRCC1-independent SSBR pathway operates when the short patch repair branch of the BER is deficient. Long patch repair is the likely mechanism, as GFP-PCNA recruitment at photo-damaged sites was normal in PARP-1(KD) cells. PARP-1 silencing elicited hyper-radiosensitivity, while radiosensitization by a PARP inhibitor reportedly occurs only in those cells treated in S phase. PARP-1 inhibition and deletion thus have different outcomes in terms of SSBR and radiosensitivity. (authors)

Molecular docking and 3D-QSAR studies on inhibitors of DNA damage signaling enzyme human PARP-1.

Science.gov (United States)

Fatima, Sabiha; Bathini, Raju; Sivan, Sree Kanth; Manga, Vijjulatha

2012-08-01

Poly (ADP-ribose) polymerase-1 (PARP-1) operates in a DNA damage signaling network. Molecular docking and three dimensional-quantitative structure activity relationship (3D-QSAR) studies were performed on human PARP-1 inhibitors. Docked conformation obtained for each molecule was used as such for 3D-QSAR analysis. Molecules were divided into a training set and a test set randomly in four different ways, partial least square analysis was performed to obtain QSAR models using the comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). Derived models showed good statistical reliability that is evident from their r², q²(loo) and r²(pred) values. To obtain a consensus for predictive ability from all the models, average regression coefficient r²(avg) was calculated. CoMFA and CoMSIA models showed a value of 0.930 and 0.936, respectively. Information obtained from the best 3D-QSAR model was applied for optimization of lead molecule and design of novel potential inhibitors.

PARP activity and inhibition in fetal and adult oligodendrocyte precursor cells: Effect on cell survival and differentiation.

Science.gov (United States)

Baldassarro, Vito A; Marchesini, Alessandra; Giardino, Luciana; Calzà, Laura

2017-07-01

Poly (ADP-ribose) polymerase (PARP) family members are ubiquitously expressed and play a key role in cellular processes, including DNA repair and cell death/survival balance. Accordingly, PARP inhibition is an emerging pharmacological strategy for cancer and neurodegenerative diseases. Consistent evidences support the critical involvement of PARP family members in cell differentiation and phenotype maturation. In this study we used an oligodendrocyte precursor cells (OPCs) enriched system derived from fetal and adult brain to investigate the role of PARP in OPCs proliferation, survival, and differentiation. The PARP inhibitors PJ34, TIQ-A and Olaparib were used as pharmacological tools. The main results of the study are: (i) PARP mRNA expression and PARP activity are much higher in fetal than in adult-derived OPCs; (ii) the culture treatment with PARP inhibitors is cytotoxic for OPCs derived from fetal, but not from adult, brain; (iii) PARP inhibition reduces cell number, according to the inhibitory potency of the compounds; (iv) PARP inhibition effect on fetal OPCs is a slow process; (v) PARP inhibition impairs OPCs maturation into myelinating OL in fetal, but not in adult cultures, according to the inhibitory potency of the compounds. These results have implications for PARP-inhibition therapies for diseases and lesions of the central nervous system, in particular for neonatal hypoxic/ischemic encephalopathy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Pathway-Enriched Gene Signature Associated with 53BP1 Response to PARP Inhibition in Triple-Negative Breast Cancer.

Science.gov (United States)

Hassan, Saima; Esch, Amanda; Liby, Tiera; Gray, Joe W; Heiser, Laura M

2017-12-01

Effective treatment of patients with triple-negative (ER-negative, PR-negative, HER2-negative) breast cancer remains a challenge. Although PARP inhibitors are being evaluated in clinical trials, biomarkers are needed to identify patients who will most benefit from anti-PARP therapy. We determined the responses of three PARP inhibitors (veliparib, olaparib, and talazoparib) in a panel of eight triple-negative breast cancer cell lines. Therapeutic responses and cellular phenotypes were elucidated using high-content imaging and quantitative immunofluorescence to assess markers of DNA damage (53BP1) and apoptosis (cleaved PARP). We determined the pharmacodynamic changes as percentage of cells positive for 53BP1, mean number of 53BP1 foci per cell, and percentage of cells positive for cleaved PARP. Inspired by traditional dose-response measures of cell viability, an EC 50 value was calculated for each cellular phenotype and each PARP inhibitor. The EC 50 values for both 53BP1 metrics strongly correlated with IC 50 values for each PARP inhibitor. Pathway enrichment analysis identified a set of DNA repair and cell cycle-associated genes that were associated with 53BP1 response following PARP inhibition. The overall accuracy of our 63 gene set in predicting response to olaparib in seven breast cancer patient-derived xenograft tumors was 86%. In triple-negative breast cancer patients who had not received anti-PARP therapy, the predicted response rate of our gene signature was 45%. These results indicate that 53BP1 is a biomarker of response to anti-PARP therapy in the laboratory, and our DNA damage response gene signature may be used to identify patients who are most likely to respond to PARP inhibition. Mol Cancer Ther; 16(12); 2892-901. ©2017 AACR . ©2017 American Association for Cancer Research.

Parp-1 genetic ablation in Ela-myc mice unveils novel roles for Parp-1 in pancreatic cancer.

Science.gov (United States)

Martínez-Bosch, Neus; Iglesias, Mar; Munné-Collado, Jessica; Martínez-Cáceres, Carlos; Moreno, Mireia; Guerra, Carmen; Yélamos, Jose; Navarro, Pilar

2014-10-01

into the clinical use of Parp-1 inhibitors. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Hyperthermia and PARP1-inhibition for sensitization of radiation and cisplatin treatment of cervical carcinoma cells

International Nuclear Information System (INIS)

Franken, Nicolaas; Oei, Arlene; Leeuwen, Caspar van; Stalpers, Lukas; Rodermond, Hans; Bel, Arjan; Kok, Petra; Crezee, Hans

2014-01-01

Ionizing radiation causes single and double strand breaks (SSBs and DSBs). DSBs are among the most critical DNA lesions and can be repaired via either non-homologous end joining (NHEJ) in which PARP1, Ku70 and DNA-PKcs are important, or homologous recombination (HR), where BRCA2 and Rad51 are essential. Hyperthermia disturbs HR by temporary inactivation of BRCA2. Cisplatin disrupts NHEJ and PARP1-inhibitor blocks Poly-(ADP-ribose)polymerase- 1, which is important in SSB repair, NHEJ and backup-NHEJ. Our goal was to investigate the additional effectiveness of hyperthermia and PARP1-inhibition on radiation and/or cisplatin treatment. Cervical carcinoma cells (SiHa) were treated at different temperature levels levels (41.0-43.0℃, PARP1-inhibitor (100 μM; NU1025), gamma-irradiation doses (0-8 Gy) or cisplatin (1'R for 1 h). Clonogenic assays were carried out to measure survival and γH2AX staining was used to visualize DSBs. To elucidate mechanisms of action expression levels of DNA repair proteins BRCA2 and DNA-PKcs were investigated after 42.0℃ (1 h) using western blot. Combined hyperthermia and radiation resulted in an increased number of γH2AX foci as compared to radiation alone. Hyperthermia treatment in combination with cisplatin and PARP1 inhibitor and with radiation and PARP1 inhibitor significantly decreased cell survival. Western blot demonstrated a decreased expression of BRCA2 protein at 30 min after hyperthermia treatment. Adding PARP1-inhibitor significantly improves the effectiveness of combined hyperthermia radiotherapy and combined hyperthermia-cisplatin treatment on cervical carcinoma cells. Hyperthermia affects DNA-DSB repair as is indicated by increased γH2AX foci numbers and decreased BRCA2 expression. (author)

Regulation of FOXO1-mediated transcription and cell proliferation by PARP-1

Energy Technology Data Exchange (ETDEWEB)

Sakamaki, Jun-ichi; Daitoku, Hiroaki; Yoshimochi, Kenji [Center for Tsukuba Advanced Research Alliance, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Miwa, Masanao [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829 (Japan); Fukamizu, Akiyoshi, E-mail: akif@tara.tsukuba.ac.jp [Center for Tsukuba Advanced Research Alliance, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan)

2009-05-08

Forkhead box O (FOXO) transcription factors play an important role in a wide range of biological processes, including cell cycle control, apoptosis, detoxification of reactive oxygen species, and gluconeogenesis through regulation of gene expression. In this study, we demonstrated that PARP-1 functions as a negative regulator of FOXO1. We showed that PARP-1 directly binds to and poly(ADP-ribosyl)ates FOXO1 protein. PARP-1 represses FOXO1-mediated expression of cell cycle inhibitor p27{sup Kip1} gene. Notably, poly(ADP-ribosyl)ation activity was not required for the repressive effect of PARP-1 on FOXO1 function. Furthermore, knockdown of PARP-1 led to a decrease in cell proliferation in a manner dependent on FOXO1 function. Chromatin immunoprecipitation experiments confirmed that PARP-1 is recruited to the p27{sup Kip1} gene promoter through a binding to FOXO1. These results suggest that PARP-1 acts as a corepressor for FOXO1, which could play an important role in proper cell proliferation by regulating p27{sup Kip1} gene expression.

PARP-1 Inhibition Is Neuroprotective in the R6/2 Mouse Model of Huntington's Disease.

Directory of Open Access Journals (Sweden)

Antonella Cardinale

Full Text Available Poly (ADP-ribose polymerase 1 (PARP-1 is a nuclear enzyme that is involved in physiological processes as DNA repair, genomic stability, and apoptosis. Moreover, published studies demonstrated that PARP-1 mediates necrotic cell death in response to excessive DNA damage under certain pathological conditions. In Huntington's disease brains, PARP immunoreactivity was described in neurons and in glial cells, thereby suggesting the involvement of apoptosis in HD. In this study, we sought to determine if the PARP-1 inhibitor exerts a neuroprotective effect in R6/2 mutant mice, which recapitulates, in many aspects, human HD. Transgenic mice were treated with the PARP-1 inhibitor INO-1001 mg/Kg daily starting from 4 weeks of age. After transcardial perfusion, histological and immunohistochemical studies were performed. We found that INO 1001-treated R6/2 mice survived longer and displayed less severe signs of neurological dysfunction than the vehicle treated ones. Primary outcome measures such as striatal atrophy, morphology of striatal neurons, neuronal intranuclear inclusions and microglial reaction confirmed a neuroprotective effect of the compound. INO-1001 was effective in significantly increasing activated CREB and BDNF in the striatal spiny neurons, which might account for the beneficial effects observed in this model. Our findings show that PARP-1 inhibition could be considered as a valid therapeutic approach for HD.

Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

Science.gov (United States)

Rank, Lisa; Veith, Sebastian; Gwosch, Eva C.; Demgenski, Janine; Ganz, Magdalena; Jongmans, Marjolijn C.; Vogel, Christopher; Fischbach, Arthur; Buerger, Stefanie; Fischer, Jan M.F.; Zubel, Tabea; Stier, Anna; Renner, Christina; Schmalz, Michael; Beneke, Sascha; Groettrup, Marcus; Kuiper, Roland P.; Bürkle, Alexander; Ferrando-May, Elisa; Mangerich, Aswin

2016-01-01

Genotoxic stress activates PARP1, resulting in the post-translational modification of proteins with poly(ADP-ribose) (PAR). We genetically deleted PARP1 in one of the most widely used human cell systems, i.e. HeLa cells, via TALEN-mediated gene targeting. After comprehensive characterization of these cells during genotoxic stress, we analyzed structure–function relationships of PARP1 by reconstituting PARP1 KO cells with a series of PARP1 variants. Firstly, we verified that the PARP1\\E988K mutant exhibits mono-ADP-ribosylation activity and we demonstrate that the PARP1\\L713F mutant is constitutively active in cells. Secondly, both mutants exhibit distinct recruitment kinetics to sites of laser-induced DNA damage, which can potentially be attributed to non-covalent PARP1–PAR interaction via several PAR binding motifs. Thirdly, both mutants had distinct functional consequences in cellular patho-physiology, i.e. PARP1\\L713F expression triggered apoptosis, whereas PARP1\\E988K reconstitution caused a DNA-damage-induced G2 arrest. Importantly, both effects could be rescued by PARP inhibitor treatment, indicating distinct cellular consequences of constitutive PARylation and mono(ADP-ribosyl)ation. Finally, we demonstrate that the cancer-associated PARP1 SNP variant (V762A) as well as a newly identified inherited PARP1 mutation (F304L\\V762A) present in a patient with pediatric colorectal carcinoma exhibit altered biochemical and cellular properties, thereby potentially supporting human carcinogenesis. Together, we establish a novel cellular model for PARylation research, by revealing strong structure–function relationships of natural and artificial PARP1 variants. PMID:27694308

Upregulation of PEDF expression by PARP inhibition contributes to the decrease in hyperglycemia-induced apoptosis in HUVECs

International Nuclear Information System (INIS)

Chen Haibing; Jia Weiping; Xu Xun; Fan Ying; Zhu Dongqing; Wu Haixiang; Xie Zhenggao; Zheng Zhi

2008-01-01

Poly(ADP-ribose)polymerase (PARP) inhibitors decrease angiogenesis through reducing vascular endothelium growth factor (VEGF) induced proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). In contrast to VEGF, pigment epithelium-derived factor (PEDF) has been demonstrated to act as a strong endogenous inhibitor of angiogenesis. Here, we show that PARP inhibition with a specific inhibitor PJ-34 or specific PARP antisense oligonucleotide upregulates hyperglycemia-induced PEDF expression in HUVECs in a dose-dependent manner. This results in the retard of activation of p38 MAP kinase and the concomitant decrease in cell apoptosis. These results give the first direct demonstration that PEDF might represent a target for PARP inhibition treatment and the effects of PEDF on endothelial cells growth are context dependent

Complementary genetic screens identify the E3 ubiquitin ligase CBLC, as a modifier of PARP inhibitor sensitivity

Czech Academy of Sciences Publication Activity Database

Frankum, J.; Moudrý, P.; Brough, R.; Hodný, Zdeněk; Ashworth, A.; Bartek, Jiří; Lord, C.J.

2015-01-01

Roč. 6, č. 13 (2015), s. 10746-10758 ISSN 1949-2553 R&D Projects: GA ČR GA13-17555S EU Projects: European Commission HEALTH-F2-2010-259893 Grant - others:Lundbeck Foundation(DK) R93-A8990; Danish Council for Independent Research(DK) DFF-1331-00262 Institutional support: RVO:68378050 Keywords : DNA damage response * ubiquitin-proteasome system * RNA interference screens * PARP inhibitors * CBLC Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.008, year: 2015

Baseline clinical predictors of antitumor response to the PARP inhibitor olaparib in germline BRCA1/2 mutated patients with advanced ovarian cancer.

Science.gov (United States)

Rafii, Saeed; Gourley, Charlie; Kumar, Rajiv; Geuna, Elena; Ern Ang, Joo; Rye, Tzyvia; Chen, Lee-May; Shapira-Frommer, Ronnie; Friedlander, Michael; Matulonis, Ursula; De Greve, Jacques; Oza, Amit M; Banerjee, Susana; Molife, L Rhoda; Gore, Martin E; Kaye, Stan B; Yap, Timothy A

2017-07-18

The PARP inhibitor olaparib was recently granted Food and Drug Administration (FDA) accelerated approval in patients with advanced BRCA1/2 mutation ovarian cancer. However, antitumor responses are observed in only approximately 40% of patients and the impact of baseline clinical factors on response to treatment remains unclear. Although platinum sensitivity has been suggested as a marker of response to PARP inhibitors, patients with platinum-resistant disease still respond to olaparib. 108 patients with advanced BRCA1/2 mutation ovarian cancers were included. The interval between the end of the most recent platinum chemotherapy and PARPi (PTPI) was used to predict response to olaparib independent of conventional definition of platinum sensitivity. RECIST complete response (CR) and partial response (PR) rates were 35% in patients with platinum-sensitive versus 13% in platinum-resistant (p<0.005). Independent of platinum sensitivity status, the RECIST CR/PR rates were 42% in patients with PTPI greater than 52 weeks and 18% in patients with PTPI less than 52 weeks (p=0.016). No association was found between baseline clinical factors such as FIGO staging, debulking surgery, BRCA1 versus BRCA2 mutations, prior history of breast cancer and prior chemotherapy for breast cancer, and the response to olaparib. We conducted an international multicenter retrospective study to investigate the association between baseline clinical characteristics of patients with advanced BRCA1/2 mutation ovarian cancers from eight different cancer centers and their antitumor response to olaparib. PTPI may be used to refine the prediction of response to PARP inhibition based on the conventional categorization of platinum sensitivity.

The dual action of poly(ADP-ribose polymerase -1 (PARP-1 inhibition in HIV-1 infection: HIV-1 LTR inhibition and diminution in Rho GTPase activity

Directory of Open Access Journals (Sweden)

Slava eRom

2015-08-01

Full Text Available The transcription of HIV-1 (HIV is regulated by complex mechanisms involving various cellular factors and virus-encoded transactivators. Poly(ADP-ribose polymerase 1 (PARP-1 inhibition has emerged recently as a potent anti-inflammatory tool, since PARP-1 is involved in the regulation of some genes through its interaction with various transcription factors. We propose a novel approach to diminish HIV replication via PARP-1 inhibition using human primary monocyte-derived macrophages (MDM as an in vitro model system. PARP-1 inhibitors were able to reduce HIV replication in MDM by 60-80% after 7 days infection. Long Terminal Repeat (LTR acts as a switch in virus replication and can be triggered by several agents such as: Tat, tumor necrosis factor α (TNFα, and phorbol 12-myristate 13-acetate (PMA. Overexpression of Tat in MDM transfected with an LTR reporter plasmid led to a 4.2-fold increase in LTR activation; PARP inhibition resulted in 70% reduction of LTR activity. LTR activity, which increased 3-fold after PMA or TNFα treatment, was reduced by PARP inhibition (by 85-95%. MDM treated with PARP inhibitors showed 90% reduction in NFκB activity (known to mediate PMA- and TNFα-induced HIV LTR activation. Cytoskeleton rearrangements are important in effective HIV-1 infection. PARP inactivation reduced actin cytoskeleton rearrangements by affecting Rho GTPase machinery. These findings suggest that HIV replication in MDM could be suppressed by PARP inhibition via NFκB suppression, diminution of LTR activation and its effects on the cytoskeleton. PARP appears to be essential for HIV replication and its inhibition may provide a potent approach to treatment of HIV infection.

Effects of PARP-1 inhibitors AG-014699 and AZD2281 on proliferation and apoptosis of human hepatoma cell line HepG2

Directory of Open Access Journals (Sweden)

DU Senrong

2015-06-01

Full Text Available ObjectiveTo observe the inhibitory and pro-apoptotic effects of two poly(ADP-ribose polymerase (PARP-1 inhibitors, AG-014699 and AZD2281, on human hepatoma HepG2 cells and preliminarily explore the mechanism by which AG-014699 induces HepG2 cell apoptosis, and to provide a new therapeutic target for hepatoma. MethodsThe effects of different concentrations of AG-014699 and AZD2281 on HepG2 cell proliferation were determined by MTT assay. The cell apoptosis rate was measured by flow cytometry. The expression levels of caspase-3 and caspase-8 were measured by Western Blot. Inter-group comparison was made by t test. ResultsBoth AG-014699 and AZD2281 suppressed HepG2 cell proliferation in a time- and dose-dependent manner. However, the sensitivity of HepG2 cells to the two PARP-1 inhibitors was different. The half-maximal inhibitory concentrations of AG-014699 and AZD2281 at 48 h determined by MTT assay were about 20 μmol/L and 400 μmol/L, respectively. Flow cytometry and Western blot were not used to evaluate the apoptosis of HepG2 cells exposed to AZD2281 to which these cells were not sensitive. HepG2 cell apoptosis could be induced by 10, 30, and 50 μmol/L AG-014699, and the highest apoptosis rate at 48 h was significantly higher than that of the control group (3100%±2.13% vs 09%±0013%, P＜0.01. Compared with those in the control group, the protein levels of caspase-3 and caspase-8 in HepG2 cells after 48-h exposure to 30, and 50 μmol/L AG-014699 increased. ConclusionThe two PARP-1 inhibitors AG-014699 and AZD2281 can inhibit the proliferation of HepG2 cells, which showed different sensitivities to the two inhibitors. AG-014699 can induce HepG2 cell apoptosis by up-regulating the protein expression of caspase-3 and caspase-8.

Targeted Radiosensitization of ETS Fusion-Positive Prostate Cancer through PARP1 Inhibition

Directory of Open Access Journals (Sweden)

Sumin Han

2013-10-01

Full Text Available ETS gene fusions, which result in overexpression of an ETS transcription factor, are considered driving mutations in approximately half of all prostate cancers. Dysregulation of ETS transcription factors is also known to exist in Ewing's sarcoma, breast cancer, and acute lymphoblastic leukemia. We previously discovered that ERG, the predominant ETS family member in prostate cancer, interacts with the DNA damage response protein poly (ADP-ribose polymerase 1 (PARP1 in human prostate cancer specimens. Therefore, we hypothesized that the ERG-PARP1 interaction may confer radiation resistance by increasing DNA repair efficiency and that this radio-resistance could be reversed through PARP1 inhibition. Using lentiviral approaches, we established isogenic models of ERG overexpression in PC3 and DU145 prostate cancer cell lines. In both cell lines, ERG overexpression increased clonogenic survival following radiation by 1.25 (±0.07 fold (mean ± SEM and also resulted in increased PARP1 activity. PARP1 inhibition with olaparib preferentially radiosensitized ERG-positive cells by a factor of 1.52 (±0.03 relative to ERG-negative cells (P < .05. Neutral and alkaline COMET assays and immunofluorescence microscopy assessing γ-H2AX foci showed increased short- and long-term efficiencies of DNA repair, respectively, following radiation that was preferentially reversed by PARP1 inhibition. These findings were verified in an in vivo xenograft model. Our findings demonstrate that ERG overexpression confers radiation resistance through increased efficiency of DNA repair following radiation that can be reversed through inhibition of PARP1. These results motivate the use of PARP1 inhibitors as radiosensitizers in patients with localized ETS fusion-positive cancers.

Poly(ADP-ribose polymerase 1 (PARP1 overexpression in human breast cancer stem cells and resistance to olaparib.

Directory of Open Access Journals (Sweden)

Marine Gilabert

Full Text Available BACKGROUND: Breast cancer stem cells (BCSCs have been recognized as playing a major role in various aspects of breast cancer biology. To identify specific biomarkers of BCSCs, we have performed comparative proteomics of BCSC-enriched and mature cancer cell populations from the human breast cancer cell line (BCL, BrCA-MZ-01. METHODS: ALDEFLUOR assay was used to sort BCSC-enriched (ALDH+ and mature cancer (ALDH- cell populations. Total proteins were extracted from both fractions and subjected to 2-Dimensional Difference In-Gel Electrophoresis (2-D DIGE. Differentially-expressed spots were excised and proteins were gel-extracted, digested and identified using MALDI-TOF MS. RESULTS: 2-D DIGE identified poly(ADP-ribose polymerase 1 (PARP1 as overexpressed in ALDH+ cells from BrCA-MZ-01. This observation was confirmed by western blot and extended to four additional human BCLs. ALDH+ cells from BRCA1-mutated HCC1937, which had the highest level of PARP1 overexpression, displayed resistance to olaparib, a specific PARP1 inhibitor. CONCLUSION: An unbiased proteomic approach identified PARP1 as upregulated in ALDH+, BCSC-enriched cells from various human BCLs, which may contribute to clinical resistance to PARP inhibitors.

Cells deficient in PARP-1 show an accelerated accumulation of DNA single strand breaks, but not AP sites, over the PARP-1-proficient cells exposed to MMS.

Science.gov (United States)

Pachkowski, Brian F; Tano, Keizo; Afonin, Valeriy; Elder, Rhoderick H; Takeda, Shunichi; Watanabe, Masami; Swenberg, James A; Nakamura, Jun

2009-12-01

Poly(ADP-ribose) polymerase-1 (PARP-1) is a base excision repair (BER) protein that binds to DNA single strand breaks (SSBs) and subsequently synthesizes and transfers poly(ADP-ribose) polymers to various nuclear proteins. Numerous biochemical studies have implicated PARP-1 as a modulator of BER; however, the role of PARP-1 in BER in living cells remains unclear partly due to lack of accurate quantitation of BER intermediates existing in cells. Since DT40 cells, chicken B lymphocytes, naturally lack PARP-2, DT40 cells allow for the investigation of the PARP-1 null phenotype without confounding by PARP-2. To test the hypothesis that PARP-1 is necessary for efficient BER during methylmethane sulfonate (MMS) exposure in vertebrate cells, intact DT40 cells and their isogenic PARP-1 null counterparts were challenged with different exposure scenarios for phenotypic characterization. With chronic exposure, PARP-1 null cells exhibited sensitivity to MMS but with an acute exposure did not accumulate base lesions or AP sites to a greater extent than wild-type cells. However, an increase in SSB content in PARP-1 null cell DNA, as indicated by glyoxal gel electrophoresis under neutral conditions, suggested the presence of BER intermediates. These data suggest that during exposure, PARP-1 impacts the stage of BER after excision of the deoxyribosephosphate moiety from the 5' end of DNA strand breaks by polymerase beta.

Radiosensitization by PARP inhibition to proton beam irradiation in cancer cells

Energy Technology Data Exchange (ETDEWEB)

Hirai, Takahisa [Department of Radiation Oncology, Juntendo University Faculty of Medicine, Bunkyo-ku, Tokyo (Japan); Division of Chemotherapy and Clinical Cancer Research, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Saito, Soichiro; Fujimori, Hiroaki [Division of Chemotherapy and Clinical Cancer Research, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Matsushita, Keiichiro; Nishio, Teiji [Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima-shi, Hiroshima (Japan); Okayasu, Ryuichi [International Open Laboratory, National Institute of Radiological Science, Chiba-shi, Chiba (Japan); Masutani, Mitsuko, E-mail: mmasutan@nagasaki-u.ac.jp [Division of Chemotherapy and Clinical Cancer Research, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Department of Frontier Life Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki (Japan)

2016-09-09

The poly(ADP-ribose) polymerase (PARP)-1 regulates DNA damage responses and promotes base excision repair. PARP inhibitors have been shown to enhance the cytotoxicity of ionizing radiation in various cancer cells and animal models. We have demonstrated that the PARP inhibitor (PARPi) AZD2281 is also an effective radiosensitizer for carbon-ion radiation; thus, we speculated that the PARPi could be applied to a wide therapeutic range of linear energy transfer (LET) radiation as a radiosensitizer. Institutes for biological experiments using proton beam are limited worldwide. This study was performed as a cooperative research at heavy ion medical accelerator in Chiba (HIMAC) in National Institute of Radiological Sciences. HIMAC can generate various ion beams; this enabled us to compare the radiosensitization effect of the PARPi on cells subjected to proton and carbon-ion beams from the same beam line. After physical optimization of proton beam irradiation, the radiosensitization effect of the PARPi was assessed in the human lung cancer cell line, A549, and the pancreatic cancer cell line, MIA PaCa-2. The effect of the PARPi, AZD2281, on radiosensitization to Bragg peak was more significant than that to entrance region. The PARPi increased the number of phosphorylated H2AX (γ-H2AX) foci and enhanced G2/M arrest after proton beam irradiation. This result supports our hypothesis that a PARPi could be applied to a wide therapeutic range of LET radiation by blocking the DNA repair response. - Highlights: • Effective radiosensitizers for particle radiation therapy have not been reported. • PARP inhibitor treatment radiosensitized after proton beam irradiation. • The sensitization at Bragg peak was greater than that at entrance region. • DSB induction and G2/M arrest is involved in the sensitization mechanism.

Cells deficient in PARP-1 show an accelerated accumulation of DNA single strand breaks, but not AP sites, over the PARP-1-proficient cells exposed to MMS

Energy Technology Data Exchange (ETDEWEB)

Pachkowski, Brian F. [Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC (United States); Tano, Keizo [Research Reactor Institute, Kyoto University, Kumatori (Japan); Afonin, Valeriy [Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC (United States); Elder, Rhoderick H. [School of Environment and Life Sciences, University of Salford, Greater Manchester (United Kingdom); Takeda, Shunichi [Department of Radiation Genetics, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto (Japan); Watanabe, Masami [Research Reactor Institute, Kyoto University, Kumatori (Japan); Swenberg, James A. [Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC (United States); Nakamura, Jun, E-mail: ynakamur@email.unc.edu [Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC (United States)

2009-12-01

Poly(ADP-ribose) polymerase-1 (PARP-1) is a base excision repair (BER) protein that binds to DNA single strand breaks (SSBs) and subsequently synthesizes and transfers poly(ADP-ribose) polymers to various nuclear proteins. Numerous biochemical studies have implicated PARP-1 as a modulator of BER; however, the role of PARP-1 in BER in living cells remains unclear partly due to lack of accurate quantitation of BER intermediates existing in cells. Since DT40 cells, chicken B lymphocytes, naturally lack PARP-2, DT40 cells allow for the investigation of the PARP-1 null phenotype without confounding by PARP-2. To test the hypothesis that PARP-1 is necessary for efficient BER during methylmethane sulfonate (MMS) exposure in vertebrate cells, intact DT40 cells and their isogenic PARP-1 null counterparts were challenged with different exposure scenarios for phenotypic characterization. With chronic exposure, PARP-1 null cells exhibited sensitivity to MMS but with an acute exposure did not accumulate base lesions or AP sites to a greater extent than wild-type cells. However, an increase in SSB content in PARP-1 null cell DNA, as indicated by glyoxal gel electrophoresis under neutral conditions, suggested the presence of BER intermediates. These data suggest that during exposure, PARP-1 impacts the stage of BER after excision of the deoxyribosephosphate moiety from the 5' end of DNA strand breaks by polymerase {beta}.

Extent of radiosensitization by the PARP inhibitor olaparib depends on its dose, the radiation dose and the integrity of the homologous recombination pathway of tumor cells

NARCIS (Netherlands)

Verhagen, Caroline V. M.; de Haan, Rosemarie; Hageman, Floor; Oostendorp, Tim P. D.; Carli, Annalisa L. E.; O'Connor, Mark J.; Jonkers, Jos; Verheij, Marcel; van den Brekel, Michiel W.; Vens, Conchita

2015-01-01

The PARP inhibitor olaparib is currently tested in clinical phase 1 trials to define safe dose levels in combination with RT. However, certain clinically relevant insights are still lacking. Here we test, while comparing to single agent activity, the olaparib dose and genetic background dependence

REV7 counteracts DNA double-strand break resection and affects PARP inhibition

NARCIS (Netherlands)

Xu, Guotai; Chapman, J. Ross; Brandsma, Inger; Yuan, Jingsong; Mistrik, Martin; Bouwman, Peter; Bartkova, Jirina; Gogola, Ewa; Warmerdam, Daniël; Barazas, Marco; Jaspers, Janneke E.; Watanabe, Kenji; Pieterse, Mark; Kersbergen, Ariena; Sol, Wendy; Celie, Patrick H. N.; Schouten, Philip C.; van den Broek, Bram; Salman, Ahmed; Nieuwland, Marja; de Rink, Iris; de Ronde, Jorma; Jalink, Kees; Boulton, Simon J.; Chen, Junjie; van Gent, Dik C.; Bartek, Jiri; Jonkers, Jos; Borst, Piet; Rottenberg, Sven

2015-01-01

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway(1). In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with

Extent of radiosensitization by the PARP inhibitor olaparib depends on its dose, the radiation dose and the integrity of the homologous recombination pathway of tumor cells

NARCIS (Netherlands)

Verhagen, C.V.M.; Haan, R. den; Hageman, F.; Oostendorp, T.P.; Carli, A.L.; O'Connor, M.J.; Jonkers, J.; Verheij, M.; Brekel, M.W. van den; Vens, C.

2015-01-01

BACKGROUND AND PURPOSE: The PARP inhibitor olaparib is currently tested in clinical phase 1 trials to define safe dose levels in combination with RT. However, certain clinically relevant insights are still lacking. Here we test, while comparing to single agent activity, the olaparib dose and genetic

Extent of radiosensitization by the PARP inhibitor olaparib depends on its dose, the radiation dose and the integrity of the homologous recombination pathway of tumor cells

NARCIS (Netherlands)

Verhagen, C.V.M.; de Haan, R.; Hageman, F.; Oostendorp, T.P.D.; Carli, A.L.E.; O'Connor, M.J.; Jonkers, J.; Verheij, M.; van den Brekel, M.W.; Vens, C.

2015-01-01

Background and purpose The PARP inhibitor olaparib is currently tested in clinical phase 1 trials to define safe dose levels in combination with RT. However, certain clinically relevant insights are still lacking. Here we test, while comparing to single agent activity, the olaparib dose and genetic

Investigation of PARP-1, PARP-2, and PARG interactomes by affinity-purification mass spectrometry

Directory of Open Access Journals (Sweden)

Isabelle Maxim

2010-04-01

Full Text Available Abstract Background Poly(ADP-ribose polymerases (PARPs catalyze the formation of poly(ADP-ribose (pADPr, a post-translational modification involved in several important biological processes, namely surveillance of genome integrity, cell cycle progression, initiation of the DNA damage response, apoptosis, and regulation of transcription. Poly(ADP-ribose glycohydrolase (PARG, on the other hand, catabolizes pADPr and thereby accounts for the transient nature of poly(ADP-ribosylation. Our investigation of the interactomes of PARP-1, PARP-2, and PARG by affinity-purification mass spectrometry (AP-MS aimed, on the one hand, to confirm current knowledge on these interactomes and, on the other hand, to discover new protein partners which could offer insights into PARPs and PARG functions. Results PARP-1, PARP-2, and PARG were immunoprecipitated from human cells, and pulled-down proteins were separated by gel electrophoresis prior to in-gel trypsin digestion. Peptides were identified by tandem mass spectrometry. Our AP-MS experiments resulted in the identifications of 179 interactions, 139 of which are novel interactions. Gene Ontology analysis of the identified protein interactors points to five biological processes in which PARP-1, PARP-2 and PARG may be involved: RNA metabolism for PARP-1, PARP-2 and PARG; DNA repair and apoptosis for PARP-1 and PARP-2; and glycolysis and cell cycle for PARP-1. Conclusions This study reveals several novel protein partners for PARP-1, PARP-2 and PARG. It provides a global view of the interactomes of these proteins as well as a roadmap to establish the systems biology of poly(ADP-ribose metabolism.

Radiosensitivity modulating factors: Role of PARP-1, PARP-2 and Cdk5 proteins and chromatin implication

International Nuclear Information System (INIS)

Boudra, M.T.

2011-12-01

an intricate network of phosphorylation and poly(ADP-ribose)ylation occurs in response to IR that involves Cdk5, PARP- 1,PARP-2 and PARG and that is probably initiated by Cdk5's kinase activity. (author)

Breast Cancer Resistance Protein (BCRP/ABCG2) and P-glycoprotein (P-GP/ABCB1) Restrict Oral Availability and Brain Accumulation of the PARP Inhibitor Rucaparib (AG-014699)

NARCIS (Netherlands)

Durmus, Selvi; Sparidans, Rolf W; van Esch, Anita; Wagenaar, Els; Beijnen, Jos H; Schinkel, Alfred H

BACKGROUND: Rucaparib is a potent, orally available, small-molecule inhibitor of poly ADP-ribose polymerase (PARP) 1 and 2. Ongoing clinical trials are assessing the efficacy of rucaparib alone or in combination with other cytotoxic drugs, mainly in breast and ovarian cancer patients with mutations

PARP-1 depletion in combination with carbon ion exposure significantly reduces MMPs activity and overall increases TIMPs expression in cultured HeLa cells

International Nuclear Information System (INIS)

Ghorai, Atanu; Sarma, Asitikantha; Chowdhury, Priyanka; Ghosh, Utpal

2016-01-01

Hadron therapy is an innovative technique where cancer cells are precisely killed leaving surrounding healthy cells least affected by high linear energy transfer (LET) radiation like carbon ion beam. Anti-metastatic effect of carbon ion exposure attracts investigators into the field of hadron biology, although details remain poor. Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors are well-known radiosensitizer and several PARP-1 inhibitors are in clinical trial. Our previous studies showed that PARP-1 depletion makes the cells more radiosensitive towards carbon ion than gamma. The purpose of the present study was to investigate combining effects of PARP-1 inhibition with carbon ion exposure to control metastatic properties in HeLa cells. Activities of matrix metalloproteinases-2, 9 (MMP-2, MMP-9) were measured using the gelatin zymography after 85 MeV carbon ion exposure or gamma irradiation (0- 4 Gy) to compare metastatic potential between PARP-1 knock down (HsiI) and control cells (H-vector - HeLa transfected with vector without shRNA construct). Expression of MMP-2, MMP-9, tissue inhibitor of MMPs such as TIMP-1, TIMP-2 and TIMP-3 were checked by immunofluorescence and western blot. Cell death by trypan blue, apoptosis and autophagy induction were studied after carbon ion exposure in each cell-type. The data was analyzed using one way ANOVA and 2-tailed paired-samples T-test. PARP-1 silencing significantly reduced MMP-2 and MMP-9 activities and carbon ion exposure further diminished their activities to less than 3 % of control H-vector. On the contrary, gamma radiation enhanced both MMP-2 and MMP-9 activities in H-vector but not in HsiI cells. The expression of MMP-2 and MMP-9 in H-vector and HsiI showed different pattern after carbon ion exposure. All three TIMPs were increased in HsiI, whereas only TIMP-1 was up-regulated in H-vector after irradiation. Notably, the expressions of all TIMPs were significantly higher in HsiI than H-vector at 4 Gy. Apoptosis was

A novel and selective poly (ADP-ribose polymerase inhibitor ameliorates chemotherapy-induced painful neuropathy.

Directory of Open Access Journals (Sweden)

Lauren E Ta

Full Text Available Chemotherapy-induced neuropathy is the principle dose limiting factor requiring discontinuation of many chemotherapeutic agents, including cisplatin and oxaliplatin. About 30 to 40% of patients receiving chemotherapy develop pain and sensory changes. Given that poly (ADP-ribose polymerase (PARP inhibition has been shown to provide neuroprotection, the current study was developed to test whether the novel PARP inhibitor compound 4a (analog of ABT-888 would attenuate pain in cisplatin and oxaliplatin-induced neuropathy in mice.An established chemotherapy-induced painful neuropathy model of two weekly cycles of 10 intraperitoneal (i.p. injections separated by 5 days rest was used to examine the therapeutic potential of the PARP inhibitor compound 4a. Behavioral testing using von Frey, paw radiant heat, cold plate, and exploratory behaviors were taken at baseline, and followed by testing at 3, 6, and 8 weeks from the beginning of drug treatment.Cisplatin-treated mice developed heat hyperalgesia and mechanical allodynia while oxaliplatin-treated mice exhibited cold hyperalgesia and mechanical allodynia. Co-administration of 50 mg/kg or 25 mg/kg compound 4a with platinum regimen, attenuated cisplatin-induced heat hyperalgesia and mechanical allodynia in a dose dependent manner. Similarly, co-administration of 50 mg/kg compound 4a attenuated oxaliplatin-induced cold hyperalgesia and mechanical allodynia. These data indicate that administration of a novel PARP inhibitor may have important applications as a therapeutic agent for human chemotherapy-induced painful neuropathy.

A novel and selective poly (ADP-ribose) polymerase inhibitor ameliorates chemotherapy-induced painful neuropathy.

Science.gov (United States)

Ta, Lauren E; Schmelzer, James D; Bieber, Allan J; Loprinzi, Charles L; Sieck, Gary C; Brederson, Jill D; Low, Philip A; Windebank, Anthony J

2013-01-01

Chemotherapy-induced neuropathy is the principle dose limiting factor requiring discontinuation of many chemotherapeutic agents, including cisplatin and oxaliplatin. About 30 to 40% of patients receiving chemotherapy develop pain and sensory changes. Given that poly (ADP-ribose) polymerase (PARP) inhibition has been shown to provide neuroprotection, the current study was developed to test whether the novel PARP inhibitor compound 4a (analog of ABT-888) would attenuate pain in cisplatin and oxaliplatin-induced neuropathy in mice. An established chemotherapy-induced painful neuropathy model of two weekly cycles of 10 intraperitoneal (i.p.) injections separated by 5 days rest was used to examine the therapeutic potential of the PARP inhibitor compound 4a. Behavioral testing using von Frey, paw radiant heat, cold plate, and exploratory behaviors were taken at baseline, and followed by testing at 3, 6, and 8 weeks from the beginning of drug treatment. Cisplatin-treated mice developed heat hyperalgesia and mechanical allodynia while oxaliplatin-treated mice exhibited cold hyperalgesia and mechanical allodynia. Co-administration of 50 mg/kg or 25 mg/kg compound 4a with platinum regimen, attenuated cisplatin-induced heat hyperalgesia and mechanical allodynia in a dose dependent manner. Similarly, co-administration of 50 mg/kg compound 4a attenuated oxaliplatin-induced cold hyperalgesia and mechanical allodynia. These data indicate that administration of a novel PARP inhibitor may have important applications as a therapeutic agent for human chemotherapy-induced painful neuropathy.

PARP-1 modulation of mTOR signaling in response to a DNA alkylating agent.

Directory of Open Access Journals (Sweden)

Chantal Ethier

Full Text Available Poly(ADP-ribose polymerase-1 (PARP-1 is widely involved in cell death responses. Depending on the degree of injury and on cell type, PARP activation may lead to autophagy, apoptosis or necrosis. In HEK293 cells exposed to the alkylating agent N-methyl-N'-nitro-N'-nitrosoguanine (MNNG, we show that PARP-1 activation triggers a necrotic cell death response. The massive poly(ADP-ribose (PAR synthesis following PARP-1 activation leads to the modulation of mTORC1 pathway. Shortly after MNNG exposure, NAD⁺ and ATP levels decrease, while AMP levels drastically increase. We characterized at the molecular level the consequences of these altered nucleotide levels. First, AMP-activated protein kinase (AMPK is activated and the mTORC1 pathway is inhibited by the phosphorylation of Raptor, in an attempt to preserve cellular energy. Phosphorylation of the mTORC1 target S6 is decreased as well as the phosphorylation of the mTORC2 component Rictor on Thr1135. Finally, Akt phosphorylation on Ser473 is lost and then, cell death by necrosis occurs. Inhibition of PARP-1 with the potent PARP inhibitor AG14361 prevents all of these events. Moreover, the antioxidant N-acetyl-L-cysteine (NAC can also abrogate all the signaling events caused by MNNG exposure suggesting that reactive oxygen species (ROS production is involved in PARP-1 activation and modulation of mTOR signaling. In this study, we show that PARP-1 activation and PAR synthesis affect the energetic status of cells, inhibit the mTORC1 signaling pathway and possibly modulate the mTORC2 complex affecting cell fate. These results provide new evidence that cell death by necrosis is orchestrated by the balance between several signaling pathways, and that PARP-1 and PAR take part in these events.

PARP-1 and PARP-2 activity in cancer-induced cachexia: potential therapeutic implications.

Science.gov (United States)

Barreiro, Esther; Gea, Joaquim

2018-01-26

Skeletal muscle dysfunction and mass loss is a characteristic feature in patients with chronic diseases including cancer and acute conditions such as critical illness. Maintenance of an adequate muscle mass is crucial for the patients' prognosis irrespective of the underlying condition. Moreover, aging-related sarcopenia may further aggravate the muscle wasting process associated with chronic diseases and cancer. Poly(adenosine diphosphate-ribose) polymerase (PARP) activation has been demonstrated to contribute to the pathophysiology of muscle mass loss and dysfunction in animal models of cancer-induced cachexia. Genetic inhibition of PARP activity attenuated the deleterious effects seen on depleted muscles in mouse models of oncologic cachexia. In the present minireview the mechanisms whereby PARP activity inhibition may improve muscle mass and performance in models of cancer-induced cachexia are discussed. Specifically, the beneficial effects of inhibition of PARP activity on attenuation of increased oxidative stress, protein catabolism, poor muscle anabolism and mitochondrial content and epigenetic modulation of muscle phenotype are reviewed in this article. Finally, the potential therapeutic strategies of pharmacological PARP activity inhibition for the treatment of cancer-induced cachexia are also being described in this review.

The multikinase inhibitor Sorafenib displays significant antiproliferative effects and induces apoptosis via caspase 3, 7 and PARP in B- and T-lymphoblastic cells

International Nuclear Information System (INIS)

Schult, Catrin; Boldt, Sonja; Wolkenhauer, Olaf; Neri, Luca Maria; Freund, Mathias; Junghanss, Christian; Dahlhaus, Meike; Ruck, Sabine; Sawitzky, Mandy; Amoroso, Francesca; Lange, Sandra; Etro, Daniela; Glass, Aenne; Fuellen, Georg

2010-01-01

Targeted therapy approaches have been successfully introduced into the treatment of several cancers. The multikinase inhibitor Sorafenib has antitumor activity in solid tumors and its effects on acute lymphoblastic leukemia (ALL) cells are still unclear. ALL cell lines (SEM, RS4;11 and Jurkat) were treated with Sorafenib alone or in combination with cytarabine, doxorubicin or RAD001. Cell count, apoptosis and necrosis rates, cell cycle distribution, protein phosphorylation and metabolic activity were determined. Sorafenib inhibited the proliferation of ALL cells by cell cycle arrest accompanied by down-regulation of CyclinD3 and CDK4. Furthermore, Sorafenib initiated apoptosis by cleavage of caspases 3, 7 and PARP. Apoptosis and necrosis rates increased significantly with most pronounced effects after 96 h. Antiproliferative effects of Sorafenib were associated with a decreased phosphorylation of Akt (Ser473 and Thr308), FoxO3A (Thr32) and 4EBP-1 (Ser65 and Thr70) as early as 0.5 h after treatment. Synergistic effects were seen when Sorafenib was combined with other cytotoxic drugs or a mTOR inhibitor emphasizing the Sorafenib effect. Sorafenib displays significant antileukemic activity in vitro by inducing cell cycle arrest and apoptosis. Furthermore, it influences PI3K/Akt/mTOR signaling in ALL cells

Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells

Science.gov (United States)

Mortusewicz, Oliver; Amé, Jean-Christophe; Leonhardt, Heinrich

2007-01-01

Genome integrity is constantly threatened by DNA lesions arising from numerous exogenous and endogenous sources. Survival depends on immediate recognition of these lesions and rapid recruitment of repair factors. Using laser microirradiation and live cell microscopy we found that the DNA-damage dependent poly(ADP-ribose) polymerases (PARP) PARP-1 and PARP-2 are recruited to DNA damage sites, however, with different kinetics and roles. With specific PARP inhibitors and mutations, we could show that the initial recruitment of PARP-1 is mediated by the DNA-binding domain. PARP-1 activation and localized poly(ADP-ribose) synthesis then generates binding sites for a second wave of PARP-1 recruitment and for the rapid accumulation of the loading platform XRCC1 at repair sites. Further PARP-1 poly(ADP-ribosyl)ation eventually initiates the release of PARP-1. We conclude that feedback regulated recruitment of PARP-1 and concomitant local poly(ADP-ribosyl)ation at DNA lesions amplifies a signal for rapid recruitment of repair factors enabling efficient restoration of genome integrity. PMID:17982172

Nicotinamide Inhibits Ethanol-Induced Caspase-3 and PARP-1 Over-activation and Subsequent Neurodegeneration in the Developing Mouse Cerebellum.

Science.gov (United States)

Ieraci, Alessandro; Herrera, Daniel G

2018-06-01

Fetal alcohol spectrum disorder (FASD) is the principal preventable cause of mental retardation in the western countries resulting from alcohol exposure during pregnancy. Ethanol-induced massive neuronal cell death occurs mainly in immature neurons during the brain growth spurt period. The cerebellum is one of the brain areas that are most sensitive to ethanol neurotoxicity. Currently, there is no effective treatment that targets the causes of these disorders and efficient treatments to counteract or reverse FASD are desirable. In this study, we investigated the effects of nicotinamide on ethanol-induced neuronal cell death in the developing cerebellum. Subcutaneous administration of ethanol in postnatal 4-day-old mice induced an over-activation of caspase-3 and PARP-1 followed by a massive neurodegeneration in the developing cerebellum. Interestingly, treatment with nicotinamide, immediately or 2 h after ethanol exposure, diminished caspase-3 and PARP-1 over-activation and reduced ethanol-induced neurodegeneration. Conversely, treatment with 3-aminobenzadine, a specific PARP-1 inhibitor, was able to completely block PARP-1 activation, but not caspase-3 activation or ethanol-induced neurodegeneration in the developing cerebellum. Our results showed that nicotinamide reduces ethanol-induced neuronal cell death and inhibits both caspase-3 and PARP-1 alcohol-induced activation in the developing cerebellum, suggesting that nicotinamide might be a promising and safe neuroprotective agent for treating FASD and other neurodegenerative disorders in the developing brain that shares similar cell death pathways.

Effects of PARP-1 Deficiency on Th1 and Th2 Cell Differentiation

Directory of Open Access Journals (Sweden)

M. Sambucci

2013-01-01

Full Text Available T cell differentiation to effector Th cells such as Th1 and Th2 requires the integration of multiple synergic and antagonist signals. Poly(ADP-ribosylation is a posttranslational modification of proteins catalyzed by Poly(ADP-ribose polymerases (PARPs. Recently, many reports showed that PARP-1, the prototypical member of the PARP family, plays a role in immune/inflammatory responses. Consistently, its enzymatic inhibition confers protection in several models of immune-mediated diseases, mainly through an inhibitory effect on NF-κB (and NFAT activation. PARP-1 regulates cell functions in many types of immune cells, including dendritic cells, macrophages, and T and B lymphocytes. Our results show that PARP-1KO cells displayed a reduced ability to differentiate in Th2 cells. Under both nonskewing and Th2-polarizing conditions, naïve CD4 cells from PARP-1KO mice generated a reduced frequency of IL-4+ cells, produced less IL-5, and expressed GATA-3 at lower levels compared with cells from wild type mice. Conversely, PARP-1 deficiency did not substantially affect differentiation to Th1 cells. Indeed, the frequency of IFN-γ+ cells as well as IFN-γ production, in nonskewing and Th1-polarizing conditions, was not affected by PARP-1 gene ablation. These findings demonstrate that PARP-1 plays a relevant role in Th2 cell differentiation and it might be a target to be exploited for the modulation of Th2-dependent immune-mediated diseases.

Edaravone abrogates LPS-induced behavioral anomalies, neuroinflammation and PARP-1.

Science.gov (United States)

Sriram, Chandra Shaker; Jangra, Ashok; Gurjar, Satendra Singh; Mohan, Pritam; Bezbaruah, Babul Kumar

2016-02-01

Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA nick-sensor enzyme that functions at the center of cellular stress response and affects the immune system at several key points, and thus modulates inflammatory diseases. Our previous study demonstrated that lipopolysaccharide (LPS)-induced depressive-like behavior in mice can be ameliorated by 3-aminobenzamide, which is a PARP-1 inhibitor. In the present study we've examined the effect of a free radical scavenger, edaravone pretreatment against LPS-induced anxiety and depressive-like behavior as well as various hippocampal biochemical parameters including PARP-1. Male Swiss albino mice were treated with edaravone (3 & 10mg/kgi.p.) once daily for 14days. On the 14th day 30min after edaravone treatment mice were challenged with LPS (1mg/kgi.p.). After 3h and 24h of LPS administration we've tested mice for anxiety and depressive-like behaviors respectively. Western blotting analysis of PARP-1 in hippocampus was carried out after 12h of LPS administration. Moreover, after 24h of LPS administration serum corticosterone, hippocampal BDNF, oxido-nitrosative stress and pro-inflammatory cytokines were estimated by ELISA. Results showed that pretreatment of edaravone (10mg/kg) ameliorates LPS-induced anxiety and depressive-like behavior. Western blotting analysis showed that LPS-induced anomalous expression of PARP-1 significantly reverses by the pretreatment of edaravone (10mg/kg). Biochemical analyses revealed that LPS significantly diminishes BDNF, increases pro-inflammatory cytokines and oxido-nitrosative stress in the hippocampus. However, pretreatment with edaravone (10mg/kg) prominently reversed all these biochemical alterations. Our study emphasized that edaravone pretreatment prevents LPS-induced anxiety and depressive-like behavior, mainly by impeding the inflammation, oxido-nitrosative stress and PARP-1 overexpression. Copyright © 2015. Published by Elsevier Inc.

PARP-1 is a key player in controlling apoptosis induced by high LET carbon ion beam and low LET gamma radiation in HeLa cells

International Nuclear Information System (INIS)

Ghorai, Atanu; Ghosh, Utpal; Bhattacharyya, Nitai P.; Sarma, Asitikantha

2014-01-01

PARP-1 inhibitors have long been used as chemo-sensitizer or radio-sensitizer and specific PARP-1 inhibitors are also in clinical trial for the treatment of various cancers. PARP-1 is not only involved in DNA repair but also plays very complex role in induction of apoptosis in postirradiation condition. Our objective is to investigate role of PARP-1 in apoptosis triggered by high LET carbon ion beam (CIB) and low LET gamma. We have treated HeLa and PARP-1 knock down HeLa (Hsil) cells with various doses of CIB and gamma. We measured DNA damage by comet assay and various apoptotic parameters such as nuclear fragmentation, activation of caspase-3,8,9, AIF translocation etc. We observed higher DNA breaks and also higher apoptosis in HsiI cells compared with HeLa cells. Both CIB and gamma treatment results G2/M arrest but unlike gamma CIB makes S-phase delay, implicating that gamma and CIB triggers different pathway after DNA damage. Cell death by CIB or by gamma increased up on knocking down of PARP-1 but increase is higher for high LET CIB compared with low LET gamma. Furthermore, expression level of PARP-1 controls the intensity of overall apoptosis in cells in post-irradiation condition. So, combination of PARP-1 inhibition with high LET CIB could be a promising tool to combat cancer. (author)

PARP-1 Interaction with and Activation by Histones and Nucleosomes.

Science.gov (United States)

Thomas, Colin; Kotova, Elena; Tulin, Alexei V

2017-01-01

Poly(ADP-ribose) Polymerase 1 (PARP-1) is an abundant chromatin associated protein, typical for most eukaryotic nuclei. The localization of PARP-1 in chromatin and its enzymatic activation involves multiple interactions of PARP-1 with nucleosomal histones, other proteins, and DNA. We report a set of methods designed to reconstitute PARP-1 regulation in vitro. These methods involve the expression of PARP-1 and PARP-1-regulating proteins using bacterial and eukaryotic systems, purification of these proteins using chromatography, testing of individual interactions in vitro, assembly of active complexes, and reconstitution of PARP-1 regulating reactions in vitro.

Genetic variants in PARP1 (rs3219090) and IRF4 (rs12203592) genes associated with melanoma susceptibility in a Spanish population

International Nuclear Information System (INIS)

Peña-Chilet, Maria; Ribas, Gloria; Blanquer-Maceiras, Maite; Ibarrola-Villava, Maider; Martinez-Cadenas, Conrado; Martin-Gonzalez, Manuel; Gomez-Fernandez, Cristina; Mayor, Matias; Aviles, Juan Antonio; Lluch, Ana

2013-01-01

Few high penetrance genes are known in Malignant Melanoma (MM), however, the involvement of low-penetrance genes such as MC1R, OCA2, ASIP, SLC45A2 and TYR has been observed. Lately, genome-wide association studies (GWAS) have been the ideal strategy to identify new common, low-penetrance susceptibility loci. In this case–control study, we try to validate in our population nine melanoma associated markers selected from published GWAS in melanoma predisposition. We genotyped the 9 markers corresponding to 8 genes (PARP1, MX2, ATM, CCND1, NADSYN1, CASP8, IRF4 and CYP2R1) in 566 cases and 347 controls from a Spanish population using KASPar probes. Genotypes were analyzed by logistic regression and adjusted by phenotypic characteristics. We confirm the protective role in MM of the rs3219090 located on the PARP1 gene (p-value 0.027). Additionally, this SNP was also associated with eye color (p-value 0.002). A second polymorphism, rs12203592, located on the IRF4 gene was associated with protection to develop MM for the dominant model (p-value 0.037). We have also observed an association of this SNP with both lentigines (p-value 0.014) and light eye color (p-value 3.76 × 10 -4 ). Furthermore, we detected a novel association with rs1485993, located on the CCND1 gene, and dark eye color (p-value 4.96 × 10 -4 ). Finally, rs1801516, located on the ATM gene, showed a trend towards a protective role in MM similar to the one firstly described in a GWAS study. To our knowledge, this is the first time that these SNPs have been associated with MM in a Spanish population. We confirmed the proposed role of rs3219090, located on the PARP1 gene, and rs12203592, located on the IRF4 gene, as protective to MM along the same lines as have previous genome-wide associated works. Finally, we have seen associations between IRF4, PARP1, and CCND1 and phenotypic characteristics, confirming previous results for the IRF4 gene and presenting novel data for the last two, suggesting that

The mechanism of action of poly (ADP-ribose) polymerases inhibitors and its application perspective

International Nuclear Information System (INIS)

Huang Xiaofei; Cao Jianping

2008-01-01

Poly (ADP-ribose) polymerases (PARP) constitute a family of enzymes involved in the regulation of many cellular processes. It plays a vital role in many physical and physiopathological processes,, In the past ten years scientists have conducted extensive research on PARP and its inhibitors, among which the role of PARP inhihitors in radiosensitization, chemopotentiation and neuroprotection have been placed close attention. There have been several PARP inhibitors entering the clinical trials, which predicts its sound application perspectives. (authors)

SU-E-T-245: MR Guided Focused Ultrasound Increased PARP Related Apoptosis On Prostate Cancer in Vivo

International Nuclear Information System (INIS)

Chen, L; Chen, X; Cvetkovic, D; Gupta, R; Yang, D; Ma, C

2014-01-01

Purpose: Our previous study demonstrated that significant tumor growth delay was observed in the mice treated with pulsed high intensity focused ultrasound (pHIFU). The purpose of this study is to understand the cell killing mechanisms of pHIFU. Methods: Prostate cancer cells (LNCaP), were grown orthotopically in 17 nude mice. Tumor-bearing mice were treated using pHIFU with an acoustic power of 25W, pulse width 100msec and 300 pulses in one sonication under MR guidance. Mutiple sonications were used to cover the whole tumor volume. Temperature (less than 40 degree centigrade in the focal spot) was monitored using MR thermometry. Animals were euthanized at pre-determined time points (n=2) after treatment: 0 hours; 6 hrs; 24 hrs; 48 hrs; 4 days and 7 days. Two tumorbearing mice were used as control. Three tumor-bearing mice were treated with radiation (RT, 2 Gy) using 6 MV photon beams. RT treated mice were euthanized at 0 hr, 6 hrs and 24 hrs. The tumors were processed for immunohistochemical (IHC) staining for PARP (a surrogate of apoptosis). A multispectral imaging analysis system was used to quantify the expression of PARP staining. Cell apoptosis was calculated based on the PARP expression level, which is the intensity of the DAB reaction. Results: Our data showed that PARP related apoptosis peaked at 48 hrs and 7 days in pHIFU treated mice, which is comparable to that for the RT group at 24 hrs. The preliminary results from this study were consistent with our previous study on tumor growth delay using pHIFU. Conclusion: Our results demonstrated that non-thermal pHIFU increased apoptotic tumor cell death through the PARP related pathway. MR guided pHIFU may have a great potential as a safe, noninvasive treatment modality for cancer therapy. This treatment modality might be able to synergize with PARP inhibitors to achieve better result

PARP-1 expression is increased in colon adenoma and carcinoma and correlates with OGG1.

Directory of Open Access Journals (Sweden)

Tomasz Dziaman

Full Text Available The ethiology of colon cancer is largely dependent on inflammation driven oxidative stress. The analysis of 8-oxodeoxyguanosine (8-oxodGuo level in leukocyte DNA of healthy controls (138 individuals, patients with benign adenomas (AD, 137 individuals and with malignant carcinomas (CRC, 169 individuals revealed a significant increase in the level of 8-oxodGuo in leukocyte DNA of AD and CRC patients in comparison to controls. The counteracting mechanism is base excision repair, in which OGG1 and PARP-1 play a key role. We investigated the level of PARP-1 and OGG1 mRNA and protein in diseased and marginal, normal tissues taken from AD and CRC patients and in leukocytes taken from the patients as well as from healthy subjects. In colon tumors the PARP-1 mRNA level was higher than in unaffected colon tissue and in polyp tissues. A high positive correlation was found between PARP-1 and OGG1 mRNA levels in all investigated tissues. This suggests reciprocal influence of PARP-1 and OGG1 on their expression and stability, and may contribute to progression of colon cancer. PARP-1 and OGG1 proteins level was several fold higher in polyps and CRC in comparison to normal colon tissues. Individuals bearing the Cys326Cys genotype of OGG1 were characterized by higher PARP-1 protein level in diseased tissues than the Ser326Cys and Ser326Ser genotypes. Aforementioned result may suggest that the diseased cells with polymorphic OGG1 recruit more PARP protein, which is necessary to remove 8-oxodGuo. Thus, patients with decreased activity of OGG1/polymorphism of the OGG1 gene and higher 8-oxodGuo level may be more susceptible to treatment with PARP-1 inhibitors.

Cardiovascular Protective Effect of Metformin and Telmisartan: Reduction of PARP1 Activity via the AMPK-PARP1 Cascade.

Directory of Open Access Journals (Sweden)

Fenqing Shang

Full Text Available Hyperglycemia and hypertension impair endothelial function in part through oxidative stress-activated poly (ADP-ribose polymerase 1 (PARP1. Biguanides and angiotensin II receptor blockers (ARBs such as metformin and telmisartan have a vascular protective effect. We used cultured vascular endothelial cells (ECs, diabetic and hypertensive rodent models, and AMPKα2-knockout mice to investigate whether metformin and telmisartan have a beneficial effect on the endothelium via AMP-activated protein kinase (AMPK phosphorylation of PARP1 and thus inhibition of PARP1 activity. The results showed that metformin and telmisartan, but not glipizide and metoprolol, activated AMPK, which phosphorylated PARP1 Ser-177 in cultured ECs and the vascular wall of rodent models. Experiments using phosphorylated/de-phosphorylated PARP1 mutants show that AMPK phosphorylation of PARP1 leads to decreased PARP1 activity and attenuated protein poly(ADP-ribosylation (PARylation, but increased endothelial nitric oxide synthase (eNOS activity and silent mating type information regulation 2 homolog 1 (SIRT1 expression. Taken together, the data presented here suggest biguanides and ARBs have a beneficial effect on the vasculature by the cascade of AMPK phosphorylation of PARP1 to inhibit PARP1 activity and protein PARylation in ECs, thereby mitigating endothelial dysfunction.

Effects of 3-AB on PARP expression of Hela cells and apoptosis and cell cycle progression of Hela cells after X-rays irradiation

International Nuclear Information System (INIS)

Du Xiang; Zhao Hongguang; Guo Wei; Gong Shouliang; Wang Wen

2007-01-01

Objective: To study the changes of apoptosis and cell cycle progression of Hela cells after the poly (ADP- ribose) polymerase (PARP) was inhibited by its inhibitor 3-aminobenzamid (3-AB) and the mechanisms of PARP interaction with Hela cells damaged by irradiation. Methods: Hela cell line was used. Flow cytometry (FCM) was used to examine the PARP expression of control and 3 AB groups at 0, 2, 4, 8, 12 h alter administration with 5 mmol·L -1 3-AB. The percentage of apoptotic cells and cell cycle progression ol control, irradiation, 3-AB plus irradiation groups were measured with FCM at 2, 8, 12, 24 h after exposure to 2 Gy irradiation following administration with 5 mmol·L -1 3-AB. Results: The percentage of Hela cells with positive expression of PARP protein decreased after administration with 3-AB and there was significant difference between 3-AB plus irradiation group and control group (P 2 cells in the 3-AB plus irradiation group were lower than those in the irradiation group (P 2 arrest induced by irradiation. (authors)

ROS-mediated PARP activity undermines mitochondrial function after permeability transition pore opening during myocardial ischemia-reperfusion.

Science.gov (United States)

Schriewer, Jacqueline M; Peek, Clara Bien; Bass, Joseph; Schumacker, Paul T

2013-04-18

Ischemia-reperfusion (I/R) studies have implicated oxidant stress, the mitochondrial permeability transition pore (mPTP), and poly(ADP-ribose) polymerase (PARP) as contributing factors in myocardial cell death. However, the interdependence of these factors in the intact, blood-perfused heart is not known. We therefore wanted to determine whether oxidant stress, mPTP opening, and PARP activity contribute to the same death pathway after myocardial I/R. A murine left anterior descending coronary artery (LAD) occlusion (30 minutes) and release (1 to 4 hours) model was employed. Experimental groups included controls and antioxidant-treated, mPTP-inhibited, or PARP-inhibited hearts. Antioxidant treatment prevented oxidative damage, mPTP opening, ATP depletion, and PARP activity, placing oxidant stress as the proximal death trigger. Genetic deletion of cyclophilin D (CypD(-/-)) prevented loss of total NAD(+) and PARP activity, and mPTP-mediated loss of mitochondrial function. Control hearts showed progressive mitochondrial depolarization and loss of ATP from 1.5 to 4 hours of reperfusion, but not outer mitochondrial membrane rupture. Neither genetic deletion of PARP-1 nor its pharmacological inhibition prevented the initial mPTP-mediated depolarization or loss of ATP, but PARP ablation did allow mitochondrial recovery by 4 hours of reperfusion. These results indicate that oxidant stress, the mPTP, and PARP activity contribute to a single death pathway after I/R in the heart. PARP activation undermines cell survival by preventing mitochondrial recovery after mPTP opening early in reperfusion. This suggests that PARP-mediated prolongation of mitochondrial depolarization contributes significantly to cell death via an energetic crisis rather than by mitochondrial outer membrane rupture.

Overcoming Resistance of Cancer Cells to PARP-1 Inhibitors with Three Different Drug Combinations.

Directory of Open Access Journals (Sweden)

Michal Yalon

Full Text Available Inhibitors of poly[ADP-ribose] polymerase 1 (PARPis show promise for treatment of cancers which lack capacity for homologous recombination repair (HRR. However, new therapeutic strategies are required in order to overcome innate and acquired resistance to these drugs and thus expand the array of cancers that could benefit from them. We show that human cancer cell lines which respond poorly to ABT-888 (a PARPi, become sensitive to it when co-treated with vorinostat (a histone deacetylase inhibitor (HDACi. Vorinostat also sensitized PARPis insensitive cancer cell lines to 6-thioguanine (6-TG-a drug that targets PARPis sensitive cells. The sensitizing effect of vorinostat was associated with increased phosphorylation of eukaryotic initiation factor (eIF 2α which in and of itself increases the sensitivity of cancer cells to ABT-888. Importantly, these drug combinations did not affect survival of normal fibroblasts and breast cells, and significantly increased the inhibition of xenograft tumor growth relative to each drug alone, without affecting the mice weight or their liver and kidney function. Our results show that combination of vorinostat and ABT-888 could potentially prove useful for treatment of cancer with innate resistance to PARPis due to active HRR machinery, while the combination of vorinostat and 6-TG could potentially overcome innate or acquired resistance to PARPis due to secondary or reversal BRCA mutations, to decreased PARP-1 level or to increased expression of multiple drug resistant proteins. Importantly, drugs which increase phosphorylation of eIF2α may mimic the sensitizing effect of vorinostat on cellular response to PARPis or to 6-TG, without activating all of its downstream effectors.

PARP1 Val762Ala polymorphism reduces enzymatic activity

International Nuclear Information System (INIS)

Wang Xiaogan; Wang Zhaoqi; Tong Weimin; Shen Yan

2007-01-01

Poly(ADP-ribose) polymerase 1 (PARP1) modifies a variety of nuclear proteins by poly(ADP-ribosyl)ation, and plays diverse roles in molecular and cellular processes. A common PARP1 single nucleotide polymorphism (SNP) at codon 762, resulting in the substitution of alanine (Ala) for valine (Val) in the catalytic domain has been implicated in susceptibility to cancer. To characterize the functional effect of this polymorphism on PARP1, we performed in vitro enzymatic analysis on PARP1-Ala762 and PARP1-Val762. We found that PARP1-Ala762 displayed 57.2% of the activity of PARP1-Val762 for auto-poly(ADP-ribosyl)ation and 61.9% of the activity of PARP1-Val762 for trans-poly(ADP-ribosyl)ation of histone H1. The kinetic characterization revealed that the K m of PARP1-Ala762 was increased to a 1.2-fold of the K m of PARP1-Val762 for trans-poly(ADP-ribosyl)ation. Thus, the PARP1 Val762Ala polymorphism reduces the enzymatic activity of PARP1 by increasing K m . This finding suggests that different levels of poly(ADP-ribosyl)ation by PARP1 might aid in understanding Cancer risk of carriers of the PARP1 Val762Ala polymorphism

Mechanistic Links Between PARP, NAD, and Brain Inflammation After TBI

Science.gov (United States)

2015-10-01

1 AWARD NUMBER: W81XWH-13-2-0091 TITLE: Mechanistic Links Between PARP, NAD , and Brain Inflammation After TBI PRINCIPAL INVESTIGATOR...COVERED 25 Sep 2014 - 24 Sep 2015 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Mechanistic Links Between PARP, NAD , and Brain Inflammation After TBI 5b. GRANT...efficacy of veliparib and NAD as agents for suppressing inflammation and improving outcomes after traumatic brain injury. The animal models include

Acquisition of Relative Interstrand Crosslinker Resistance and PARP Inhibitor Sensitivity in Fanconi Anemia Head and Neck Cancers.

Science.gov (United States)

Lombardi, Anne J; Hoskins, Elizabeth E; Foglesong, Grant D; Wikenheiser-Brokamp, Kathryn A; Wiesmüller, Lisa; Hanenberg, Helmut; Andreassen, Paul R; Jacobs, Allison J; Olson, Susan B; Keeble, Winifred W; Hays, Laura E; Wells, Susanne I

2015-04-15

Fanconi anemia is an inherited disorder associated with a constitutional defect in the Fanconi anemia DNA repair machinery that is essential for resolution of DNA interstrand crosslinks. Individuals with Fanconi anemia are predisposed to formation of head and neck squamous cell carcinomas (HNSCC) at a young age. Prognosis is poor, partly due to patient intolerance of chemotherapy and radiation requiring dose reduction, which may lead to early recurrence of disease. Using HNSCC cell lines derived from the tumors of patients with Fanconi anemia, and murine HNSCC cell lines derived from the tumors of wild-type and Fancc(-/-) mice, we sought to define Fanconi anemia-dependent chemosensitivity and DNA repair characteristics. We utilized DNA repair reporter assays to explore the preference of Fanconi anemia HNSCC cells for non-homologous end joining (NHEJ). Surprisingly, interstrand crosslinker (ICL) sensitivity was not necessarily Fanconi anemia-dependent in human or murine cell systems. Our results suggest that the increased Ku-dependent NHEJ that is expected in Fanconi anemia cells did not mediate relative ICL resistance. ICL exposure resulted in increased DNA damage sensing and repair by PARP in Fanconi anemia-deficient cells. Moreover, human and murine Fanconi anemia HNSCC cells were sensitive to PARP inhibition, and sensitivity of human cells was attenuated by Fanconi anemia gene complementation. The observed reliance upon PARP-mediated mechanisms reveals a means by which Fanconi anemia HNSCCs can acquire relative resistance to the ICL-based chemotherapy that is a foundation of HNSCC treatment, as well as a potential target for overcoming chemoresistance in the chemosensitive individual. ©2015 American Association for Cancer Research.

UPP mediated Diabetic Retinopathy via ROS/PARP and NF-κB inflammatory factor pathways.

Science.gov (United States)

Luo, D-W; Zheng, Z; Wang, H; Fan, Y; Chen, F; Sun, Y; Wang, W-J; Sun, T; Xu, X

2015-01-01

Diabetic retinopathy (DR) is a leading cause of blindness in adults at working age. Human diabetic retinopathy is characterized by the basement membrane thick, pericytes loss, microaneurysms formation, retina neovascularization and vitreous hemorrhage. To investigate whether UPP activated ROS/PARP and NF-κB inflammatory factor pathways in Diabetic Retinopathy, human retinal endothelial cells (HRECs) and rats with streptozotocin-induced diabetes were used to determine the effect of UPP on ROS generation, cell apoptosis, mitochondrial membrane potential (ΔΨm) and inflammatory factor protein expression, through flow cytometry assay, immunohistochemistry, Real-time PCR, Western blot analysis and ELISA. The levels of ROS and apoptosis and the expressions of UPP (Ub and E3) and inflammatory factor protein were increased in high glucose-induced HRECs and retina of diabetic rats, while ΔΨm was decreased. The UPP inhibitor and UbshRNA could attenuate these effects through inhibiting the pathway of ROS/PARP and the expression of NF-κB inflammatory factors, and the increased UPP was a result of high glucose-induced increase of ROS generation and NF-κBp65 expression, accompanied with the decrease of ΔΨm. Clinical study showed the overexpression of UPP and detachment of epiretinal membranes in proliferative DR (PDR) patients. It has been indicated that the pathogenic effect of UPP on DR was involved in the increase of ROS generation and NF-κB expression, which associated with the ROS/PARP and NF-κB inflammatory factor pathways. Our study supports a new insight for further application of UPP inhibitor in DR treatment.

Functional characterisation of an Arabidopsis gene strongly induced by ionising radiation: the gene coding the poly(ADP-ribose)polymerase-1 (AthPARP-1)

International Nuclear Information System (INIS)

Doucet-Chabeaud, G.

2000-01-01

Arabidopsis thaliana, the model-system in plant genetics, has been used to study the responses to DNA damage, experimentally introduced by γ-irradiation. We have characterised a radiation-induced gene coding a 111 kDa protein, AthPARP-1, homologous to the human poly(ADP-ribose)polymerase-1 (hPARP-1). As hPARP-1 is composed by three functional domain with characteristic motifs, AthPARP-1 binds to DNA bearing single-strand breaks and shows DNA damage-dependent poly(ADP-ribosyl)ation. The preferential expression of AthPARP-1 in mitotically active tissues is in agreement with a potential role in the maintenance of genome integrity during DNA replication, as proposed for its human counterpart. Transcriptional gene activation by ionising radiation of AthPARP-1 and AthPARP-2 genes is to date plant specific activation. Our expression analyses after exposure to various stress indicate that 1) AthPARP-1 and AthPARP-2 play an important role in the response to DNA lesions, particularly they are activated by genotoxic agents implicating the BER DNA repair pathway 2) AthPARP-2 gene seems to play an additional role in the signal transduction induced by oxidative stress 3) the observed expression profile of AthPARP-1 is in favour of the regulation of AthPARP-1 gene expression at the level of transcription and translation. This mode of regulation of AthPARP-1 protein biosynthesis, clearly distinct from that observed in animals, needs the implication of a so far unidentified transcription factor that is activated by the presence of DNA lesions. The major outcome of this work resides in the isolation and characterisation of such new transcription factor, which will provide new insight on the regulation of plant gene expression by genotoxic stress. (author) [fr

Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from dihydropyridophthalazinone

Energy Technology Data Exchange (ETDEWEB)

Aoyagi-Scharber, Mika, E-mail: maoyagi@bmrn.com [BioMarin Pharmaceutical Inc., 105 Digital Drive, Novato, CA 94949 (United States); Gardberg, Anna S. [Emerald BioStructures, 7869 NE Day Road West, Bainbridge Island, WA 98110 (United States); Yip, Bryan K.; Wang, Bing; Shen, Yuqiao; Fitzpatrick, Paul A. [BioMarin Pharmaceutical Inc., 105 Digital Drive, Novato, CA 94949 (United States)

2014-08-29

BMN 673, a novel PARP1/2 inhibitor in clinical development with substantial tumor cytotoxicity, forms extensive hydrogen-bonding and π-stacking in the nicotinamide pocket, with its unique disubstituted scaffold extending towards the less conserved edges of the pocket. These interactions might provide structural insight into the ability of BMN 673 to both inhibit catalysis and affect DNA-binding activity. Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2), which are involved in DNA damage response, are targets of anticancer therapeutics. BMN 673 is a novel PARP1/2 inhibitor with substantially increased PARP-mediated tumor cytotoxicity and is now in later-stage clinical development for BRCA-deficient breast cancers. In co-crystal structures, BMN 673 is anchored to the nicotinamide-binding pocket via an extensive network of hydrogen-bonding and π-stacking interactions, including those mediated by active-site water molecules. The novel di-branched scaffold of BMN 673 extends the binding interactions towards the outer edges of the pocket, which exhibit the least sequence homology among PARP enzymes. The crystallographic structural analyses reported here therefore not only provide critical insights into the molecular basis for the exceptionally high potency of the clinical development candidate BMN 673, but also new opportunities for increasing inhibitor selectivity.

Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from dihydropyridophthalazinone

International Nuclear Information System (INIS)

Aoyagi-Scharber, Mika; Gardberg, Anna S.; Yip, Bryan K.; Wang, Bing; Shen, Yuqiao; Fitzpatrick, Paul A.

2014-01-01

BMN 673, a novel PARP1/2 inhibitor in clinical development with substantial tumor cytotoxicity, forms extensive hydrogen-bonding and π-stacking in the nicotinamide pocket, with its unique disubstituted scaffold extending towards the less conserved edges of the pocket. These interactions might provide structural insight into the ability of BMN 673 to both inhibit catalysis and affect DNA-binding activity. Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2), which are involved in DNA damage response, are targets of anticancer therapeutics. BMN 673 is a novel PARP1/2 inhibitor with substantially increased PARP-mediated tumor cytotoxicity and is now in later-stage clinical development for BRCA-deficient breast cancers. In co-crystal structures, BMN 673 is anchored to the nicotinamide-binding pocket via an extensive network of hydrogen-bonding and π-stacking interactions, including those mediated by active-site water molecules. The novel di-branched scaffold of BMN 673 extends the binding interactions towards the outer edges of the pocket, which exhibit the least sequence homology among PARP enzymes. The crystallographic structural analyses reported here therefore not only provide critical insights into the molecular basis for the exceptionally high potency of the clinical development candidate BMN 673, but also new opportunities for increasing inhibitor selectivity

PARP2 Is the Predominant Poly(ADP-Ribose Polymerase in Arabidopsis DNA Damage and Immune Responses.

Directory of Open Access Journals (Sweden)

Junqi Song

2015-05-01

Full Text Available Poly (ADP-ribose polymerases (PARPs catalyze the transfer of multiple poly(ADP-ribose units onto target proteins. Poly(ADP-ribosylation plays a crucial role in a variety of cellular processes including, most prominently, auto-activation of PARP at sites of DNA breaks to activate DNA repair processes. In humans, PARP1 (the founding and most characterized member of the PARP family accounts for more than 90% of overall cellular PARP activity in response to DNA damage. We have found that, in contrast with animals, in Arabidopsis thaliana PARP2 (At4g02390, rather than PARP1 (At2g31320, makes the greatest contribution to PARP activity and organismal viability in response to genotoxic stresses caused by bleomycin, mitomycin C or gamma-radiation. Plant PARP2 proteins carry SAP DNA binding motifs rather than the zinc finger domains common in plant and animal PARP1 proteins. PARP2 also makes stronger contributions than PARP1 to plant immune responses including restriction of pathogenic Pseudomonas syringae pv. tomato growth and reduction of infection-associated DNA double-strand break abundance. For poly(ADP-ribose glycohydrolase (PARG enzymes, we find that Arabidopsis PARG1 and not PARG2 is the major contributor to poly(ADP-ribose removal from acceptor proteins. The activity or abundance of PARP2 is influenced by PARP1 and PARG1. PARP2 and PARP1 physically interact with each other, and with PARG1 and PARG2, suggesting relatively direct regulatory interactions among these mediators of the balance of poly(ADP-ribosylation. As with plant PARP2, plant PARG proteins are also structurally distinct from their animal counterparts. Hence core aspects of plant poly(ADP-ribosylation are mediated by substantially different enzymes than in animals, suggesting the likelihood of substantial differences in regulation.

Arsenite binding-induced zinc loss from PARP-1 is equivalent to zinc deficiency in reducing PARP-1 activity, leading to inhibition of DNA repair

Energy Technology Data Exchange (ETDEWEB)

Sun, Xi; Zhou, Xixi [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States); Du, Libo [Center for Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Liu, Wenlan [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States); Liu, Yang [Center for Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Hudson, Laurie G. [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States); Liu, Ke Jian, E-mail: kliu@salud.unm.edu [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States)

2014-01-15

Inhibition of DNA repair is a recognized mechanism for arsenic enhancement of ultraviolet radiation-induced DNA damage and carcinogenesis. Poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger DNA repair protein, has been identified as a sensitive molecular target for arsenic. The zinc finger domains of PARP-1 protein function as a critical structure in DNA recognition and binding. Since cellular poly(ADP-ribosyl)ation capacity has been positively correlated with zinc status in cells, we hypothesize that arsenite binding-induced zinc loss from PARP-1 is equivalent to zinc deficiency in reducing PARP-1 activity, leading to inhibition of DNA repair. To test this hypothesis, we compared the effects of arsenite exposure with zinc deficiency, created by using the membrane-permeable zinc chelator TPEN, on 8-OHdG formation, PARP-1 activity and zinc binding to PARP-1 in HaCat cells. Our results show that arsenite exposure and zinc deficiency had similar effects on PARP-1 protein, whereas supplemental zinc reversed these effects. To investigate the molecular mechanism of zinc loss induced by arsenite, ICP-AES, near UV spectroscopy, fluorescence, and circular dichroism spectroscopy were utilized to examine arsenite binding and occupation of a peptide representing the first zinc finger of PARP-1. We found that arsenite binding as well as zinc loss altered the conformation of zinc finger structure which functionally leads to PARP-1 inhibition. These findings suggest that arsenite binding to PARP-1 protein created similar adverse biological effects as zinc deficiency, which establishes the molecular mechanism for zinc supplementation as a potentially effective treatment to reverse the detrimental outcomes of arsenic exposure. - Highlights: • Arsenite binding is equivalent to zinc deficiency in reducing PARP-1 function. • Zinc reverses arsenic inhibition of PARP-1 activity and enhancement of DNA damage. • Arsenite binding and zinc loss alter the conformation of zinc finger

Arsenite binding-induced zinc loss from PARP-1 is equivalent to zinc deficiency in reducing PARP-1 activity, leading to inhibition of DNA repair

International Nuclear Information System (INIS)

Sun, Xi; Zhou, Xixi; Du, Libo; Liu, Wenlan; Liu, Yang; Hudson, Laurie G.; Liu, Ke Jian

2014-01-01

Inhibition of DNA repair is a recognized mechanism for arsenic enhancement of ultraviolet radiation-induced DNA damage and carcinogenesis. Poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger DNA repair protein, has been identified as a sensitive molecular target for arsenic. The zinc finger domains of PARP-1 protein function as a critical structure in DNA recognition and binding. Since cellular poly(ADP-ribosyl)ation capacity has been positively correlated with zinc status in cells, we hypothesize that arsenite binding-induced zinc loss from PARP-1 is equivalent to zinc deficiency in reducing PARP-1 activity, leading to inhibition of DNA repair. To test this hypothesis, we compared the effects of arsenite exposure with zinc deficiency, created by using the membrane-permeable zinc chelator TPEN, on 8-OHdG formation, PARP-1 activity and zinc binding to PARP-1 in HaCat cells. Our results show that arsenite exposure and zinc deficiency had similar effects on PARP-1 protein, whereas supplemental zinc reversed these effects. To investigate the molecular mechanism of zinc loss induced by arsenite, ICP-AES, near UV spectroscopy, fluorescence, and circular dichroism spectroscopy were utilized to examine arsenite binding and occupation of a peptide representing the first zinc finger of PARP-1. We found that arsenite binding as well as zinc loss altered the conformation of zinc finger structure which functionally leads to PARP-1 inhibition. These findings suggest that arsenite binding to PARP-1 protein created similar adverse biological effects as zinc deficiency, which establishes the molecular mechanism for zinc supplementation as a potentially effective treatment to reverse the detrimental outcomes of arsenic exposure. - Highlights: • Arsenite binding is equivalent to zinc deficiency in reducing PARP-1 function. • Zinc reverses arsenic inhibition of PARP-1 activity and enhancement of DNA damage. • Arsenite binding and zinc loss alter the conformation of zinc finger

The NAD+/PARP1/SIRT1 Axis in Aging.

Science.gov (United States)

Mendelsohn, Andrew R; Larrick, James W

2017-06-01

NAD+ levels decline with age in diverse animals from Caenorhabditis elegans to mice. Raising NAD+ levels by dietary supplementation with NAD+ precursors, nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN), improves mitochondrial function and muscle and neural and melanocyte stem cell function in mice, as well as increases murine life span. Decreased NAD+ levels with age reduce SIRT1 function and reduce the mitochondrial unfolded protein response, which can be overcome by NR supplementation. Decreased NAD+ levels cause NAD+-binding protein DBC1 to form a complex with PARP1, inhibiting poly(adenosine diphosphate-ribose) polymerase (PARP) catalytic activity. Old mice have increased amounts of DBC1-PARP1 complexes, lower PARP activity, increased DNA damage, and reduced nonhomologous end joining and homologous recombination repair. DBC1-PARP1 complexes in old mice can be broken by increasing NAD+ levels through treatment with NMN, reducing DNA damage and restoring PARP activity to youthful levels. The mechanism of declining NAD+ levels and its fundamental importance to aging are yet to be elucidated. There is a correlation of PARP activity with mammalian life span that suggests that NAD+/SIRT1/PARP1 may be more significant than the modest effects on life span observed for NR supplementation in old mice. The NAD+/PARP1/SIRT1 axis may link NAD+ levels and DNA damage with the apparent epigenomic DNA methylation clocks that have been described.

Effects of niacin restriction on sirtuin and PARP responses to photodamage in human skin.

Directory of Open Access Journals (Sweden)

Claudia A Benavente

Full Text Available Sirtuins (SIRTs and poly(ADP-ribose polymerases (PARPs, NAD(+-dependent enzymes, link cellular energy status with responses to environmental stresses. Skin is frequently exposed to the DNA damaging effects of UV irradiation, a known etiology in skin cancer. Thus, understanding the defense mechanisms in response to UV, including the role of SIRTs and PARPs, may be important in developing skin cancer prevention strategies. Here, we report expression of the seven SIRT family members in human skin. SIRTs gene expressions are progressively upregulated in A431 epidermoid carcinoma cells (SIRTs1 and 3, actinic keratoses (SIRTs 2, 3, 5, 6, and 7 and squamous cell carcinoma (SIRTs 1-7. Photodamage induces dynamic changes in SIRT expression with upregulation of both SIRT1 and SIRT4 mRNAs. Specific losses of SIRT proteins occur early after photodamage followed by accumulation later, especially for SIRT4. Niacin restriction, which decreases NAD(+, the sirtuin substrate, results in an increase in acetylated proteins, upregulation of SIRTs 2 and 4, increased inherent DNA damage, alterations in SIRT responses to photodamage, abrogation of PARP activation following photodamage, and increased sensitivity to photodamage that is completely reversed by repleting niacin. These data support the hypothesis that SIRTs and PARPs play important roles in resistance to photodamage and identify specific SIRTs that respond to photodamage and may be targets for skin cancer prevention.

The inhibition of PARP but not EGFR results in the radiosensitization of HPV/p16-positive HNSCC cell lines

International Nuclear Information System (INIS)

Güster, Julian David; Weissleder, Stephanie Valerie; Busch, Chia-Jung; Kriegs, Malte; Petersen, Cordula; Knecht, Rainald; Dikomey, Ekkehard; Rieckmann, Thorsten

2014-01-01

Background and purpose: HPV-negative and HPV-positive HNSCC comprise distinct tumor entities with different biological characteristics. Specific regimens for the comparably well curable HPV-positive entity that reduce side effects without compromising outcome have yet to be established. Therefore, we tested here whether the inhibition of EGFR or PARP may be used to specifically enhance the radiosensitivity of HPV-positive HNSCC cells. Materials and methods: Experiments were performed with five HPV/p16-positive HNSCC cell lines. Inhibitors used were cetuximab, olaparib and PF-00477736. The respective inhibition of EGFR, PARP and Chk1 was evaluated by Western blot, immunofluorescence analysis and assessment of cell cycle distribution. Cell survival was assessed by colony formation assay. Results: Inhibition of EGFR by cetuximab failed to radiosensitize any of the HPV-positive HNSCC cell lines tested. In contrast, PARP-inhibition resulted in a substantial radiosensitization of all strains, with the sensitization being further enhanced by the additional inhibition of Chk1. Conclusions: PARP-inhibition effectively radiosensitizes HPV-positive HNSCC cells and may therefore represent a viable alternative to chemotherapy possibly even allowing for a reduction in radiation dose. For the latter, PARP-inhibition may be combined with the inhibition of Chk1. In contrast, the inhibition of EGFR cannot be expected to radiosensitize HPV-positive HNSCC through the modulation of cellular radiosensitivity

Glycyrrhetinic acid and its derivatives as inhibitors of poly(ADP-ribosepolymerases 1 and 2, apurinic/apyrimidinic endonuclease 1 and DNA polymerase β

Directory of Open Access Journals (Sweden)

Salakhutdinov N. F.

2012-06-01

Full Text Available Aim. For strengthening the efficiency of monofunctional alkylating antineoplastic drugs it is important to lower the capacity of base excision repair (BER system which corrects the majority of DNA damages caused by these reagents. The objective was to create inhibitors of the key BER enzymes (PARP1, PARP2, DNA polymerase β, and APE1 by the directed modification of glycyrrhetinic acid (GA. Methods. Amides of GA were produced from the GA acetate by formation of the corresponding acyl chloride, amidation with the appropriate amine and subsequent deacylation. Small library of 2-cyano substituted derivatives of GA methyl esters was obtained by the structural modification of GA framework and carboxylic acid group. The inhibitory capacity of the compounds was estimated by comparison of the enzyme activities in specific tests in the presence of compounds versus their absence. Results. None of tested compounds inhibits PARP1 significantly. Unmodified GA and its morpholinic derivative were shown to be weak inhibitors of PARP2. The derivatives of GA containing keto-group in 11 triterpene framework were shown to be moderate inhibitors of pol β. Compound 3, containing 12-oxo-9(11-en moiety in the ring C, was shown to be a single inhibitor of APE1 among all compounds studied. Conclusions. The class of GA derivatives, selective pol β inhibitors, was found out. The selective inhibitor of APE1 and weak selective inhibitor of PARP2 were also revealed.

Phase I, Dose-Escalation, Two-Part Trial of the PARP Inhibitor Talazoparib in Patients with Advanced Germline BRCA1/2 Mutations and Selected Sporadic Cancers.

Science.gov (United States)

de Bono, Johann; Ramanathan, Ramesh K; Mina, Lida; Chugh, Rashmi; Glaspy, John; Rafii, Saeed; Kaye, Stan; Sachdev, Jasgit; Heymach, John; Smith, David C; Henshaw, Joshua W; Herriott, Ashleigh; Patterson, Miranda; Curtin, Nicola J; Byers, Lauren Averett; Wainberg, Zev A

2017-06-01

Talazoparib inhibits PARP catalytic activity, trapping PARP1 on damaged DNA and causing cell death in BRCA1/2 -mutated cells. We evaluated talazoparib therapy in this two-part, phase I, first-in-human trial. Antitumor activity, MTD, pharmacokinetics, and pharmacodynamics of once-daily talazoparib were determined in an open-label, multicenter, dose-escalation study (NCT01286987). The MTD was 1.0 mg/day, with an elimination half-life of 50 hours. Treatment-related adverse events included fatigue (26/71 patients; 37%) and anemia (25/71 patients; 35%). Grade 3 to 4 adverse events included anemia (17/71 patients; 24%) and thrombocytopenia (13/71 patients; 18%). Sustained PARP inhibition was observed at doses ≥0.60 mg/day. At 1.0 mg/day, confirmed responses were observed in 7 of 14 (50%) and 5 of 12 (42%) patients with BRCA mutation-associated breast and ovarian cancers, respectively, and in patients with pancreatic and small cell lung cancer. Talazoparib demonstrated single-agent antitumor activity and was well tolerated in patients at the recommended dose of 1.0 mg/day. Significance: In this clinical trial, we show that talazoparib has single-agent antitumor activity and a tolerable safety profile. At its recommended phase II dose of 1.0 mg/day, confirmed responses were observed in patients with BRCA mutation-associated breast and ovarian cancers and in patients with pancreatic and small cell lung cancer. Cancer Discov; 7(6); 620-9. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 539 . ©2017 American Association for Cancer Research.

PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration

Directory of Open Access Journals (Sweden)

Alexander Jonathan S

2010-12-01

Full Text Available Abstract The normal function of poly (ADP-ribose polymerase-1 (PARP-1 is the routine repair of DNA damage by adding poly (ADP ribose polymers in response to a variety of cellular stresses. Recently, it has become widely appreciated that PARP-1 also participates in diverse physiological and pathological functions from cell survival to several forms of cell death and has been implicated in gene transcription, immune responses, inflammation, learning, memory, synaptic functions, angiogenesis and aging. In the CNS, PARP inhibition attenuates injury in pathologies like cerebral ischemia, trauma and excitotoxicity demonstrating a central role of PARP-1 in these pathologies. PARP-1 is also a preferred substrate for several 'suicidal' proteases and the proteolytic action of suicidal proteases (caspases, calpains, cathepsins, granzymes and matrix metalloproteinases (MMPs on PARP-1 produces several specific proteolytic cleavage fragments with different molecular weights. These PARP-1 signature fragments are recognized biomarkers for specific patterns of protease activity in unique cell death programs. This review focuses on specific suicidal proteases active towards PARP-1 to generate signature PARP-1 fragments that can identify key proteases and particular forms of cell death involved in pathophysiology. The roles played by some of the PARP-1 fragments and their associated binding partners in the control of different forms of cell death are also discussed.

Poly(ADP-ribose polymerase (PARP-1 is not involved in DNA double-strand break recovery

Directory of Open Access Journals (Sweden)

Fernet Marie

2003-07-01

Full Text Available Abstract Background The cytotoxicity and the rejoining of DNA double-strand breaks induced by γ-rays, H2O2 and neocarzinostatin, were investigated in normal and PARP-1 knockout mouse 3T3 fibroblasts to determine the role of poly(ADP-ribose polymerase (PARP-1 in DNA double-strand break repair. Results PARP-1-/- were considerably more sensitive than PARP-1+/+ 3T3s to induced cell kill by γ-rays and H2O2. However, the two cell lines did not show any significant difference in the susceptibility to neocarzinostatin below 1.5 nM drug. Restoration of PARP-1 expression in PARP-1-/- 3T3s by retroviral transfection of the full PARP-1 cDNA did not induce any change in neocarzinostatin response. Moreover the incidence and the rejoining kinetics of neocarzinostatin-induced DNA double-strand breaks were identical in PARP-1+/+ and PARP-1-/- 3T3s. Poly(ADP-ribose synthesis following γ-rays and H2O2 was observed in PARP-1-proficient cells only. In contrast neocarzinostatin, even at supra-lethal concentration, was unable to initiate PARP-1 activation yet it induced H2AX histone phosphorylation in both PARP1+/+ and PARP-1-/- 3T3s as efficiently as γ-rays and H2O2. Conclusions The results show that PARP-1 is not a major determinant of DNA double-strand break recovery with either strand break rejoining or cell survival as an endpoint. Even though both PARP-1 and ATM activation are major determinants of the cell response to γ-rays and H2O2, data suggest that PARP-1-dependent poly(ADP-ribose synthesis and ATM-dependent H2AX phosphorylation, are not inter-related in the repair pathway of neocarzinostatin-induced DNA double-strand breaks.

PARPs database: A LIMS systems for protein-protein interaction data mining or laboratory information management system

Directory of Open Access Journals (Sweden)

Picard-Cloutier Aude

2007-12-01

Full Text Available Abstract Background In the "post-genome" era, mass spectrometry (MS has become an important method for the analysis of proteins and the rapid advancement of this technique, in combination with other proteomics methods, results in an increasing amount of proteome data. This data must be archived and analysed using specialized bioinformatics tools. Description We herein describe "PARPs database," a data analysis and management pipeline for liquid chromatography tandem mass spectrometry (LC-MS/MS proteomics. PARPs database is a web-based tool whose features include experiment annotation, protein database searching, protein sequence management, as well as data-mining of the peptides and proteins identified. Conclusion Using this pipeline, we have successfully identified several interactions of biological significance between PARP-1 and other proteins, namely RFC-1, 2, 3, 4 and 5.

The BRCA1-Δ11q Alternative Splice Isoform Bypasses Germline Mutations and Promotes Therapeutic Resistance to PARP Inhibition and Cisplatin

DEFF Research Database (Denmark)

Wang, Yifan; Bernhardy, Andrea J; Cruz, Cristina

2016-01-01

Breast and ovarian cancer patients harboring BRCA1/2 germline mutations have clinically benefitted from therapy with PARP inhibitor (PARPi) or platinum compounds, but acquired resistance limits clinical impact. In this study, we investigated the impact of mutations on BRCA1 isoform expression and...

Deficiency in Poly(ADP-ribose Polymerase-1 (PARP-1 Accelerates Aging and Spontaneous Carcinogenesis in Mice

Directory of Open Access Journals (Sweden)

Tatiana S. Piskunova

2008-01-01

Full Text Available Genetic and biochemical studies have shown that PARP-1 and poly(ADP-ribosylation play an important role in DNA repair, genomic stability, cell death, inflammation, telomere maintenance, and suppressing tumorigenesis, suggesting that the homeostasis of poly(ADP-ribosylation and PARP-1 may also play an important role in aging. Here we show that PARP-1−/− mice exhibit a reduction of life span and a significant increase of population aging rate. Analysis of noninvasive parameters, including body weight gain, body temperature, estrous function, behavior, and a number of biochemical indices suggests the acceleration of biological aging in PARP-1−/− mice. The incidence of spontaneous tumors in both PARP-1−/− and PARP-1+/+ groups is similar; however, malignant tumors including uterine tumors, lung adenocarcinomas and hepatocellular carcinomas, develop at a significantly higher frequency in PARP-1−/− mice than PARP-1+/+ mice (72% and 49%, resp.; < .05. In addition, spontaneous tumors appear earlier in PARP-1−/− mice compared to the wild type group. Histopathological studies revealed a wide spectrum of tumors in uterus, ovaries, liver, lungs, mammary gland, soft tissues, and lymphoid organs in both groups of the mice. These results demonstrate that inactivation of DNA repair gene PARP-1 in mice leads to acceleration of aging, shortened life span, and increased spontaneous carcinogenesis.

The Rothmund-Thomson gene product RECQL4 localizes to the nucleolus in response to oxidative stress

International Nuclear Information System (INIS)

Woo, Leslie L.; Futami, Kazunobu; Shimamoto, Akira; Furuichi, Yasuhiro; Frank, Karen M.

2006-01-01

Mutations in the RECQL4 helicase gene have been linked to Rothmund-Thomson syndrome (RTS), which is characterized by poikiloderma, growth deficiency, and a predisposition to cancer. Examination of RECQL4 subcellular localization in live cells demonstrated a nucleoplasmic pattern and, to a lesser degree, staining in nucleoli. Analysis of RECQL4-GFP deletion mutants revealed two nuclear localization regions in the N-terminal region of RECQL4 and a nucleolar localization signal at amino acids 376-386. RECQL4 localization did not change after treatment with the DNA-damaging agents bleomycin, etoposide, UV irradiation and γ irradiation, in contrast to the Bloom and Werner syndrome helicases that relocate to distinct nuclear foci after damage. However, in a significant number of cells exposed to hydrogen peroxide or streptonigrin, RECQL4 accumulated in nucleoli. Using a T7 phage display screen, we determined that RECQL4 interacts with poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme that promotes genomic integrity through its involvement in DNA repair and signaling pathways. The RECQL4 nucleolar localization was inhibited by pretreatment with a PARP-1 inhibitor. The C-terminal portion of RECQL4 was found to be an in vitro substrate for PARP-1. These results demonstrate changes in the intracellular localization of RECQL4 in response to oxidative stress and identify an interaction between RECQL4 and PARP-1

A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells

Science.gov (United States)

Nüske, Stefan; Scholz, Armin M.; Bogner, Jacqueline; Ruf, Benjamin; Zolghadr, Kourosh; Drexler, Sophie E.; Drexler, Guido A.; Girst, Stefanie; Greubel, Christoph; Reindl, Judith; Siebenwirth, Christian; Romer, Tina; Friedl, Anna A.; Rothbauer, Ulrich

2016-01-01

Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair. PMID:26950694

A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells.

Directory of Open Access Journals (Sweden)

Andrea Buchfellner

Full Text Available Poly(ADP-ribose polymerase 1 (PARP1 is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa, termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.

A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells.

Science.gov (United States)

Buchfellner, Andrea; Yurlova, Larisa; Nüske, Stefan; Scholz, Armin M; Bogner, Jacqueline; Ruf, Benjamin; Zolghadr, Kourosh; Drexler, Sophie E; Drexler, Guido A; Girst, Stefanie; Greubel, Christoph; Reindl, Judith; Siebenwirth, Christian; Romer, Tina; Friedl, Anna A; Rothbauer, Ulrich

2016-01-01

Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.

Antitumor efficacy of PKI-587, a highly potent dual PI3K/mTOR kinase inhibitor.

Science.gov (United States)

Mallon, Robert; Feldberg, Larry R; Lucas, Judy; Chaudhary, Inder; Dehnhardt, Christoph; Santos, Efren Delos; Chen, Zecheng; dos Santos, Osvaldo; Ayral-Kaloustian, Semiramis; Venkatesan, Aranapakam; Hollander, Irwin

2011-05-15

The aim of this study was to show preclinical efficacy and clinical development potential of PKI-587, a dual phosphoinositide 3-kinase (PI3K)/mTOR inhibitor. In vitro class 1 PI3K enzyme and human tumor cell growth inhibition assays and in vivo five tumor xenograft models were used to show efficacy. In vitro, PKI-587 potently inhibited class I PI3Ks (IC(50) vs. PI3K-α = 0.4 nmol/L), PI3K-α mutants, and mTOR. PKI-587 inhibited growth of 50 diverse human tumor cell lines at IC(50) values of less than 100 nmol/L. PKI-587 suppressed phosphorylation of PI3K/mTOR effectors (e.g., Akt), and induced apoptosis in human tumor cell lines with elevated PI3K/mTOR signaling. MDA-MB-361 [breast; HER2(+), PIK3CA mutant (E545K)] was particularly sensitive to this effect, with cleaved PARP, an apoptosis marker, induced by 30 nmol/L PKI-587 at 4 hours. In vivo, PKI-587 inhibited tumor growth in breast (MDA-MB-361, BT474), colon (HCT116), lung (H1975), and glioma (U87MG) xenograft models. In MDA-MB-361 tumors, PKI-587 (25 mg/kg, single dose i.v.) suppressed Akt phosphorylation [at threonine(T)308 and serine(S)473] for up to 36 hours, with cleaved PARP (cPARP) evident up to 18 hours. PKI-587 at 25 mg/kg (once weekly) shrank large (∼1,000 mm(3)) MDA-MB-361 tumors and suppressed tumor regrowth. Tumor regression correlated with suppression of phosphorylated Akt in the MDA-MB-361 model. PKI-587 also caused regression in other tumor models, and efficacy was enhanced when given in combination with PD0325901 (MEK 1/2 inhibitor), irinotecan (topoisomerase I inhibitor), or HKI-272 (neratinib, HER2 inhibitor). Significant antitumor efficacy and a favorable pharmacokinetic/safety profile justified phase 1 clinical evaluation of PKI-587. ©2011 AACR.

A Phase 1 trial of the poly(ADP-ribose) polymerase inhibitor olaparib (AZD2281) in combination with the anti-angiogenic cediranib (AZD2171) in recurrent epithelial ovarian or triple-negative breast cancer.

Science.gov (United States)

Liu, Joyce F; Tolaney, Sara M; Birrer, Michael; Fleming, Gini F; Buss, Mary K; Dahlberg, Suzanne E; Lee, Hang; Whalen, Christin; Tyburski, Karin; Winer, Eric; Ivy, Percy; Matulonis, Ursula A

2013-09-01

Poly(ADP-ribose) polymerase (PARP)-inhibitors and anti-angiogenics have activity in recurrent ovarian and breast cancer; however, the effect of combined therapy against PARP and angiogenesis in this population has not been reported. We investigated the toxicities and recommended phase 2 dosing (RP2D) of the combination of cediranib, a multitargeted inhibitor of vascular endothelial growth factor receptor (VEGFR)-1/2/3 and olaparib, a PARP-inhibitor (NCT01116648). Cediranib tablets once daily and olaparib capsules twice daily were administered orally in a standard 3+3 dose escalation design. Patients with recurrent ovarian or metastatic triple-negative breast cancer were eligible. Patients had measurable disease by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 or met Gynecologic Cancer InterGroup (GCIG) CA125 criteria. No prior PARP-inhibitors or anti-angiogenics in the recurrent setting were allowed. 28 patients (20 ovarian, 8 breast) enrolled to 4 dose levels. 2 dose limiting toxicities (DLTs) (1 grade 4 neutropenia ≥ 4 days; 1 grade 4 thrombocytopenia) occurred at the highest dose level (cediranib 30 mg daily; olaparib 400 mg twice daily [BID]). The RP2D was cediranib 30 mg daily and olaparib 200 mg BID. Grade 3 or higher toxicities occurred in 75% of patients, and included grade 3 hypertension (25%) and grade 3 fatigue (18%). One grade 3 bowel obstruction occurred. The overall response rate (ORR) in the 18 RECIST-evaluable ovarian cancer patients was 44%, with a clinical benefit rate (ORR plus stable disease (SD) > 24 weeks) of 61%. None of the seven evaluable breast cancer patients achieved clinical response; two patients had stable disease for > 24 weeks. The combination of cediranib and olaparib has haematologic DLTs and anticipated class toxicities, with promising evidence of activity in ovarian cancer patients. Copyright © 2013 Elsevier Ltd. All rights reserved.

Sensitization to radiation and alkylating agents by inhibitors of poly(ADP-ribose) polymerase is enhanced in cells deficient in DNA double-strand break repair.

Science.gov (United States)

Löser, Dana A; Shibata, Atsushi; Shibata, Akiko K; Woodbine, Lisa J; Jeggo, Penny A; Chalmers, Anthony J

2010-06-01

As single agents, chemical inhibitors of poly(ADP-ribose) polymerase (PARP) are nontoxic and have clinical efficacy against BRCA1- and BRCA2-deficient tumors. PARP inhibitors also enhance the cytotoxicity of ionizing radiation and alkylating agents but will only improve clinical outcomes if tumor sensitization exceeds effects on normal tissues. It is unclear how tumor DNA repair proficiency affects the degree of sensitization. We have previously shown that the radiosensitizing effect of PARP inhibition requires DNA replication and will therefore affect rapidly proliferating tumors more than normal tissues. Because many tumors exhibit defective DNA repair, we investigated the impact of double-strand break (DSB) repair integrity on the sensitizing effects of the PARP inhibitor olaparib. Sensitization to ionizing radiation and the alkylating agent methylmethane sulfonate was enhanced in DSB repair-deficient cells. In Artemis(-/-) and ATM(-/-) mouse embryo fibroblasts, sensitization was replication dependent and associated with defective repair of replication-associated damage. Radiosensitization of Ligase IV(-/-) mouse embryo fibroblasts was independent of DNA replication and is explained by inhibition of "alternative" end joining. After methylmethane sulfonate treatment, PARP inhibition promoted replication-independent accumulation of DSB, repair of which required Ligase IV. Our findings predict that the sensitizing effects of PARP inhibitors will be more pronounced in rapidly dividing and/or DNA repair defective tumors than normal tissues and show their potential to enhance the therapeutic ratio achieved by conventional DNA-damaging agents.

Characterization of Novel Cytoplasmic PARP in the Brain of Octopus vulgaris

Science.gov (United States)

DE LISA, EMILIA; DE MAIO, ANNA; MOROZ, LEONID L.; MOCCIA, FRANCESCO; MENNELLA, MARIA ROSARIA FARAONE; DI COSMO, ANNA

2014-01-01

Recent investigation has focused on the participation of the poly (ADP-ribose) polymerase (PARP) reaction in the invertebrate central nervous system (CNS) during the process of long-term memory (LTM). In this paper, we characterize, localize, and assign a possible role to a cytoplasmic PARP in the brain of Octopus vulgaris. PARP activity was assayed in optic lobes, supraesophageal mass, and optic nerves. The highest levels of enzyme were found in the cytoplasmic fraction. Hyper-activation of the enzyme was detected in Octopus brain after visual discrimination training. Finally, cytoplasmic PARP was found to inhibit Octopus vulgaris actin polymerization. We propose that the cytoplasmic PARP plays a role in vivo to induce the cytoskeletonal reorganization that occurs during learning-induced neuronal plasticity. PMID:22815366

PARP-1 serves as a novel molecular marker for hepatocellular carcinoma in a Southern Chinese Zhuang population.

Science.gov (United States)

Li, Jiatong; Dou, Dongwei; Li, Ping; Luo, Wenqi; Lv, Wenxin; Zhang, Chengdong; Song, Xiaowei; Yang, Yuan; Zhang, Yuening; Xu, Yanzhen; Xiao, Feifan; Wei, Yan; Qin, Jian; Li, Hongtao; Yang, Xiaoli

2017-07-01

PARP-1 (poly(ADP-ribose) polymerase-1) plays an important role in tumorigenesis. Since its effects on different populations are varied, this study investigated the impact of PARP-1 on primary hepatocellular carcinoma in a Southern Chinese Zhuang population. We assessed the global PARP-1 messenger RNA expression in patients with hepatocellular carcinoma using The Cancer Genome Atlas dataset. Increased PARP-1 expression, related to alpha-fetoprotein level, was observed. The area under the receiver operating characteristic curve value was 0.833. Kaplan-Meier survival curves indicated that higher PARP-1 expression was not correlated with poorer overall survival and recurrence-free survival. In a Zhuang population, PARP-1 messenger RNA and protein levels were increased in the hepatocellular carcinoma tissue and its adjacent liver tissues as assessed by quantitative polymerase chain reaction, immunohistochemistry, and western blotting. Higher PARP-1 level was associated with a higher tumor stage (p  0.05). Further analysis suggested that H2AX, a PARP-1 protein interaction partner, was coordinated with PARP-1 in hepatocellular carcinoma tumorigenesis. Overall, some new characteristics of PARP-1 expression were noted in the Zhuang population. PARP-1 is a novel promising diagnostic marker for hepatocellular carcinoma in the Southern Chinese Zhuang population.

Protective effect of the poly(ADP-ribose polymerase inhibitor PJ34 on mitochondrial depolarization-mediated cell death in hepatocellular carcinoma cells involves attenuation of c-Jun N-terminal kinase-2 and protein kinase B/Akt activation

Directory of Open Access Journals (Sweden)

Radnai Balazs

2012-05-01

Full Text Available Abstract Background 2,4-Dimethoxyphenyl-E-4-arylidene-3-isochromanone (IK11 was previously described to induce apoptotic death of A431 tumor cells. In this report, we investigated the molecular action of IK11 in the HepG2 human hepatocellular carcinoma cell line to increase our knowledge of the role of poly (ADP-ribose-polymerase (PARP, protein kinase B/Akt and mitogen activated protein kinase (MAPK activation in the survival and death of tumor cells and to highlight the possible role of PARP-inhibitors in co-treatments with different cytotoxic agents in cancer therapy. Results We found that sublethal concentrations of IK11 prevented proliferation, migration and entry of the cells into their G2 phase. At higher concentrations, IK11 induced reactive oxygen species (ROS production, mitochondrial membrane depolarization, activation of c-Jun N-terminal kinase 2 (JNK2, and substantial loss of HepG2 cells. ROS production appeared marginal in mediating the cytotoxicity of IK11 since N-acetyl cysteine was unable to prevent it. However, the PARP inhibitor PJ34, although not a ROS scavenger, strongly inhibited both IK11-induced ROS production and cell death. JNK2 activation seemed to be a major mediator of the effect of IK11 since inhibition of JNK resulted in a substantial cytoprotection while inhibitors of the other kinases failed to do so. Inhibition of Akt slightly diminished the effect of IK11, while the JNK and Akt inhibitor and ROS scavenger trans-resveratrol completely protected against it. Conclusions These results indicate significant involvement of PARP, a marginal role of ROS and a pro-apoptotic role of Akt in this system, and raise attention to a novel mechanism that should be considered when cancer therapy is augmented with PARP-inhibition, namely the cytoprotection by inhibition of JNK2.

Theophylline prevents NAD+ depletion via PARP-1 inhibition in human pulmonary epithelial cells

International Nuclear Information System (INIS)

Moonen, Harald J.J.; Geraets, Liesbeth; Vaarhorst, Anika; Bast, Aalt; Wouters, Emiel F.M.; Hageman, Geja J.

2005-01-01

Oxidative DNA damage, as occurs during exacerbations in chronic obstructive pulmonary disease (COPD), highly activates the nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-1). This can lead to cellular depletion of its substrate NAD + , resulting in an energy crisis and ultimately in cell death. Inhibition of PARP-1 results in preservation of the intracellular NAD + pool, and of NAD + -dependent cellular processes. In this study, PARP-1 activation by hydrogen peroxide decreased intracellular NAD + levels in human pulmonary epithelial cells, which was found to be prevented in a dose-dependent manner by theophylline, a widely used compound in the treatment of COPD. This enzyme inhibition by theophylline was confirmed in an ELISA using purified human PARP-1 and was found to be competitive by nature. These findings provide new mechanistic insights into the therapeutic effect of theophylline in oxidative stress-induced lung pathologies

ROS-induced DNA damage and PARP-1 are required for optimal induction of starvation-induced autophagy

DEFF Research Database (Denmark)

Rodríguez-Vargas, José Manuel; Ruiz-Magaña, María José; Ruiz-Ruiz, Carmen

2012-01-01

, leading to ATP depletion (an early event after nutrient deprivation). The absence of PARP-1 blunted AMPK activation and prevented the complete loss of mTOR activity, leading to a delay in autophagy. PARP-1 depletion favors apoptosis in starved cells, suggesting a pro-survival role of autophagy and PARP-1...

Autoimmune response to PARP and BRCA1/BRCA2 in cancer

Science.gov (United States)

Zhu, Qing; Han, Su-Xia; Zhou, Cong-Ya; Cai, Meng-Jiao; Dai, Li-Ping; Zhang, Jian-Ying

2015-01-01

Purpose To determine the role of autoantibodies to PARP1 and BRCA1/BRCA2 which were involved in the synthetic lethal interaction in cancer. Methods Enzyme-Linked Immunosorbent Assay (ELISA) was used to detect autoantibodies to PARP1 and BRCA1/BRCA2 in 618 serum samples including 131 from breast cancer, 94 from lung cancer, 34 from ovarian cancer, 107 from prostate cancer, 76 from liver cancer, 41 from pancreatic cancer and 135 from normal individuals. The positive sera with ELISA were confirmed by Western blot. Immunohistochemistry was used to examine the expression of PARP1 and BRCA1/BRCA2 in breast cancer. Results Autoantibody frequency to PARP1, BRCA1, and BRCA2 in cancer varied from 0% to 50%. When the sera from cancer patients were tested for the presence of autoantibodies to PARP1 and BRCA1/BRCA2, the autoantibody responses slightly decreased and the positive autoantibody reactions varied from 0% to 50.0%. This was significantly higher autoantibody responses to PARP1 and BRCA1/BRCA2 (especially to PARP1 and BRCA1) in ovarian cancer and breast cancer compared to normal control sera (P cancer was different (P cancers have different profiles of autoantibodies. The autoantibodies to proteins involving the synthetic lethal interactions would be novel serological biomarker in some selective cancers. PMID:25865228

Regulation of HFE expression by poly(ADP-ribose) polymerase-1 (PARP1) through an inverted repeat DNA sequence in the distal promoter.

Science.gov (United States)

Pelham, Christopher; Jimenez, Tamara; Rodova, Marianna; Rudolph, Angela; Chipps, Elizabeth; Islam, M Rafiq

2013-12-01

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron overload among Caucasians of northern European descent. Over 85% of all cases with HH are due to mutations in the hemochromatosis protein (HFE) involved in iron metabolism. Although the importance in iron homeostasis is well recognized, the mechanism of sensing and regulating iron absorption by HFE, especially in the absence of iron response element in its gene, is not fully understood. In this report, we have identified an inverted repeat sequence (ATGGTcttACCTA) within 1700bp (-1675/+35) of the HFE promoter capable to form cruciform structure that binds PARP1 and strongly represses HFE promoter. Knockdown of PARP1 increases HFE mRNA and protein. Similarly, hemin or FeCl3 treatments resulted in increase in HFE expression by reducing nuclear PARP1 pool via its apoptosis induced cleavage, leading to upregulation of the iron regulatory hormone hepcidin mRNA. Thus, PARP1 binding to the inverted repeat sequence on the HFE promoter may serve as a novel iron sensing mechanism as increased iron level can trigger PARP1 cleavage and relief of HFE transcriptional repression. © 2013.

PARP-1: Friend or Foe of DNA Damage and Repair in Tumorigenesis?

Energy Technology Data Exchange (ETDEWEB)

Swindall, Amanda F.; Stanley, Jennifer A. [Department of Radiation Oncology Comprehensive Cancer Center, University of Alabama at Birmingham School of Medicine, 176F HSROC Suite 2232B, 1700 6th Avenue South, Birmingham, AL 35249 (United States); Yang, Eddy S., E-mail: eyang@uab.edu [Department of Radiation Oncology Comprehensive Cancer Center, University of Alabama at Birmingham School of Medicine, 176F HSROC Suite 2232B, 1700 6th Avenue South, Birmingham, AL 35249 (United States); Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35249 (United States); Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35249 (United States)

2013-07-26

Oxidative stress induced by reactive oxygen species can result in DNA damage within cells and subsequently increase risk for carcinogenesis. This may be averted by repair of DNA damage through the base or nucleotide excision repair (BER/NER) pathways. PARP, a BER protein, is known for its role in DNA-repair. However, multiple lesions can occur within a small range of DNA, known as oxidative clustered DNA lesions (OCDLs), which are difficult to repair and may lead to the more severe DNA double-strand break (DSB). Inefficient DSB repair can then result in increased mutagenesis and neoplastic transformation. OCDLs occur more frequently within a variety of tumor tissues. Interestingly, PARP is highly expressed in several human cancers. Additionally, chronic inflammation may contribute to tumorigenesis through ROS-induced DNA damage. Furthermore, PARP can modulate inflammation through interaction with NFκB and regulating the expression of inflammatory signaling molecules. Thus, the upregulation of PARP may present a double-edged sword. PARP is needed to repair ROS-induced DNA lesions, but PARP expression may lead to increased inflammation via upregulation of NFκB signaling. Here, we discuss the role of PARP in the repair of oxidative damage versus the formation of OCDLs and speculate on the feasibility of PARP inhibition for the treatment and prevention of cancers by exploiting its role in inflammation.

PARP-1: Friend or Foe of DNA Damage and Repair in Tumorigenesis?

International Nuclear Information System (INIS)

Swindall, Amanda F.; Stanley, Jennifer A.; Yang, Eddy S.

2013-01-01

Oxidative stress induced by reactive oxygen species can result in DNA damage within cells and subsequently increase risk for carcinogenesis. This may be averted by repair of DNA damage through the base or nucleotide excision repair (BER/NER) pathways. PARP, a BER protein, is known for its role in DNA-repair. However, multiple lesions can occur within a small range of DNA, known as oxidative clustered DNA lesions (OCDLs), which are difficult to repair and may lead to the more severe DNA double-strand break (DSB). Inefficient DSB repair can then result in increased mutagenesis and neoplastic transformation. OCDLs occur more frequently within a variety of tumor tissues. Interestingly, PARP is highly expressed in several human cancers. Additionally, chronic inflammation may contribute to tumorigenesis through ROS-induced DNA damage. Furthermore, PARP can modulate inflammation through interaction with NFκB and regulating the expression of inflammatory signaling molecules. Thus, the upregulation of PARP may present a double-edged sword. PARP is needed to repair ROS-induced DNA lesions, but PARP expression may lead to increased inflammation via upregulation of NFκB signaling. Here, we discuss the role of PARP in the repair of oxidative damage versus the formation of OCDLs and speculate on the feasibility of PARP inhibition for the treatment and prevention of cancers by exploiting its role in inflammation

Low ATM protein expression and depletion of p53 correlates with olaparib sensitivity in gastric cancer cell lines

Science.gov (United States)

Kubota, Eiji; Williamson, Christopher T; Ye, Ruiqiong; Elegbede, Anifat; Peterson, Lars; Lees-Miller, Susan P; Bebb, D Gwyn

2014-01-01

Small-molecule inhibitors of poly (ADP-ribose) polymerase (PARP) have shown considerable promise in the treatment of homologous recombination (HR)-defective tumors, such as BRCA1- and BRCA2-deficient breast and ovarian cancers. We previously reported that mantle cell lymphoma cells with deficiency in ataxia telangiectasia mutated (ATM) are sensitive to PARP-1 inhibitors in vitro and in vivo. Here, we report that PARP inhibitors can potentially target ATM deficiency arising in a solid malignancy. We show that ATM protein expression varies between gastric cancer cell lines, with NUGC4 having significantly reduced protein levels. Significant correlation was found between ATM protein expression and sensitivity to the PARP inhibitor olaparib, with NUGC4 being the most sensitive. Moreover, reducing ATM kinase activity using a small-molecule inhibitor (KU55933) or shRNA-mediated depletion of ATM protein enhanced olaparib sensitivity in gastric cancer cell lines with depletion or inactivation of p53. Our results demonstrate that ATM is a potential predictive biomarker for PARP-1 inhibitor activity in gastric cancer harboring disruption of p53, and that combined inhibition of ATM and PARP-1 is a rational strategy for expanding the utility of PARP-1 inhibitors to gastric cancer with p53 disruption. PMID:24841718

Accelerated Aging during Chronic Oxidative Stress: A Role for PARP-1

Directory of Open Access Journals (Sweden)

Daniëlle M. P. H. J. Boesten

2013-01-01

Full Text Available Oxidative stress plays a major role in the pathophysiology of chronic inflammatory disease and it has also been linked to accelerated telomere shortening. Telomeres are specialized structures at the ends of linear chromosomes that protect these ends from degradation and fusion. Telomeres shorten with each cell division eventually leading to cellular senescence. Research has shown that poly(ADP-ribose polymerase-1 (PARP-1 and subtelomeric methylation play a role in telomere stability. We hypothesized that PARP-1 plays a role in accelerated aging in chronic inflammatory diseases due to its role as coactivator of NF-κb and AP-1. Therefore we evaluated the effect of chronic PARP-1 inhibition (by fisetin and minocycline in human fibroblasts (HF cultured under normal conditions and under conditions of chronic oxidative stress, induced by tert-butyl hydroperoxide (t-BHP. Results showed that PARP-1 inhibition under normal culturing conditions accelerated the rate of telomere shortening. However, under conditions of chronic oxidative stress, PARP-1 inhibition did not show accelerated telomere shortening. We also observed a strong correlation between telomere length and subtelomeric methylation status of HF cells. We conclude that chronic PARP-1 inhibition appears to be beneficial in conditions of chronic oxidative stress but may be detrimental under relatively normal conditions.

MicroRNAs Modulate Oxidative Stress in Hypertension through PARP-1 Regulation

Directory of Open Access Journals (Sweden)

Douglas F. Dluzen

2017-01-01

Full Text Available Oxidative stress is thought to contribute to aging and age-related diseases, such as cardiovascular and neurodegenerative diseases, and is a risk factor for systemic arterial hypertension. Previously, we reported differential mRNA and microRNA (miRNA expression between African American (AA and white women with hypertension. Here, we found that the poly-(ADP-ribose polymerase 1 (PARP-1, a DNA damage sensor protein involved in DNA repair and other cellular processes, is upregulated in AA women with hypertension. To explore this mechanism, we identified two miRNAs, miR-103a-2-5p and miR-585-5p, that are differentially expressed with hypertension and were predicted to target PARP1. Through overexpression of each miRNA-downregulated PARP-1 mRNA and protein levels and using heterologous luciferase reporter assays, we demonstrate that miR-103a-2-5p and miR-585-5p regulate PARP1 through binding within the coding region. Given the important role of PARP-1 in DNA repair, we assessed whether overexpression of miR-103a-2-5p or miR-585-5p affected DNA damage and cell survival. Overexpression of these miRNAs enhanced DNA damage and decreased both cell survival and colony formation. These findings highlight the role for PARP-1 in regulating oxidative DNA damage in hypertension and identify important new miRNA regulators of PARP-1 expression. These insights may provide additional avenues to understand hypertension health disparities.

Development of potent ALK inhibitor and its molecular inhibitory mechanism against NSCLC harboring EML4-ALK proteins

Energy Technology Data Exchange (ETDEWEB)

Kang, Chung Hyo [Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600 (Korea, Republic of); College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Yun, Jeong In [Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600 (Korea, Republic of); Lee, Kwangho [Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600 (Korea, Republic of); Medicinal & Pharmaceutical Chemistry, Korea University of Science and Technology, Daejeon 305-350 (Korea, Republic of); Lee, Chong Ock; Lee, Heung Kyoung [Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600 (Korea, Republic of); Yun, Chang-Soo; Hwang, Jong Yeon; Cho, Sung Yun; Jung, Heejung; Kim, Pilho [Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600 (Korea, Republic of); Medicinal & Pharmaceutical Chemistry, Korea University of Science and Technology, Daejeon 305-350 (Korea, Republic of); Ha, Jae Du; Jeon, Jeong Hee; Choi, Sang Un [Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600 (Korea, Republic of); Jeong, Hye Gwang [College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Kim, Hyoung Rae, E-mail: hyungrk@krict.re.kr [Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600 (Korea, Republic of); Park, Chi Hoon, E-mail: chpark@krict.re.kr [Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600 (Korea, Republic of); Medicinal & Pharmaceutical Chemistry, Korea University of Science and Technology, Daejeon 305-350 (Korea, Republic of)

2015-08-28

Here, we show the newly synthesized and potent ALK inhibitor having similar scaffold to KRCA-0008, which was reported previously, and its molecular mechanism against cancer cells harboring EML4-ALK fusion protein. Through ALK wild type enzyme assay, we selected two compounds, KRCA-0080 and KRCA-0087, which have trifluoromethyl instead of chloride in R2 position. We characterized these newly synthesized compounds by in vitro and in vivo assays. Enzyme assay shows that KRCA-0080 is more potent against various ALK mutants, including L1196M, G1202R, T1151-L1152insT, and C1156Y, which are seen in crizotinib-resistant patients, than KRCA-0008 is. Cell based assays demonstrate our compounds downregulate the cellular signaling, such as Akt and Erk, by suppressing ALK activity to inhibit the proliferation of the cells harboring EML4-ALK. Interestingly, our compounds induced strong G1/S arrest in H3122 cells leading to the apoptosis, which is proved by PARP-1 cleavage. In vivo H3122 xenograft assay, we found that KRCA-0080 shows significant reduction in tumor size compared to crizotinib and KRCA-0008 by 15–20%. Conclusively, we report a potent ALK inhibitor which shows significant in vivo efficacy as well as excellent inhibitory activity against various ALK mutants. - Highlights: • We synthesized KRCA-0008 derivatives having trifluoromethyl instead of chloride. • KRCA-0080 shows superior activity against several ALK mutants to KRCA-0008. • Cellular assays show our ALK inhibitors suppress only EML4-ALK positive cells. • Our ALK inhibitors induce G1/S arrest to lead apoptosis in H3122 cells. • KRCA-0080 has superior in vivo efficacy to crizotinib and KRCA-0008 by 15–20%.

Structure/function analysis of PARP-1 in oxidative and nitrosative stress-induced monomeric ADPR formation.

Directory of Open Access Journals (Sweden)

Ben Buelow

2009-07-01

Full Text Available Poly adenosine diphosphate-ribose polymerase-1 (PARP-1 is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR, and formation of the soluble 2(nd messenger monomeric adenosine diphosphate-ribose (mADPR. Previous studies have delineated specific roles for several of PARP-1's structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1's BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2(nd messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.

Unfolding of core nucleosomes by PARP-1 revealed by spFRET microscopy

Directory of Open Access Journals (Sweden)

Daniel Sultanov

2017-01-01

Full Text Available DNA accessibility to various protein complexes is essential for various processes in the cell and is affected by nucleosome structure and dynamics. Protein factor PARP-1 (poly(ADP-ribose polymerase 1 increases the accessibility of DNA in chromatin to repair proteins and transcriptional machinery, but the mechanism and extent of this chromatin reorganization are unknown. Here we report on the effects of PARP-1 on single nucleosomes revealed by spFRET (single-particle Förster Resonance Energy Transfer microscopy. PARP-1 binding to a double-strand break in the vicinity of a nucleosome results in a significant increase of the distance between the adjacent gyres of nucleosomal DNA. This partial uncoiling of the entire nucleosomal DNA occurs without apparent loss of histones and is reversed after poly(ADP-ribosylation of PARP-1. Thus PARP-1-nucleosome interactions result in reversible, partial uncoiling of the entire nucleosomal DNA.

PARP-1 como regulador del ciclo celular

OpenAIRE

Iglesias Vázquez, Pablo

2015-01-01

En el presente estudio hemos querido investigar las implicaciones biológicas de la interacción PARP-1/E2F-1 en escenarios en los que el factor de transcripción E2F-1 resulta de gran importancia como son el desarrollo embrionario y la oncogénesis. En este respecto, hemos demostrado que tanto PJ34, inhibidor de la actividad enzimática de PARP, como gosipol, inhibidor de las interacciones proteína-proteína, son capaces de reducir la actividad transcripcional de E2F-1 y la proli...

Increased transcript level of poly(ADP-ribose) polymerase (PARP-1) in human tricuspid compared with bicuspid aortic valves correlates with the stenosis severity

International Nuclear Information System (INIS)

Nagy, Edit; Caidahl, Kenneth; Franco-Cereceda, Anders; Bäck, Magnus

2012-01-01

Highlights: ► Oxidative stress has been implicated in the pathomechanism of calcific aortic valve stenosis. ► We assessed the transcript levels for PARP-1 (poly(ADP-ribose) polymerase), acts as a DNA damage nick sensor in stenotic valves. ► Early stage of diseased tricuspid valves exhibited higher mRNA levels for PARP-1 compared to bicuspid valves. ► The mRNA levels for PARP-1 inversely correlated with the clinical stenosis severity in tricuspid valves. ► Our data demonstrated that DNA damage pathways might be associated with stenosis severity only in tricuspid valves. -- Abstract: Oxidative stress may contribute to the hemodynamic progression of aortic valve stenosis, and is associated with activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) 1. The aim of the present study was to assess the transcriptional profile and the topological distribution of PARP-1 in human aortic valves, and its relation to the stenosis severity. Human stenotic aortic valves were obtained from 46 patients undergoing aortic valve replacement surgery and used for mRNA extraction followed by quantitative real-time PCR to correlate the PARP-1 expression levels with the non invasive hemodynamic parameters quantifying the stenosis severity. Primary isolated valvular interstitial cells (VICs) were used to explore the effects of cytokines and leukotriene C 4 (LTC 4 ) on valvular PARP-1 expression. The thickened areas of stenotic valves with tricuspid morphology expressed significantly higher levels of PARP-1 mRNA compared with the corresponding part of bicuspid valves (0.501 vs 0.243, P = 0.01). Furthermore, the quantitative gene expression levels of PARP-1 were inversely correlated with the aortic valve area (AVA) (r = −0.46, P = 0.0469) and AVA indexed for body surface area (BSA) (r = −0.498; P = 0.0298) only in tricuspid aortic valves. LTC 4 (1 nM) significantly elevated the mRNA levels of PARP-1 by 2.38-fold in VICs. Taken together, these data suggest that

Carbon ion beam triggers both caspase-dependent and caspase-independent pathway of apoptosis in HeLa and status of PARP-1 controls intensity of apoptosis.

Science.gov (United States)

Ghorai, Atanu; Sarma, Asitikantha; Bhattacharyya, Nitai P; Ghosh, Utpal

2015-04-01

High linear energy transfer (LET) carbon ion beam (CIB) is becoming very promising tool for various cancer treatments and is more efficient than conventional low LET gamma or X-rays to kill malignant or radio-resistant cells, although detailed mechanism of cell death is still unknown. Poly (ADP-ribose) polymerase-1 (PARP-1) is a key player in DNA repair and its inhibitors are well-known as radio-sensitizer for low LET radiation. The objective of our study was to find mechanism(s) of induction of apoptosis by CIB and role of PARP-1 in CIB-induced apoptosis. We observed overall higher apoptosis in PARP-1 knocked down HeLa cells (HsiI) compared with negative control H-vector cells after irradiation with CIB (0-4 Gy). CIB activated both intrinsic and extrinsic pathways of apoptosis via caspase-9 and caspase-8 activation respectively, followed by caspase-3 activation, apoptotic body, nucleosomal ladder formation and sub-G1 accumulation. Apoptosis inducing factor translocation into nucleus in H-vector but not in HsiI cells after CIB irradiation contributed caspase-independent apoptosis. Higher p53 expression was observed in HsiI cells compared with H-vector after exposure with CIB. Notably, we observed about 37 % fall of mitochondrial membrane potential, activation of caspase-9 and caspase-3 and mild activation of caspase-8 without any detectable apoptotic body formation in un-irradiated HsiI cells. We conclude that reduction of PARP-1 expression activates apoptotic signals via intrinsic and extrinsic pathways in un-irradiated cells. CIB irradiation further intensified both intrinsic and extrinsic pathways of apoptosis synergistically along with up-regulation of p53 in HsiI cells resulting overall higher apoptosis in HsiI than H-vector.

Hyperactivation of PARP triggers nonhomologous end-joining in repair-deficient mouse fibroblasts.

Directory of Open Access Journals (Sweden)

Natalie R Gassman

Full Text Available Regulation of poly(ADP-ribose (PAR synthesis and turnover is critical to determining cell fate after genotoxic stress. Hyperactivation of PAR synthesis by poly(ADP-ribose polymerase-1 (PARP-1 occurs when cells deficient in DNA repair are exposed to genotoxic agents; however, the function of this hyperactivation has not been adequately explained. Here, we examine PAR synthesis in mouse fibroblasts deficient in the base excision repair enzyme DNA polymerase β (pol β. The extent and duration of PARP-1 activation was measured after exposure to either the DNA alkylating agent, methyl methanesulfonate (MMS, or to low energy laser-induced DNA damage. There was strong DNA damage-induced hyperactivation of PARP-1 in pol β nullcells, but not in wild-type cells. In the case of MMS treatment, PAR synthesis did not lead to cell death in the pol β null cells, but instead resulted in increased PARylation of the nonhomologous end-joining (NHEJ protein Ku70 and increased association of Ku70 with PARP-1. Inhibition of the NHEJ factor DNA-PK, under conditions of MMS-induced PARP-1 hyperactivation, enhanced necrotic cell death. These data suggest that PARP-1 hyperactivation is a protective mechanism triggering the classical-NHEJ DNA repair pathway when the primary alkylated base damage repair pathway is compromised.

Differential sensitivities of cellular XPA and PARP-1 to arsenite inhibition and zinc rescue.

Science.gov (United States)

Ding, Xiaofeng; Zhou, Xixi; Cooper, Karen L; Huestis, Juliana; Hudson, Laurie G; Liu, Ke Jian

2017-09-15

Arsenite directly binds to the zinc finger domains of the DNA repair protein poly (ADP ribose) polymerase (PARP)-1, and inhibits PARP-1 activity in the base excision repair (BER) pathway. PARP inhibition by arsenite enhances ultraviolet radiation (UVR)-induced DNA damage in keratinocytes, and the increase in DNA damage is reduced by zinc supplementation. However, little is known about the effects of arsenite and zinc on the zinc finger nucleotide excision repair (NER) protein xeroderma pigmentosum group A (XPA). In this study, we investigated the difference in response to arsenite exposure between XPA and PARP-1, and the differential effectiveness of zinc supplementation in restoring protein DNA binding and DNA damage repair. Arsenite targeted both XPA and PARP-1 in human keratinocytes, resulting in zinc loss from each protein and a pronounced decrease in XPA and PARP-1 binding to chromatin as demonstrated by Chip-on-Western assays. Zinc effectively restored DNA binding of PARP-1 and XPA to chromatin when zinc concentrations were equal to those of arsenite. In contrast, zinc was more effective in rescuing arsenite-augmented direct UVR-induced DNA damage than oxidative DNA damage. Taken together, our findings indicate that arsenite interferes with PARP-1 and XPA binding to chromatin, and that zinc supplementation fully restores DNA binding activity to both proteins in the cellular context. Interestingly, rescue of arsenite-inhibited DNA damage repair by supplemental zinc was more sensitive for DNA damage repaired by the XPA-associated NER pathway than for the PARP-1-dependent BER pathway. This study expands our understanding of arsenite's role in DNA repair inhibition and co-carcinogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Antitumor efficacy profile of PKI-402, a dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor.

Science.gov (United States)

Mallon, Robert; Hollander, Irwin; Feldberg, Larry; Lucas, Judy; Soloveva, Veronica; Venkatesan, Aranapakam; Dehnhardt, Christoph; Delos Santos, Efren; Chen, Zecheng; Dos Santos, Osvaldo; Ayral-Kaloustian, Semiramis; Gibbons, Jay

2010-04-01

PKI-402 is a selective, reversible, ATP-competitive, equipotent inhibitor of class I phosphatidylinositol 3-kinases (PI3K), including PI3K-alpha mutants, and mammalian target of rapamycin (mTOR; IC(50) versus PI3K-alpha = 2 nmol/L). PKI-402 inhibited growth of human tumor cell lines derived from breast, brain (glioma), pancreas, and non-small cell lung cancer tissue and suppressed phosphorylation of PI3K and mTOR effector proteins (e.g., Akt at T308) at concentrations that matched those that inhibited cell growth. In MDA-MB-361 [breast: Her2(+) and PIK3CA mutant (E545K)], 30 nmol/L PKI-402 induced cleaved poly(ADP-ribose) polymerase (PARP), a marker for apoptosis. In vivo, PKI-402 inhibited tumor growth in MDA-MB-361, glioma (U87MG), and lung (A549) xenograft models. In MDA-MB-361, PKI-402 at 100 mg/kg (daily for 5 days, one round) reduced initial tumor volume of 260 mm(3) to 129 mm(3) and prevented tumor regrowth for 70 days. In MDA-MB-361 tumors, PKI-402 (100 mg/kg, single dose) suppressed Akt phosphorylation (at T308) and induced cleaved PARP. Suppression of phosphorylated Akt (p-Akt) was complete at 8 hours and still evident at 24 hours. Cleaved PARP was evident at 8 and 24 hours. In normal tissue (heart and lung), PKI-402 (100 mg/kg) had minimal effect on p-Akt, with no detectable cleaved PARP. Preferential accumulation of PKI-402 in tumor tissue was observed. Complete, sustained suppression of Akt phosphorylation may cause tumor regression in MDA-MB-361 and other xenograft models. We are testing whether dual PI3K/mTOR inhibitors can durably suppress p-Akt, induce cleaved PARP, and cause tumor regression in a diverse set of human tumor xenograft models. Mol Cancer Ther; 9(4); 976-84. (c)2010 AACR.

PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

International Nuclear Information System (INIS)

Liang, Ya-Chen; Hsu, Chiao-Yu; Yao, Ya-Li; Yang, Wen-Ming

2013-01-01

Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently of its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression

Parp1 protects against Aag-dependent alkylation-induced nephrotoxicity in a sex-dependent manner.

Science.gov (United States)

Calvo, Jennifer A; Allocca, Mariacarmela; Fake, Kimberly R; Muthupalani, Sureshkumar; Corrigan, Joshua J; Bronson, Roderick T; Samson, Leona D

2016-07-19

Nephrotoxicity is a common toxic side-effect of chemotherapeutic alkylating agents. Although the base excision repair (BER) pathway is essential in repairing DNA alkylation damage, under certain conditions the initiation of BER produces toxic repair intermediates that damage healthy tissues. We have shown that the alkyladenine DNA glycosylase, Aag (a.k.a. Mpg), an enzyme that initiates BER, mediates alkylation-induced whole-animal lethality and cytotoxicity in the pancreas, spleen, retina, and cerebellum, but not in the kidney. Cytotoxicity in both wild-type and Aag-transgenic mice (AagTg) was abrogated in the absence of Poly(ADP-ribose) polymerase-1 (Parp1). Here we report that Parp1-deficient mice expressing increased Aag (AagTg/Parp1-/-) develop sex-dependent kidney failure upon exposure to the alkylating agent, methyl methanesulfonate (MMS), and suffer increased whole-animal lethality compared to AagTg and wild-type mice. Macroscopic, histological, electron microscopic and immunohistochemical analyses revealed morphological kidney damage including dilated tubules, proteinaceous casts, vacuolation, collapse of the glomerular tuft, and deterioration of podocyte structure. Moreover, mice exhibited clinical signs of kidney disease indicating functional damage, including elevated blood nitrogen urea and creatinine, hypoproteinemia and proteinuria. Pharmacological Parp inhibition in AagTg mice also resulted in sensitivity to MMS-induced nephrotoxicity. These findings provide in vivo evidence that Parp1 modulates Aag-dependent MMS-induced nephrotoxicity in a sex-dependent manner and highlight the critical roles that Aag-initiated BER and Parp1 may play in determining the side-effects of chemotherapeutic alkylating agents.

Expression of DNA Damage Response Molecules PARP1, γH2AX, BRCA1, and BRCA2 Predicts Poor Survival of Breast Carcinoma Patients

Directory of Open Access Journals (Sweden)

See-Hyoung Park

2015-08-01

Full Text Available BACKGROUND: Poly(ADP-ribose polymerase 1 (PARP1, γH2AX, BRCA1, and BRCA2 are conventional molecular indicators of DNA damage in cells and are often overexpressed in various cancers. In this study, we aimed, using immunohistochemical detection, whether the co-expression of PARP1, γH2AX, BRCA1, and BRCA2 in breast carcinoma (BCA tissue can provide more reliable prediction of survival of BCA patients. MATERIALS AND METHODS: We investigated immunohistochemical expression and prognostic significance of the expression of PARP1, γH2AX, BRCA1, and BRCA2 in 192 cases of BCAs. RESULTS: The expression of these four molecules predicted earlier distant metastatic relapse, shorter overall survival (OS, and relapse-free survival (RFS by univariate analysis. Multivariate analysis revealed the expression of PARP1, γH2AX, and BRCA2 as independent poor prognostic indicators of OS and RFS. In addition, the combined expressional pattern of BRCA1, BRCA2, PARP1, and γH2AX (CSbbph was an additional independent prognostic predictor for OS (P < .001 and RFS (P < .001. The 10-year OS rate was 95% in the CSbbph-low (CSbbph scores 0 and 1 subgroup, but that was only 35% in the CSbbph-high (CSbbph score 4 subgroup. CONCLUSION: This study has demonstrated that the individual and combined expression patterns of PARP1, γH2AX, BRCA1, and BRCA2 could be helpful in determining an accurate prognosis for BCA patients and for the selection of BCA patients who could potentially benefit from anti-PARP1 therapy with a combination of genotoxic chemotherapeutic agents.

Reduced estradiol-induced vasodilation and poly-(ADP-ribose) polymerase (PARP) activity in the aortas of rats with experimental polycystic ovary syndrome (PCOS).

Science.gov (United States)

Masszi, Gabriella; Horvath, Eszter Maria; Tarszabo, Robert; Benko, Rita; Novak, Agnes; Buday, Anna; Tokes, Anna-Maria; Nadasy, Gyorgy L; Hamar, Peter; Benyó, Zoltán; Varbiro, Szabolcs

2013-01-01

Polycystic ovary syndrome (PCOS) is a complex endocrine disorder characterized by hyperandrogenism and insulin resistance, both of which have been connected to atherosclerosis. Indeed, an increased risk of clinical manifestations of arterial vascular diseases has been described in PCOS. On the other hand endothelial dysfunction can be detected early on, before atherosclerosis develops. Thus we assumed that vascular dysfunction is also related directly to the hormonal imbalance rather than to its metabolic consequences. To detect early functional changes, we applied a novel rodent model of PCOS: rats were either sham operated or hyperandrogenism was achieved by implanting subcutaneous pellets of dihydrotestosterone (DHT). After ten weeks, myograph measurements were performed on isolated aortic rings. Previously we described an increased contractility to norepinephrine (NE). Here we found a reduced immediate relaxation to estradiol treatment in pre-contracted aortic rings from hyperandrogenic rats. Although the administration of vitamin D3 along with DHT reduced responsiveness to NE, it did not restore relaxation to estradiol. Poly-(ADP-ribose) polymerase (PARP) activity was assessed by poly-ADP-ribose immunostaining. Increased PAR staining in ovaries and circulating leukocytes from DHT rats showed enhanced DNA damage, which was reduced by concomitant vitamin D3 treatment. Surprisingly, PAR staining was reduced in both the endothelium and vascular smooth muscle cells of the aorta rings from hyperandrogenic rats. Thus in the early phase of PCOS, vascular tone is already shifted towards vasoconstriction, characterized by reduced vasorelaxation and vascular dysfunction is concomitant with altered PARP activity. Based on our findings, PARP inhibitors might have a future perspective in restoring metabolic disorders in PCOS.

Tumor suppressive effect of PARP1 and FOXO3A in gastric cancers and its clinical implications

Science.gov (United States)

Yoon, Sarah; Jo, Yuna; Kwon, So Mee; Kim, Kyoung Min; Kwon, Keun Sang; Kim, Chan Young; Woo, Hyun Goo

2015-01-01

Poly (ADP-ribose) polymerase1 (PARP1) has been reported as a possible target for chemotherapy in many cancer types. However, its action mechanisms and clinical implications for gastric cancer survival are not yet fully understood. Here, we investigated the effect of PARP1 inhibition in the growth of gastric cancer cells. PARP1 inhibition by Olaparib or PARP1 siRNA could significantly attenuate growth and colony formation of gastric cancer cells, and which were mediated through induction of G2/M cell cycle arrest but not apoptosis. FOXO3A expression was induced by PARP1 inhibition, suggesting that FOXO3A might be one of downstream target of the PARP1 effect on gastric cancer cell growth. In addition, by performing tissue microarrays on the 166 cases of gastric cancer patients, we could observe that the expression status of PARP1 and FOXO3A were significantly associated with overall survival (OS) and relapse-free survival (RFS). Strikingly, combined expression status of PARP1 and FOXO3A showed better prediction for patient's clinical outcomes. The patient group with PARP1+/FOXO3A− expression had the worst prognosis while the patient group with PARP1−/FOXO3A+ had the most favorable prognosis (OS: P = 6.0 × 10−9, RFS: P = 2.2 × 10−8). In conclusion, we suggest that PARP1 and FOXO3A play critical roles in gastric cancer progression, and might have therapeutic and/or diagnostic potential in clinic. PMID:26540566

Telomere shortening is associated to TRF1 and PARP1 overexpression in Duchenne muscular dystrophy.

Science.gov (United States)

Aguennouz, M'Hammed; Vita, Gian Luca; Messina, Sonia; Cama, Annamaria; Lanzano, Natalia; Ciranni, Annamaria; Rodolico, Carmelo; Di Giorgio, Rosa Maria; Vita, Giuseppe

2011-12-01

Telomere shortening is thought to contribute to premature senescence of satellite cells in Duchenne muscular dystrophy (DMD) muscle. Telomeric repeat binding factor-1 (TRF1) and poly (ADP-ribose) polymerase-1 (PARP1) are proteins known to modulate telomerase reverse transcriptase (TERT) activity, which controls telomere elongation. Here we show that an age-dependent telomere shortening occurs in DMD muscles and is associated to overexpression of mRNA and protein levels of TRF1 and PARP1. TERT expression and activity are detectable in normal control muscles and they slightly increase in DMD. This is the first demonstration of TRF1 and PARP1 overexpression in DMD muscles. They can be directly involved in replicative senescence of satellite cells and/or in the pathogenetic cascade through a cross-talk with oxidative stress and inflammatory response. Modulation of these events by TRF1 or PARP1 inhibition might represent a novel strategy for treatment of DMD and other muscular dystrophies. Copyright © 2010 Elsevier Inc. All rights reserved.

Targeting EGFR induced oxidative stress by PARP1 inhibition in glioblastoma therapy.

Science.gov (United States)

Nitta, Masayuki; Kozono, David; Kennedy, Richard; Stommel, Jayne; Ng, Kimberly; Zinn, Pascal O; Kushwaha, Deepa; Kesari, Santosh; Inda, Maria-del-Mar; Wykosky, Jill; Furnari, Frank; Hoadley, Katherine A; Chin, Lynda; DePinho, Ronald A; Cavenee, Webster K; D'Andrea, Alan; Chen, Clark C

2010-05-24

Despite the critical role of Epidermal Growth Factor Receptor (EGFR) in glioblastoma pathogenesis, EGFR targeted therapies have achieved limited clinical efficacy. Here we propose an alternate therapeutic strategy based on the conceptual framework of non-oncogene addiction. A directed RNAi screen revealed that glioblastoma cells over-expressing EGFRvIII, an oncogenic variant of EGFR, become hyper-dependent on a variety of DNA repair genes. Among these, there was an enrichment of Base Excision Repair (BER) genes required for the repair of Reactive Oxygen Species (ROS)-induced DNA damage, including poly-ADP ribose polymerase 1 (PARP1). Subsequent studies revealed that EGFRvIII over-expression in glioblastoma cells caused increased levels of ROS, DNA strand break accumulation, and genome instability. In a panel of primary glioblastoma lines, sensitivity to PARP1 inhibition correlated with the levels of EGFR activation and oxidative stress. Gene expression analysis indicated that reduced expression of BER genes in glioblastomas with high EGFR expression correlated with improved patient survival. These observations suggest that oxidative stress secondary to EGFR hyper-activation necessitates increased cellular reliance on PARP1 mediated BER, and offer critical insights into clinical trial design.

Targeting EGFR induced oxidative stress by PARP1 inhibition in glioblastoma therapy.

Directory of Open Access Journals (Sweden)

Masayuki Nitta

Full Text Available Despite the critical role of Epidermal Growth Factor Receptor (EGFR in glioblastoma pathogenesis, EGFR targeted therapies have achieved limited clinical efficacy. Here we propose an alternate therapeutic strategy based on the conceptual framework of non-oncogene addiction. A directed RNAi screen revealed that glioblastoma cells over-expressing EGFRvIII, an oncogenic variant of EGFR, become hyper-dependent on a variety of DNA repair genes. Among these, there was an enrichment of Base Excision Repair (BER genes required for the repair of Reactive Oxygen Species (ROS-induced DNA damage, including poly-ADP ribose polymerase 1 (PARP1. Subsequent studies revealed that EGFRvIII over-expression in glioblastoma cells caused increased levels of ROS, DNA strand break accumulation, and genome instability. In a panel of primary glioblastoma lines, sensitivity to PARP1 inhibition correlated with the levels of EGFR activation and oxidative stress. Gene expression analysis indicated that reduced expression of BER genes in glioblastomas with high EGFR expression correlated with improved patient survival. These observations suggest that oxidative stress secondary to EGFR hyper-activation necessitates increased cellular reliance on PARP1 mediated BER, and offer critical insights into clinical trial design.

The discovery and the structural basis of an imidazo[4,5-b]pyridine-based p21-activated kinase 4 inhibitor.

Science.gov (United States)

Park, Jeung Kuk; Kim, Sunmin; Han, Yu Jin; Kim, Seong Hwan; Kang, Nam Sook; Lee, Hyuk; Park, SangYoun

2016-06-01

p21-Activated kinases (PAKs) which belong to the family of ste20 serine/threonine protein kinases regulate cytoskeletal reorganization, cell motility, cell proliferation, and oncogenic transformation which are all related to the cellular functions during cancer induction and metastasis. The fact that PAK mutations are detected in multiple tumor tissues makes PAKs a novel therapeutic drug target. In this study, an imidazo[4,5-b]pyridine-based PAK4 inhibitor, KY-04045 (6-Bromo-2-(3-isopropyl-1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-b]pyridine), was discovered using a virtual site-directed fragment-based drug design and was validated using an inhibition assay. Although PAK4 affinity to KY-04045 seems much weaker than that of the reported PAK4 inhibitors, the location of KY-04045 is clearly defined in the structure of PAK4 co-crystallized with KY-04045. The crystal structure illustrates that the pyrazole and imidazopyridine rings of KY-04045 are sufficient for mediating PAK4 hinge loop interaction. Hence, we believe that KY-04045 can be exploited as a basic building block in designing novel imidazo[4,5-b]pyridine-based PAK4 inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

P2X7 receptor-mediated PARP1 activity regulates astroglial death in the rat hippocampus following status epilepticus

Directory of Open Access Journals (Sweden)

Ji Yang eKim

2015-09-01

Full Text Available Poly(ADP-ribose polymerase-1 (PARP1 plays a regulatory role in apoptosis, necrosis, and other cellular processes after injury. Recently, we revealed that PARP1 regulates the differential neuronal/astroglial responses to pilocarpine-induced status epilepticus (SE in the distinct brain regions. In addition, P2X7 receptor (P2X7R, an ATP-gated ion channel, activation accelerates astroglial apoptosis, while it attenuates clasmatodendrosis (lysosome-derived autophagic astroglial death. Therefore, we investigated whether P2X7R regulates regional specific astroglial PARP1 expression/activation in response to SE. In the present study, P2X7R activation exacerbates SE-induced astroglial apoptosis, while P2X7R inhibition attenuates it accompanied by increasing PARP1 activity in the molecular layer of the dentate gyrus following SE. In the CA1 region, however, P2X7R inhibition deteriorates SE-induced clasmatodendrosis via PARP1 activation following SE. Taken together, our findings suggest that P2X7R function may affect SE-induced astroglial death by regulating PARP1 activation/expression in regional-specific manner. Therefore, the selective modulation of P2X7R-mediated PARP1 functions may be a considerable strategy for controls in various types of cell deaths.

Resistance to BET Inhibitor Leads to Alternative Therapeutic Vulnerabilities in Castration-Resistant Prostate Cancer.

Science.gov (United States)

Pawar, Aishwarya; Gollavilli, Paradesi Naidu; Wang, Shaomeng; Asangani, Irfan A

2018-02-27

BRD4 plays a major role in the transcription networks orchestrated by androgen receptor (AR) in castration-resistant prostate cancer (CRPC). Several BET inhibitors (BETi) that displace BRD4 from chromatin are being evaluated in clinical trials for CRPC. Here, we describe mechanisms of acquired resistance to BETi that are amenable to targeted therapies in CRPC. BETi-resistant CRPC cells displayed cross-resistance to a variety of BETi in the absence of gatekeeper mutations, exhibited reduced chromatin-bound BRD4, and were less sensitive to BRD4 degraders/knockdown, suggesting a BRD4-independent transcription program. Transcriptomic analysis revealed reactivation of AR signaling due to CDK9-mediated phosphorylation of AR, resulting in sensitivity to CDK9 inhibitors and enzalutamide. Additionally, increased DNA damage associated with PRC2-mediated transcriptional silencing of DDR genes was observed, leading to PARP inhibitor sensitivity. Collectively, our results identify the therapeutic limitation of BETi as a monotherapy; however, our BETi resistance data suggest unique opportunities for combination therapies in treating CRPC. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

The nuclear protein Poly(ADP-ribose) polymerase 3 (AtPARP3) is required for seed storability in Arabidopsis thaliana.

Science.gov (United States)

Rissel, D; Losch, J; Peiter, E

2014-11-01

The deterioration of seeds during prolonged storage results in a reduction of viability and germination rate. DNA damage is one of the major cellular defects associated with seed deterioration. It is provoked by the formation of reactive oxygen species (ROS) even in the quiescent state of the desiccated seed. In contrast to other stages of seed life, DNA repair during storage is hindered through the low seed water content; thereby DNA lesions can accumulate. To allow subsequent seedling development, DNA repair has thus to be initiated immediately upon imbibition. Poly(ADP-ribose) polymerases (PARPs) are important components in the DNA damage response in humans. Arabidopsis thaliana contains three homologues to the human HsPARP1 protein. Of these three, only AtPARP3 was very highly expressed in seeds. Histochemical GUS staining of embryos and endosperm layers revealed strong promoter activity of AtPARP3 during all steps of germination. This coincided with high ROS activity and indicated a role of the nuclear-localised AtPARP3 in DNA repair during germination. Accordingly, stored parp3-1 mutant seeds lacking AtPARP3 expression displayed a delay in germination as compared to Col-0 wild-type seeds. A controlled deterioration test showed that the mutant seeds were hypersensitive to unfavourable storage conditions. The results demonstrate that AtPARP3 is an important component of seed storability and viability. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Differential transactivation by orphan nuclear receptor NOR1 and its fusion gene product EWS/NOR1: possible involvement of poly(ADP-ribose) polymerase I, PARP-1.

Science.gov (United States)

Ohkura, Naganari; Nagamura, Yuko; Tsukada, Toshihiko

2008-10-15

In extraskeletal myxoid chondrosarcoma, a chromosomal translocation creates a gene fusion between EWS and an orphan nuclear receptor, NOR1. The resulting fusion protein EWS/NOR1 has been believed to lead to malignant transformation by functioning as a transactivator for NOR1-target genes. By comparing the gene expression profiles of NOR1- and EWS/NOR1-overexpressing cells, we found that they largely shared up-regulated genes, but no significant correlation was observed with respect to the transactivation levels of each gene. In addition, the proteins associated with NOR1 and EWS/NOR1 were mostly the same in these cells. The results suggest that these proteins differentially transactivate overlapping target genes through a similar transcriptional machinery. To clarify the mechanisms underlying the transcriptional divergence between NOR1 and EWS/NOR1, we searched for alternatively associated proteins, and identified poly(ADP-ribose) polymerase I (PARP-1) as an NOR1-specific binding protein. Consistent with its binding properties, PARP-1 acted as a transcriptional repressor of NOR1, but not EWS/NOR1, in a luciferase reporter assay employing PARP-1(-/-) fibroblasts. Interestingly, suppressive activity of PARP-1 was observed in a DNA response element-specific manner, and in a subtype-specific manner toward the NR4A family (Nur77, Nurr1, and NOR1), suggesting that PARP-1 plays a role in the diversity of transcriptional regulation mediated by the NR4A family in normal cells. Altogether, our findings suggest that NOR1 and EWS/NOR1 regulate overlapping target genes differently by utilizing associated proteins, including PARP-1; and that EWS/NOR1 may acquire oncogenic activities by avoiding (or gaining) transcription factor-specific modulation by the associated proteins. (c) 2008 Wiley-Liss, Inc.

BZLF1 Expression of EBV is correlated with PARP1 Regulation on Nasopharyngeal Carcinoma Tissues

Directory of Open Access Journals (Sweden)

Wahyu nur laili fajri, Ahmad Rofi'i, Fatchiyah Fatchiyah

2013-04-01

Full Text Available Nasopharyngeal carcinomas (NPC is a cancer that arises in the epithelial tissue that covers the inside of the nasopharyngeal mucosa and nasopharynx. Infected Epstein Barr Virus (EBV cell in a latent infection associated with the expression of nine latent proteins. Latent Membrane Protein 1 (LMP1 is one of latent proteins, and mayor EBV oncoprotein, with functions including virus growth, and to activate BamHI-Z Leftward Reading Frame 1 (BZLF1-EBV, which can inhibit p53 to induce apoptotic resistance, metastasis, and immune modulation. The body will respond to the expansion of EBV infection with activation of Poly(ADP-ribosePolymerase-1 (PARP1. The objective of study is to observe the expression of BZLF1 and determine PARP1 regulation in nasopharyngeal tissues. NPC-T2, NPC-T3 and polyp tissues slides are from Ulin Hospital, Banjarmasin. To characterize the necrotic cells such as pyknosis, karyorrhexsis, and karyolysis, histological slides were stained by HE that the necrotic cells measured by using a BX-53 microscope (Olympus with CellSens Standard software. Tissues slides were stained by using immunofluorohistochemistry with EBV-BZLF1 antibody-Mouse anti-EBV monoclonal antibody against Goat anti-mouse IgG-FITC and anti-PARP1 antibody (MC-10 against Goat anti-mouse IgG labeled Rhodamin. The expression intensities were measured by Confocal Laser Scanning Microscope (Olympus. The percentage number of necrotic cells and BZLF1 and PARP1 expression intensity were analyzed using SPSS 16.0 by one-way ANOVA test with α = 0.05, beside that we use correlate and regression analyze. The research showed that the amount of karryorhexis higher than pyknosis and karyolysis in both tissues. BZLF1 expression 1.79 INT/sel (in polyp, 2.76 INT/sel (NPC Type 2 and 4.36 INT/sel (NPC Type 3, PARP1 expression 2.25 INT/sel (in polyp, 3.31 INT/sel (NPC Type 2, dan 5.93 INT/sel (NPC Type 3.The high of intensity of expression BZLF1 induced the increasing of PARP1 expression

Dipeptidyl peptidase-4 inhibitor induced angioedema - an overlooked and potentially lethal adverse drug reaction?

DEFF Research Database (Denmark)

Scott, Susanne Irene; Andersen, Michelle Fog; Aagaard, Lise

2018-01-01

to vasodilatation and increase in vascular permeability in the capillaries. Objective To assess the risk and pathomechanism of angioedema due to inhibition of dipeptidyl peptidase-4 inhibitors when used as monotherapy and in combination with angiotensin converting enzyme-inhibitors. Method PubMed, Embase......, the Cochrane Library, PubMed Central, Web of Science, Google Scholar and clinicaltrials.gov were searched using different combinations of keywords "angioedema", "dipeptidyl peptidase 4", "dipeptidyl peptidase 4 inhibitors", "gliptins", "bradykinin", "substance P" and "angiotensin converting enzyme...

Reduced estradiol-induced vasodilation and poly-(ADP-ribose polymerase (PARP activity in the aortas of rats with experimental polycystic ovary syndrome (PCOS.

Directory of Open Access Journals (Sweden)

Gabriella Masszi

Full Text Available Polycystic ovary syndrome (PCOS is a complex endocrine disorder characterized by hyperandrogenism and insulin resistance, both of which have been connected to atherosclerosis. Indeed, an increased risk of clinical manifestations of arterial vascular diseases has been described in PCOS. On the other hand endothelial dysfunction can be detected early on, before atherosclerosis develops. Thus we assumed that vascular dysfunction is also related directly to the hormonal imbalance rather than to its metabolic consequences. To detect early functional changes, we applied a novel rodent model of PCOS: rats were either sham operated or hyperandrogenism was achieved by implanting subcutaneous pellets of dihydrotestosterone (DHT. After ten weeks, myograph measurements were performed on isolated aortic rings. Previously we described an increased contractility to norepinephrine (NE. Here we found a reduced immediate relaxation to estradiol treatment in pre-contracted aortic rings from hyperandrogenic rats. Although the administration of vitamin D3 along with DHT reduced responsiveness to NE, it did not restore relaxation to estradiol. Poly-(ADP-ribose polymerase (PARP activity was assessed by poly-ADP-ribose immunostaining. Increased PAR staining in ovaries and circulating leukocytes from DHT rats showed enhanced DNA damage, which was reduced by concomitant vitamin D3 treatment. Surprisingly, PAR staining was reduced in both the endothelium and vascular smooth muscle cells of the aorta rings from hyperandrogenic rats. Thus in the early phase of PCOS, vascular tone is already shifted towards vasoconstriction, characterized by reduced vasorelaxation and vascular dysfunction is concomitant with altered PARP activity. Based on our findings, PARP inhibitors might have a future perspective in restoring metabolic disorders in PCOS.

A Sensitive and Robust Ultra HPLC Assay with Tandem Mass Spectrometric Detection for the Quantitation of the PARP Inhibitor Olaparib (AZD2281 in Human Plasma for Pharmacokinetic Application

Directory of Open Access Journals (Sweden)

Jeffrey Roth

2014-06-01

Full Text Available Olaparib (AZD2281 is an orally active PARP-1 inhibitor, primarily effective against cancers with BRCA1/2 mutations. It is currently in Phase III development and has previously been investigated in numerous clinical trials, both as a single agent and in combination with chemotherapy. Despite this widespread testing, there is only one published method that provides assay details and stability studies for olaparib alone. A more sensitive uHPLC-MS/MS method for the quantification of olaparib in human plasma was developed, increasing the range of quantification at both ends (0.5–50,000 ng/mL compared to previously published methods (10–5,000 ng/mL. The wider range encompasses CMAX levels produced by typical olaparib doses and permits better pharmacokinetic modeling of olaparib elimination. This assay also utilizes a shorter analytical runtime, allowing for more rapid quantification and reduced use of reagents. A liquid-liquid extraction was followed by chromatographic separation on a Waters UPLC® BEH C18 column (2.1 × 50 mm, 1.7 µm and mass spectrometric detection. The mass transitions m/z 435.4→281.1 and m/z 443.2→281.1 were used for olaparib and the internal standard [2H8]-olaparib, respectively. The assay proved to be accurate (<9% deviation and precise (CV < 11%. Stability studies showed that olaparib is stable at room temperature for 24 h. in whole blood, at 4 °C for 24 h post-extraction, at −80 °C in plasma for at least 19 months, and through three freeze-thaw cycles. This method proved to be robust for measuring olaparib levels in clinical samples from a Phase I trial.

Studies of the expression of human poly(ADP-ribose) polymerase-1 in Saccharomyces cerevisiae and identification of PARP-1 substrates by yeast proteome microarray screening.

Science.gov (United States)

Tao, Zhihua; Gao, Peng; Liu, Hung-Wen

2009-12-15

Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.

Influence of MLH1 on colon cancer sensitivity to poly(ADP-ribose) polymerase inhibitor combined with irinotecan.

Science.gov (United States)

Tentori, Lucio; Leonetti, Carlo; Muzi, Alessia; Dorio, Annalisa Susanna; Porru, Manuela; Dolci, Susanna; Campolo, Federica; Vernole, Patrizia; Lacal, Pedro Miguel; Praz, Françoise; Graziani, Grazia

2013-07-01

Poly(ADP-ribose) polymerase inhibitors (PARPi) are currently evaluated in clinical trials in combination with topoisomerase I (Top1) inhibitors against a variety of cancers, including colon carcinoma. Since the mismatch repair component MLH1 is defective in 10-15% of colorectal cancers we have investigated whether MLH1 affects response to the Top1 inhibitor irinotecan, alone or in combination with PARPi. To this end, the colon cancer cell lines HCT116, carrying MLH1 mutations on chromosome 3 and HCT116 in which the wild-type MLH1 gene was replaced via chromosomal transfer (HCT116+3) or by transfection of the corresponding MLH1 cDNA (HCT116 1-2) were used. HCT116 cells or HCT116+3 cells stably silenced for PARP-1 expression were also analysed. The results of in vitro and in vivo experiments indicated that MLH1, together with low levels of Top1, contributed to colon cancer resistance to irinotecan. In the MLH1-proficient cells SN-38, the active metabolite of irinotecan, induced lower levels of DNA damage than in MLH1-deficient cells, as shown by the weaker induction of γ-H2AX and p53 phosphorylation. The presence of MLH1 contributed to induce of prompt Chk1 phosphorylation, restoring G2/M cell cycle checkpoint and repair of DNA damage. On the contrary, in the absence of MLH1, HCT116 cells showed minor Chk1 phosphorylation and underwent apoptosis. Remarkably, inhibition of PARP function by PARPi or by PARP-1 gene silencing always increased the antitumor activity of irinotecan, even in the presence of low PARP-1 expression.

Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)

International Nuclear Information System (INIS)

Okita, Naoyuki; Ashizawa, Daisuke; Ohta, Ryo; Abe, Hideaki; Tanuma, Sei-ichi

2010-01-01

Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (V max ) and the michaelis constant (k m ) of PARG reaction were 4.46 μM and 128.33 μmol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 μM. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors.

Hypoxia/reoxygenation stress signals an increase in organic anion transporting polypeptide 1a4 (Oatp1a4) at the blood-brain barrier: relevance to CNS drug delivery.

Science.gov (United States)

Thompson, Brandon J; Sanchez-Covarrubias, Lucy; Slosky, Lauren M; Zhang, Yifeng; Laracuente, Mei-li; Ronaldson, Patrick T

2014-04-01

Cerebral hypoxia and subsequent reoxygenation stress (H/R) is a component of several diseases. One approach that may enable neural tissue rescue after H/R is central nervous system (CNS) delivery of drugs with brain protective effects such as 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (i.e., statins). Our present in vivo data show that atorvastatin, a commonly prescribed statin, attenuates poly (ADP-ribose) polymerase (PARP) cleavage in the brain after H/R, suggesting neuroprotective efficacy. However, atorvastatin use as a CNS therapeutic is limited by poor blood-brain barrier (BBB) penetration. Therefore, we examined regulation and functional expression of the known statin transporter organic anion transporting polypeptide 1a4 (Oatp1a4) at the BBB under H/R conditions. In rat brain microvessels, H/R (6% O2, 60 minutes followed by 21% O2, 10 minutes) increased Oatp1a4 expression. Brain uptake of taurocholate (i.e., Oap1a4 probe substrate) and atorvastatin were reduced by Oatp inhibitors (i.e., estrone-3-sulfate and fexofenadine), suggesting involvement of Oatp1a4 in brain drug delivery. Pharmacological inhibition of transforming growth factor-β (TGF-β)/activin receptor-like kinase 5 (ALK5) signaling with the selective inhibitor SB431542 increased Oatp1a4 functional expression, suggesting a role for TGF-β/ALK5 signaling in Oatp1a4 regulation. Taken together, our novel data show that targeting an endogenous BBB drug uptake transporter (i.e., Oatp1a4) may be a viable approach for optimizing CNS drug delivery for treatment of diseases with an H/R component.

Role of PARP activity in lung cancer-induced cachexia: Effects on muscle oxidative stress, proteolysis, anabolic markers, and phenotype.

Science.gov (United States)

Chacon-Cabrera, Alba; Mateu-Jimenez, Mercè; Langohr, Klaus; Fermoselle, Clara; García-Arumí, Elena; Andreu, Antoni L; Yelamos, Jose; Barreiro, Esther

2017-12-01

Strategies to treat cachexia are still at its infancy. Enhanced muscle protein breakdown and ubiquitin-proteasome system are common features of cachexia associated with chronic conditions including lung cancer (LC). Poly(ADP-ribose) polymerases (PARP), which play a major role in chromatin structure regulation, also underlie maintenance of muscle metabolism and body composition. We hypothesized that protein catabolism, proteolytic markers, muscle fiber phenotype, and muscle anabolism may improve in respiratory and limb muscles of LC-cachectic Parp-1-deficient (Parp-1 -/- ) and Parp-2 -/- mice. In diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) bearing mice (wild type, Parp-1 -/- , and Parp-2 -/- ), PARP activity (ADP-ribose polymers, pADPr), redox balance, muscle fiber phenotype, apoptotic nuclei, tyrosine release, protein ubiquitination, muscle-specific E3 ligases, NF-κB signaling pathway, markers of muscle anabolism (Akt, mTOR, p70S6K, and mitochondrial DNA) were evaluated along with body and muscle weights, and limb muscle force. Compared to wild type cachectic animals, in both respiratory and limb muscles of Parp-1 -/- and Parp-2 -/- cachectic mice: cancer induced-muscle wasting characterized by increased PARP activity, protein oxidation, tyrosine release, and ubiquitin-proteasome system (total protein ubiquitination, atrogin-1, and 20S proteasome C8 subunit) were blunted, the reduction in contractile myosin and atrophy of the fibers was attenuated, while no effects were seen in other structural features (inflammatory cells, internal or apoptotic nuclei), and markers of muscle anabolism partly improved. Activation of either PARP-1 or -2 is likely to play a role in muscle protein catabolism via oxidative stress, NF-κB signaling, and enhanced proteasomal degradation in cancer-induced cachexia. Therapeutic potential of PARP activity inhibition deserves attention. © 2017 Wiley Periodicals, Inc.

Differential expression of PARP1 mRNA in leucocytes of patients ...

Indian Academy of Sciences (India)

P. 2011 Differential expression of PARP1 mRNA in leucocytes of patients with Down's syndrome. J. Genet. ... of Alzheimer disease at an earlier age than subjects with- ... family and personal informed consent. .... In effect, they report that.

DIPEPTIDYL PEPTIDASE 4 (DPP-4 INHIBITORS FOR THE TREATMENT OF TYPE 2 DIABETES MELLITUS

Directory of Open Access Journals (Sweden)

Erna Kristin

2016-12-01

Diabetes mellitus (DM merupakan penyakit kronis yang menyebabkan sekitar 1,5 juta kematian pada tahun 2012 menurut Organisasi Kesehatan Dunia (WHO. DM tipe 2 (DMT2 banyaknya 90% dari keseluruhan DM di seluruh dunia. Prevalensi DMT2 meningkat karena obesitas. Pedoman klinis merekomendasikan penggunaan metformin sebagai pengobatan lini pertama kecuali ada kontraindikasi, maka bisa diikuti dengan penambahan 1 atau 2 OADs, seperti sulfonilurea (SU, inhibitor alpha-glucosidase, atau thiazolidinediones (TZD. Baru-baru ini, obat baru golongan dipeptidyl peptidase 4 (DPP-4 inhibitor telah ditambahkan ke algoritma pengobatan. Dipeptidyl peptidase 4 (DPP-4 inhibitor inhibitor adalah kelas obat antidiabetes oral yang menghambat DPP-4 enzim. Sitagliptin, saxagliptin, vildagliptin dan linagliptin yang merupakan golongan dipeptidyl peptidase-4 (DPP-4 inhibitor tersedia untuk pengobatan diabetes tipe 2 di Indonesia dan banyak negara lainnya. DPP-4 inhibitor memiliki khasiat glikemik yang setara. DPP-4 inhibitor menghasilkan peningkatan moderat hemoglobin terglikasi (A1C. Namun uji coba head-to-head jumlahnya terbatas, dan tidak ada data tentang penggunaan penggunaan jangka panjang (lebih dari dua tahun keamanan, kematian, komplikasi diabetes, atau kualitas-hidup pasien. Meskipun DPP-inhibitor tidak digunakan sebagai terapi awal untuk mayoritas pasien dengan diabetes tipe 2, DPP-4 inhibitor dapat digunakan sebagai terapi tambahan di tipe 2 pasien diabetes yang tidak toleran, ada kontraindikasi, atau tidak terkontrol dengan penggunaan metformin, sulfonilurea, atau thiazolidinediones. Peran sebenarnya dari DPP-4 inhibitor di antara beberapa obat lainnya untuk DMT2 tidak begitu jelas. Hanya ada sejumlah kecil studi jangka panjang pada DPP-4 inhibitor menilai penurunan glikemik, kemanjuran, kejadian kardiovaskular, kematian, atau keamanan. Pada pasien dengan gagal ginjal (perkiraan laju filtrasi glomerulus [eGFR] <30 mL / menit kronis dapat menggunakan DPP-4 inhibitor, linagliptin

Aag DNA glycosylase promotes alkylation-induced tissue damage mediated by Parp1.

Science.gov (United States)

Calvo, Jennifer A; Moroski-Erkul, Catherine A; Lake, Annabelle; Eichinger, Lindsey W; Shah, Dharini; Jhun, Iny; Limsirichai, Prajit; Bronson, Roderick T; Christiani, David C; Meira, Lisiane B; Samson, Leona D

2013-04-01

Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic β-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag⁻/⁻ mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage.

Aag DNA glycosylase promotes alkylation-induced tissue damage mediated by Parp1.

Directory of Open Access Journals (Sweden)

Jennifer A Calvo

2013-04-01

Full Text Available Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic β-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag⁻/⁻ mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage.

The role of hnRPUL1 involved in DNA damage response is related to PARP1.

Directory of Open Access Journals (Sweden)

Zehui Hong

Full Text Available Heterogeneous nuclear ribonucleoprotein U-like 1 (hnRPUL1 -also known as adenovirus early region 1B-associated proteins 5 (E1B-AP5 - plays a role in RNA metabolism. Recently, hnRPUL1 has also been shown to be involved in DNA damage response, but the function of hnRPUL1 in response to DNA damage remains unclear. Here, we have demonstrated that hnRPUL1 is associated with PARP1 and recruited to DNA double-strand breaks (DSBs sites in a PARP1-mediated poly (ADP-ribosyl ation dependent manner. In turn, hnRPUL1 knockdown enhances the recruitment of PARP1 to DSBs sites. Specifically, we showed that hnRPUL1 is also implicated in the transcriptional regulation of PARP1 gene. Thus, we propose hnRPUL1 as a new component related to PARP1 in DNA damage response and repair.

PARP-1 and YY1 are important novel regulators of CXCL12 gene transcription in rat pancreatic beta cells.

Directory of Open Access Journals (Sweden)

Jelena Marković

Full Text Available Despite significant progress, the molecular mechanisms responsible for pancreatic beta cell depletion and development of diabetes remain poorly defined. At present, there is no preventive measure against diabetes. The positive impact of CXCL12 expression on the pancreatic beta cell prosurvival phenotype initiated this study. Our aim was to provide novel insight into the regulation of rat CXCL12 gene (Cxcl12 transcription. The roles of poly(ADP-ribose polymerase-1 (PARP-1 and transcription factor Yin Yang 1 (YY1 in Cxcl12 transcription were studied by examining their in vitro and in vivo binding affinities for the Cxcl12 promoter in a pancreatic beta cell line by the electrophoretic mobility shift assay and chromatin immunoprecipitation. The regulatory activities of PARP-1 and YY1 were assessed in transfection experiments using a reporter vector with a Cxcl12 promoter sequence driving luciferase gene expression. Experimental evidence for PARP-1 and YY1 revealed their trans-acting potential, wherein PARP-1 displayed an inhibitory, and YY1 a strong activating effect on Cxcl12 transcription. Streptozotocin (STZ-induced general toxicity in pancreatic beta cells was followed by changes in Cxcl12 promoter regulation. PARP-1 binding to the Cxcl12 promoter during basal and in STZ-compromised conditions led us to conclude that PARP-1 regulates constitutive Cxcl12 expression. During the early stage of oxidative stress, YY1 exhibited less affinity toward the Cxcl12 promoter while PARP-1 displayed strong binding. These interactions were accompanied by Cxcl12 downregulation. In the later stages of oxidative stress and intensive pancreatic beta cell injury, YY1 was highly expressed and firmly bound to Cxcl12 promoter in contrast to PARP-1. These interactions resulted in higher Cxcl12 expression. The observed ability of PARP-1 to downregulate, and of YY1 to upregulate Cxcl12 promoter activity anticipates corresponding effects in the natural context where the

Assessment of SLX4 Mutations in Hereditary Breast Cancers.

Directory of Open Access Journals (Sweden)

Sohela Shah

Full Text Available SLX4 encodes a DNA repair protein that regulates three structure-specific endonucleases and is necessary for resistance to DNA crosslinking agents, topoisomerase I and poly (ADP-ribose polymerase (PARP inhibitors. Recent studies have reported mutations in SLX4 in a new subtype of Fanconi anemia (FA, FA-P. Monoallelic defects in several FA genes are known to confer susceptibility to breast and ovarian cancers.To determine if SLX4 is involved in breast cancer susceptibility, we sequenced the entire SLX4 coding region in 738 (270 Jewish and 468 non-Jewish breast cancer patients with 2 or more family members affected by breast cancer and no known BRCA1 or BRCA2 mutations. We found a novel nonsense (c.2469G>A, p.W823* mutation in one patient. In addition, we also found 51 missense variants [13 novel, 23 rare (MAF1%], of which 22 (5 novel and 17 rare were predicted to be damaging by Polyphen2 (score = 0.65-1. We performed functional complementation studies using p.W823* and 5 SLX4 variants (4 novel and 1 rare cDNAs in a human SLX4-null fibroblast cell line, RA3331. While wild type SLX4 and all the other variants fully rescued the sensitivity to mitomycin C (MMC, campthothecin (CPT, and PARP inhibitor (Olaparib the p.W823* SLX4 mutant failed to do so.Loss-of-function mutations in SLX4 may contribute to the development of breast cancer in very rare cases.

Melatonin regulates PARP1 to control the senescence-associated secretory phenotype (SASP) in human fetal lung fibroblast cells.

Science.gov (United States)

Yu, Songtao; Wang, Xiaojiao; Geng, Peiliang; Tang, Xudong; Xiang, Lisha; Lu, Xin; Li, Jianjun; Ruan, Zhihua; Chen, Jianfang; Xie, Ganfeng; Wang, Zhe; Ou, Juanjuan; Peng, Yuan; Luo, Xi; Zhang, Xuan; Dong, Yan; Pang, Xueli; Miao, Hongming; Chen, Hongshan; Liang, Houjie

2017-08-01

Cellular senescence is an important tumor-suppressive mechanism. However, acquisition of a senescence-associated secretory phenotype (SASP) in senescent cells has deleterious effects on the tissue microenvironment and, paradoxically, promotes tumor progression. In a drug screen, we identified melatonin as a novel SASP suppressor in human cells. Strikingly, melatonin blunts global SASP gene expression upon oncogene-induced senescence (OIS). Moreover, poly(ADP-ribose) polymerase-1 (PARP-1), a sensor of DNA damage, was identified as a new melatonin-dependent regulator of SASP gene induction upon OIS. Here, we report two different but potentially coherent epigenetic strategies for melatonin regulation of SASP. The interaction between the telomeric repeat-containing RNA (TERRA) and PARP-1 stimulates the SASP, which was attenuated by 67.9% (illustrated by the case of IL8) by treatment with melatonin. Through binding to macroH2A1.1, PARP-1 recruits CREB-binding protein (CBP) to mediate acetylation of H2BK120, which positively regulates the expression of target SASP genes, and this process is interrupted by melatonin. Consequently, the findings provide novel insight into melatonin's epigenetic role via modulating PARP-1 in suppression of SASP gene expression in OIS-induced senescent cells. Our studies identify melatonin as a novel anti-SASP molecule, define PARP-1 as a new target by which melatonin regulates SASP, and establish a new epigenetic paradigm for a pharmacological mechanism by which melatonin interrupts PARP-1 interaction with the telomeric long noncoding RNA(lncRNA) or chromatin. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The Nonglycemic Actions of Dipeptidyl Peptidase-4 Inhibitors

Directory of Open Access Journals (Sweden)

Na-Hyung Kim

2014-01-01

Full Text Available A cell surface serine protease, dipeptidyl peptidase 4 (DPP-4, cleaves dipeptide from peptides containing proline or alanine in the N-terminal penultimate position. Two important incretin hormones, glucagon-like peptide-1 (GLP-1 and glucose-dependent insulinotropic peptide (GIP, enhance meal-stimulated insulin secretion from pancreatic β-cells, but are inactivated by DPP-4. Diabetes and hyperglycemia increase the DPP-4 protein level and enzymatic activity in blood and tissues. In addition, multiple other functions of DPP-4 suggest that DPP-4 inhibitor, a new class of antidiabetic agents, may have pleiotropic effects. Studies have shown that DPP-4 itself is involved in the inflammatory signaling pathway, the stimulation of vascular smooth cell proliferation, and the stimulation of oxidative stress in various cells. DPP-4 inhibitor ameliorates these pathophysiologic processes and has been shown to have cardiovascular protective effects in both in vitro and in vivo experiments. However, in recent randomized clinical trials, DPP-4 inhibitor therapy in high risk patients with type 2 diabetes did not show cardiovascular protective effects. Some concerns on the actions of DPP-4 inhibitor include sympathetic activation and neuropeptide Y-mediated vascular responses. Further studies are required to fully characterize the cardiovascular effects of DPP-4 inhibitor.

Cycling Towards Progress: Ribociclib, CDK 4/6 inhibitor for Breast Cancer.

Science.gov (United States)

Spring, Laura; Bardia, Aditya

2018-04-23

Ribociclib is an orally active, highly selective inhibitor of cyclin-dependent kinase (CDK) 4 and 6. It is the second CDK 4/6 inhibitor approved for hormone receptor-positive breast cancer. The addition of ribociclib to an aromatase inhibitor has resulted in marked improvements in progression-free survival for patients with metastatic breast cancer. Copyright ©2018, American Association for Cancer Research.

Comparison of newly developed anti-bone morphogenetic protein 4 llama-derived antibodies with commercially available BMP4 inhibitors.

Science.gov (United States)

Calpe, Silvia; Correia, Ana C P; Sancho-Serra, Maria Del Carmen; Krishnadath, Kausilia K

2016-01-01

Due to improved understanding of the role of bone morphogenetic protein 4 (BMP4) in an increasing number of diseases, the development of selective inhibitors of BMP4 is an attractive therapeutic option. The currently available BMP4 inhibitors are not suitable as therapeutics because of their low specificity and low effectiveness. Here, we compared newly generated anti-BMP4 llama-derived antibodies (VHHs) with 3 different types of commercially available BMP4 inhibitors, natural antagonists, small molecule BMPR inhibitors and conventional anti-BMP4 monoclonal antibodies. We found that the anti-BMP4 VHHs were as effective as the natural antagonist or small molecule inhibitors, but had higher specificity. We also showed that commercial anti-BMP4 antibodies were inferior in terms of both specificity and effectiveness. These findings might result from the fact that the VHHs C4C4 and C8C8 target a small region within the BMPR1 epitope of BMP4, whereas the commercial antibodies target other areas of the BMP4 molecule. Our results show that the newly developed anti-BMP4 VHHs are promising antibodies with better specificity and effectivity for inhibition of BMP4, making them an attractive tool for research and for therapeutic applications.

A comparison of effects of DPP-4 inhibitor and SGLT2 inhibitor on lipid profile in patients with type 2 diabetes.

Science.gov (United States)

Cha, Seon-Ah; Park, Yong-Moon; Yun, Jae-Seung; Lim, Tae-Seok; Song, Ki-Ho; Yoo, Ki-Dong; Ahn, Yu-Bae; Ko, Seung-Hyun

2017-04-13

Previous studies suggest that dipeptidyl peptidase-4 (DPP-4) inhibitors and sodium glucose cotransporter 2 (SGLT2) inhibitors have different effects on the lipid profile in patients with type 2 diabetes. We investigated the effects of DPP-4 inhibitors and SGLT2 inhibitors on the lipid profile in patients with type 2 diabetes. From January 2013 to December 2015, a total of 228 patients with type 2 diabetes who were receiving a DPP-4 inhibitor or SGLT2 inhibitor as add-on therapy to metformin and/or a sulfonylurea were consecutively enrolled. We compared the effects of DPP-4 inhibitors and SGLT2 inhibitors on the lipid profile at baseline and after 24 weeks of treatment. To compare lipid parameters between the two groups, we used the analysis of covariance (ANCOVA). A total of 184 patients completed follow-up (mean age: 53.1 ± 6.9 years, mean duration of diabetes: 7.1 ± 5.7 years). From baseline to 24 weeks, HDL-cholesterol (HDL-C) levels were increased by 0.5 (95% CI, -0.9 to 2.0) mg/dl with a DPP-4 inhibitor and by 5.1 (95% CI, 3.0 to 7.1) mg/dl with an SGLT2 inhibitor (p = 0.001). LDL-cholesterol (LDL-C) levels were reduced by 8.4 (95% CI, -14.0 to -2.8) mg/dl with a DPP-4 inhibitor, but increased by 1.3 (95% CI, -5.1 to 7.6) mg/dl with an SGLT2 inhibitor (p = 0.046). There was no significant difference in the mean hemoglobin A1c (8.3 ± 1.1 vs. 8.0 ± 0.9%, p = 0.110) and in the change of total cholesterol (TC) (p = 0.836), triglyceride (TG) (p = 0.867), apolipoprotein A (p = 0.726), apolipoprotein B (p = 0.660), and lipoprotein (a) (p = 0.991) between the DPP-4 inhibitor and the SGLT2 inhibitor. The SGLT2 inhibitor was associated with a significant increase in HDL-C and LDL-C after 24 weeks of SGLT2 inhibitor treatment in patients with type 2 diabetes compared with those with DPP-4 inhibitor treatment in this study. This study was conducted by retrospective medical record review.

Phosphodiesterase 4 inhibitors for chronic obstructive pulmonary disease.

Science.gov (United States)

Chong, Jimmy; Leung, Bonnie; Poole, Phillippa

2017-09-19

Chronic obstructive pulmonary disease (COPD) is associated with cough, sputum production or dyspnoea and a reduction in lung function, quality of life and life expectancy. Apart from smoking cessation, there are no other treatments that slow lung function decline. Roflumilast and cilomilast are oral phosphodiesterase 4 (PDE 4 ) inhibitors proposed to reduce the airway inflammation and bronchoconstriction seen in COPD. This is an update of a Cochrane review first published in 2011 and updated in 2013. To evaluate the efficacy and safety of oral PDE 4 inhibitors in the management of stable COPD. We identified randomised controlled trials (RCTs) from the Cochrane Airways Trials Register (date of last search October 2016). We found other trials from web-based clinical trials registers. We included RCTs if they compared oral PDE 4 inhibitors with placebo in people with COPD. We allowed co-administration of standard COPD therapy. One review author extracted data and a second review author checked the data. We reported pooled data in Review Manager as mean differences (MD), standardised mean differences (SMD) or odds ratios (OR). We converted the odds ratios into absolute treatment effects in a 'Summary of findings' table. Thirty-four separate RCTs studying roflumilast (20 trials with 17,627 participants) or cilomilast (14 trials with 6457 participants) met the inclusion criteria, with a duration of between six weeks and one year. These included people across international study centres with moderate to very severe COPD (Global Initiative for Chronic Obstructive Lung Disease (GOLD) grades II-IV), with a mean age of 64 years.We considered that the methodological quality of the 34 published and unpublished trials was acceptable overall. Treatment with a PDE 4 inhibitor was associated with a significant improvement in forced expiratory volume in one second (FEV 1 ) over the trial period compared with placebo (MD 51.53 mL, 95% confidence interval (CI) 43.17 to 59.90, 27

Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS

Directory of Open Access Journals (Sweden)

Valovka Taras

2008-07-01

Full Text Available Abstract Background The enzymes responsible for the synthesis of poly-ADP-ribose are named poly-ADP-ribose polymerases (PARP. PARP-2 is a nuclear protein, which regulates a variety of cellular functions that are mainly controlled by protein-protein interactions. A previously described non-conventional bipartite nuclear localization sequence (NLS lies in the amino-terminal DNA binding domain of PARP-2 between amino acids 1–69; however, this targeting sequence has not been experimentally examined or validated. Results Using a site-directed mutagenesis approach, we found that lysines 19 and 20, located within a previously described bipartite NLS, are not required for nuclear localization of PARP-2. In contrast, lysine 36, which is located within a predicted classical monopartite NLS, was required for PARP-2 nuclear localization. While wild type PARP-2 interacted with importin α3 and to a very weak extent with importin α1 and importin α5, the mutant PARP-2 (K36R did not interact with importin α3, providing a molecular explanation why PARP-2 (K36R is not targeted to the nucleus. Conclusion Our results provide strong evidence that lysine 36 of PARP-2 is a critical residue for proper nuclear targeting of PARP-2 and consequently for the execution of its biological functions.

Renal Effects of DPP-4 Inhibitors: A Focus on Microalbuminuria

Directory of Open Access Journals (Sweden)

Martin Haluzík

2013-01-01

Full Text Available Incretin-based therapies represent one of the most promising options in type 2 diabetes treatment owing to their good effectiveness with low risk of hypoglycemia and no weight gain. Other numerous potential beneficial effects of incretin-based therapies have been suggested based mostly on experimental and small clinical studies including its beta-cell- and vasculo-protective actions. One of the recently emerged interesting features of dipeptidyl peptidase-4 (DPP-4 inhibitors is its possible protective effect on the diabetic kidney disease. Here, we review the renal effects of DPP-4 inhibitors with special focus on its influence on the onset and progression of microalbuminuria, as presence of microalbuminuria represents an important early sign of kidney damage and is also associated with increased risk of hypoglycemia and cardiovascular complications. Mechanisms underlying possible nephroprotective properties of DPP-4 inhibitors include reduction of oxidative stress and inflammation and improvement of endothelial dysfunction. Effects of DPP-4 inhibitors may be both glucagon-like peptide-1 (GLP-1 dependent and independent. Ongoing prospective studies focused on the nephroprotective effects of DPP-4 inhibitors will further clarify its possible role in the prevention/attenuation of diabetic kidney disease beyond its glucose lowering properties.

Wound healing effects of dipeptidyl peptidase-4 inhibitors: An emerging concept in management of diabetic foot ulcer-A review.

Science.gov (United States)

Saboo, Apoorva; Rathnayake, Ayeshmanthe; Vangaveti, Venkat N; Malabu, Usman H

2016-01-01

Dipeptidyl peptidase-4 (DPP-4) inhibitors have a well-known effect on glycaemic control in patients with diabetes but little is known on their wound healing role in this group of population. This paper reviews the effects of DPP-4 inhibitors on wound healing of diabetic foot ulcers. Published data on effects and mechanism of DDP-4 inhibitors on wound healing were derived from Medline, PubMed and Google Scholar search of English language literature from 1994 to 2014 using the key words such as "DPP-4 inhibitors", "endothelial healing" "diabetes" and "chronic ulcers". DPP-4 inhibitors show a potential benefit in processes of wound healing in diabetic chronic foot ulcers. The enzyme inhibitors promote recruitment of endothelial progenitor cells and allow the final scaffolding of wounds. Furthermore DPP-4 inhibitors augment angiogenesis and have widespread effects on optimising the immune response to persistent hypoxia in chronic diabetes wounds. DPP-4 inhibitors show promise in the local wound healing of diabetic foot ulcers in addition to its already established glycaemic control. In the light of high rate of amputations due to non-healing ulcers with profound psychological and economical liability, more investigations on the usefulness of DPP-4 inhibitors in the high risk diabetes population are needed. Copyright © 2015 Diabetes India. Published by Elsevier Ltd. All rights reserved.

[Gene Expression Profile of Apoptosis in Leukemia Cells Induced by Hsp90 Selective inhibitor 17-AAG].

Science.gov (United States)

Wang, Na-Na; Li, Zhi-Heng; Tao, Yan-Fang; Xu, Li-Xiao; Pan, Jian; Hu, Shao-Yan

2016-06-01

To investigate the apoptotic effects of Hsp90 selective inhibitor 17-AAG on human leukemia HL-60 and NB4 cells and analyse its possible mechanism. CCK-8 assay was used to quantify the growth inhibition of cells after exposure to 17-AAG for 24 hours. Flow cytometrve with annexin V/propidium iodide staining was used to detect apoptosis of leukemia cells. Then Western blot was used to detect the activation of apoptosis related protein caspase-3 and PARP level. Gene expression profile of NB4 cells treated with 17-AAG was analyzed with real-time PCR arrays. The inhibition of leukemia cell proliferation displayed a dose-dependent manner. Annexin V assay, cell cycle analysis and activation of PARP demonstrate that 17-AAG induced apoptosis leukemia cells. Real-time PCR array analysis showed that expression of 56 genes significantly up-regulated and expression of 23 genes were significantly down-regulated after 17-AAG treatment. The 17-AAG can inhibit the proliferation and induce the apoptosis of leukemia cells. After leukemia cells are treated with 17-AAG, the significant changes of apoptosis-related genes occured, and the cell apoptosis occurs via activating apoptosis related signaling pathway.

Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity, Leading to the Under Replication of DNA.

Directory of Open Access Journals (Sweden)

Simon Gemble

2015-07-01

Full Text Available Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at "difficult-to-replicate" sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS, a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3'-5' DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.

The Prognostic Value of BRCA1 and PARP Expression in Epithelial Ovarian Carcinoma

DEFF Research Database (Denmark)

Hjortkjær, Mette; Waldstrøm, Marianne; Jakobsen, Anders

2017-01-01

BRCA1/2 mutation status in epithelial ovarian cancer (EOC) presently relies on genetic testing which is resource consuming. Immunohistochemistry is cheap, fairly reproducible, and may identify gene product alterations due to both germline and somatic mutations and other defects along the BRCA gene...... tissue from 170 patients with EOC was stained immunohistochemically with BRCA1 and PARP antibodies. Semiquantitative analyses were performed to determine loss of, equivocal, and retained BRCA1 and high versus low PARP protein expression. These parameters were analyzed for relation with patient...

Poly (ADP-ribose polymerase plays an important role in intermittent hypoxia-induced cell death in rat cerebellar granule cells

Directory of Open Access Journals (Sweden)

Chiu Sheng-Chun

2012-03-01

Full Text Available Abstract Background Episodic cessation of airflow during sleep in patients with sleep apnea syndrome results in intermittent hypoxia (IH. Our aim was to investigate the effects of IH on cerebellar granule cells and to identify the mechanism of IH-induced cell death. Methods Cerebellar granule cells were freshly prepared from neonatal Sprague-Dawley rats. IH was created by culturing the cerebellar granule cells in the incubators with oscillating O2 concentration at 20% and 5% every 30 min for 1-4 days. The results of this study are based on image analysis using a confocal microscope and associated software. Cellular oxidative stress increased with increase in IH. In addition, the occurrence of cell death (apoptosis and necrosis increased as the duration of IH increased, but decreased in the presence of an iron chelator (phenanthroline or poly (ADP-ribose polymerase (PARP inhibitors [3-aminobenzamide (3-AB and DPQ]. The fluorescence of caspase-3 remained the same regardless of the duration of IH, and Western blots did not detect activation of caspase-3. However, IH increased the ratio of apoptosis-inducing factor (AIF translocation to the nucleus, while PARP inhibitors (3-AB reduced this ratio. Results According to our findings, IH increased oxidative stress and subsequently leading to cell death. This effect was at least partially mediated by PARP activation, resulting in ATP depletion, calpain activation leading to AIF translocation to the nucleus. Conclusions We suggest that IH induces cell death in rat primary cerebellar granule cells by stimulating oxidative stress PARP-mediated calpain and AIF activation.

A Systematic Analysis of Factors Localized to Damaged Chromatin Reveals PARP-Dependent Recruitment of Transcription Factors.

Science.gov (United States)

Izhar, Lior; Adamson, Britt; Ciccia, Alberto; Lewis, Jedd; Pontano-Vaites, Laura; Leng, Yumei; Liang, Anthony C; Westbrook, Thomas F; Harper, J Wade; Elledge, Stephen J

2015-06-09

Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the amyotrophic lateral sclerosis (ALS) candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a poly-(ADP-ribose) polymerase (PARP)-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors; > 70% of randomly tested transcription factors localized to sites of DNA damage, and of these, ∼90% were PARP dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding-domain dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

A Systematic Analysis of Factors Localized to Damaged Chromatin Reveals PARP-Dependent Recruitment of Transcription Factors

Directory of Open Access Journals (Sweden)

Lior Izhar

2015-06-01

Full Text Available Localization to sites of DNA damage is a hallmark of DNA damage response (DDR proteins. To identify DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the amyotrophic lateral sclerosis (ALS candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a poly-(ADP-ribose polymerase (PARP-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors; > 70% of randomly tested transcription factors localized to sites of DNA damage, and of these, ∼90% were PARP dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding-domain dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins.

Defective mitophagy in XPA via PARP-1 hyperactivation and NAD(+)/SIRT1 reduction

DEFF Research Database (Denmark)

Fang, Evandro Fei; Scheibye-Knudsen, Morten; Brace, Lear E

2014-01-01

with excessive cleavage of PINK1 and increased mitochondrial membrane potential. The mitochondrial abnormalities appear to be caused by decreased activation of the NAD(+)-SIRT1-PGC-1α axis triggered by hyperactivation of the DNA damage sensor PARP-1. This phenotype is rescued by PARP-1 inhibition...... or by supplementation with NAD(+) precursors that also rescue the lifespan defect in xpa-1 nematodes. Importantly, this pathogenesis appears common to ataxia-telangiectasia and Cockayne syndrome, two other DNA repair disorders with neurodegeneration, but absent in XPC, a DNA repair disorder without neurodegeneration...

Poly ADP-ribose polymerase-1 as a potential therapeutic target in Merkel cell carcinoma.

Science.gov (United States)

Ferrarotto, Renata; Cardnell, Robert; Su, Shirley; Diao, Lixia; Eterovic, A Karina; Prieto, Victor; Morrisson, William H; Wang, Jing; Kies, Merrill S; Glisson, Bonnie S; Byers, Lauren Averett; Bell, Diana

2018-03-23

Patients with metastatic Merkel cell carcinoma are treated similarly to small cell lung cancer (SCLC). Poly ADP-ribose polymerase-1 (PARP1) is overexpressed in SCLC and response to PARP inhibitors have been reported in patients with SCLC. Our study explores PARP as a therapeutic target in Merkel cell carcinoma. We evaluated PARP1 expression and Merkel cell polyomavirus (MCPyV) in 19 patients with Merkel cell carcinoma. Target exome-sequencing was performed in 14 samples. Sensitivity to olaparib was tested in 4 Merkel cell carcinoma cell lines. Most Merkel cell carcinomas (74%) express PARP1 at high levels. Mutations in DNA-damage repair genes were identified in 9 samples (64%), occurred exclusively in head neck primaries, and correlated with TP53/RB1 mutations. The TP53/RB1 mutations were more frequent in MCPyV-negative tumors. Sensitivity to olaparib was seen in the Merkel cell carcinoma line with highest PARP1 expression. Based on PARP1 overexpression, DNA-damage repair gene mutations, platinum sensitivity, and activity of olaparib in a Merkel cell carcinoma line, clinical trials with PARP inhibitors are warranted in Merkel cell carcinoma. © 2018 Wiley Periodicals, Inc.

4-Hydroxyphenylpyruvate Dioxygenase Inhibitors: From Chemical Biology to Agrochemicals.

Science.gov (United States)

Ndikuryayo, Ferdinand; Moosavi, Behrooz; Yang, Wen-Chao; Yang, Guang-Fu

2017-10-04

The development of new herbicides is receiving considerable attention to control weed biotypes resistant to current herbicides. Consequently, new enzymes are always desired as targets for herbicide discovery. 4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) is an enzyme engaged in photosynthetic activity and catalyzes the transformation of 4-hydroxyphenylpyruvic acid (HPPA) into homogentisic acid (HGA). HPPD inhibitors constitute a promising area of discovery and development of innovative herbicides with some advantages, including excellent crop selectivity, low application rates, and broad-spectrum weed control. HPPD inhibitors have been investigated for agrochemical interests, and some of them have already been commercialized as herbicides. In this review, we mainly focus on the chemical biology of HPPD, discovery of new potential inhibitors, and strategies for engineering transgenic crops resistant to current HPPD-inhibiting herbicides. The conclusion raises some relevant gaps for future research directions.

Alkylation induced cerebellar degeneration dependent on Aag and Parp1 does not occur via previously established cell death mechanisms.

Directory of Open Access Journals (Sweden)

Carrie M Margulies

Full Text Available Alkylating agents are ubiquitous in our internal and external environments, causing DNA damage that contributes to mutations and cell death that can result in aging, tissue degeneration and cancer. Repair of methylated DNA bases occurs primarily through the base excision repair (BER pathway, a multi-enzyme pathway initiated by the alkyladenine DNA glycosylase (Aag, also known as Mpg. Previous work demonstrated that mice treated with the alkylating agent methyl methanesulfonate (MMS undergo cerebellar degeneration in an Aag-dependent manner, whereby increased BER initiation by Aag causes increased tissue damage that is dependent on activation of poly (ADP-ribose polymerase 1 (Parp1. Here, we dissect the molecular mechanism of cerebellar granule neuron (CGN sensitivity to MMS using primary ex vivo neuronal cultures. We first established a high-throughput fluorescent imaging method to assess primary neuron sensitivity to treatment with DNA damaging agents. Next, we verified that the alkylation sensitivity of CGNs is an intrinsic phenotype that accurately recapitulates the in vivo dependency of alkylation-induced CGN cell death on Aag and Parp1 activity. Finally, we show that MMS-induced CGN toxicity is independent of all the cellular events that have previously been associated with Parp-mediated toxicity, including mitochondrial depolarization, AIF translocation, calcium fluxes, and NAD+ consumption. We therefore believe that further investigation is needed to adequately describe all varieties of Parp-mediated cell death.

Alkylation induced cerebellar degeneration dependent on Aag and Parp1 does not occur via previously established cell death mechanisms.

Science.gov (United States)

Margulies, Carrie M; Chaim, Isaac Alexander; Mazumder, Aprotim; Criscione, June; Samson, Leona D

2017-01-01

Alkylating agents are ubiquitous in our internal and external environments, causing DNA damage that contributes to mutations and cell death that can result in aging, tissue degeneration and cancer. Repair of methylated DNA bases occurs primarily through the base excision repair (BER) pathway, a multi-enzyme pathway initiated by the alkyladenine DNA glycosylase (Aag, also known as Mpg). Previous work demonstrated that mice treated with the alkylating agent methyl methanesulfonate (MMS) undergo cerebellar degeneration in an Aag-dependent manner, whereby increased BER initiation by Aag causes increased tissue damage that is dependent on activation of poly (ADP-ribose) polymerase 1 (Parp1). Here, we dissect the molecular mechanism of cerebellar granule neuron (CGN) sensitivity to MMS using primary ex vivo neuronal cultures. We first established a high-throughput fluorescent imaging method to assess primary neuron sensitivity to treatment with DNA damaging agents. Next, we verified that the alkylation sensitivity of CGNs is an intrinsic phenotype that accurately recapitulates the in vivo dependency of alkylation-induced CGN cell death on Aag and Parp1 activity. Finally, we show that MMS-induced CGN toxicity is independent of all the cellular events that have previously been associated with Parp-mediated toxicity, including mitochondrial depolarization, AIF translocation, calcium fluxes, and NAD+ consumption. We therefore believe that further investigation is needed to adequately describe all varieties of Parp-mediated cell death.

Combining the ABL1 kinase inhibitor ponatinib and the histone deacetylase inhibitor vorinostat: a potential treatment for BCR-ABL-positive leukemia.

Science.gov (United States)

Okabe, Seiichi; Tauchi, Tetsuzo; Kimura, Shinya; Maekawa, Taira; Kitahara, Toshihiko; Tanaka, Yoko; Ohyashiki, Kazuma

2014-01-01

Resistance to imatinib (Gleevec®) in cancer cells is frequently because of acquired point mutations in the kinase domain of BCR-ABL. Ponatinib, also known as AP24534, is an oral multi-targeted tyrosine kinase inhibitor (TKI), and it has been investigated in a pivotal phase 2 clinical trial. The histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid) has been evaluated for its significant clinical activity in hematological malignancies. Thus, treatments combining ABL TKIs with additional drugs may be a promising strategy in the treatment of leukemia. In the current study, we analyzed the efficacy of ponatinib and vorinostat treatment by using BCR-ABL-positive cell lines. Treatment with ponatinib for 72 h inhibited cell growth and induced apoptosis in K562 cells in a dose-dependent manner. We found that ponatinib potently inhibited the growth of Ba/F3 cells ectopically expressing BCR-ABL T315I mutation. Upon BCR-ABL phosphorylation, Crk-L was decreased, and poly (ADP-ribose) polymerase (PARP) was activated in a dose-dependent manner. Combined treatment of Ba/F3 T315I mutant cells with vorinostat and ponatinib resulted in significantly increased cytotoxicity. Additionally, the intracellular signaling of ponatinib and vorinostat was examined. Caspase 3 and PARP activation increased after combination treatment with ponatinib and vorinostat. Moreover, an increase in the phosphorylation levels of γH2A.X was observed. Previously established ponatinib-resistant Ba/F3 cells were also resistant to imatinib, nilotinib, and dasatinib. We investigated the difference in the efficacy of ponatinib and vorinostat by using ponatinib-resistant Ba/F3 cells. Combined treatment of ponatinib-resistant cells with ponatinib and vorinostat caused a significant increase in cytotoxicity. Thus, combined administration of ponatinib and vorinostat may be a powerful strategy against BCR-ABL mutant cells and could enhance the cytotoxic effects of ponatinib in those BCR

PARP-1 Variant Rs1136410 Confers Protection against Coronary Artery Disease in a Chinese Han Population: A Two-Stage Case-Control Study Involving 5643 Subjects

Directory of Open Access Journals (Sweden)

Xue-bin Wang

2017-11-01

Full Text Available Inhibition of poly(ADP-ribose polymerase (PARP may protect against coronary artery disease (CAD in animal models, and rs1136410, a non-synonymous single nucleotide polymorphism (SNP in PARP-1, has a potential impact on PARP activities in vitro. This two-stage case-control study, involving 2803 CAD patients and 2840 controls, aimed to investigate the associations of PARP-1 rs1136410 with CAD development, lipid levels, PARP activities, 8-hydroxy-2′-dexyguanosine (8-OHdG, and interleukin (IL-6 levels in a Chinese Han population. Assuming a recessive model, the variant genotype GG of SNP rs1136410 showed a significantly inverse association with CAD risk (adjusted odds ratio (OR = 0.73, P < 0.001, left main coronary artery (LMCA lesions (P = 0.003, vessel scores (P = 0.003, and modified Gensini scores (P < 0.001. There were significant correlations of SNP rs1136410 with higher levels of total cholesterol (TC and lower levels of high-density lipoprotein cholesterol (HDL-c. In gene-environment interaction analyses, participants with the variant genotype GG, but without smoking habit, type 2 diabetes mellitus, and hyperlipidemia, conferred an 84% (P < 0.001 decreased risk of CAD. The genotype-phenotype correlation analyses further supported the functional roles of SNP rs1136410 in decreasing PARP activities and 8-OHdG levels. Taken together, our data suggest that SNP rs1136410 may confer protection against CAD through modulation of PARP activities and gene-environment interactions in a Chinese Han population.

The neuroprotective effect of nicotine in Parkinson’s disease models is associated with inhibiting PARP-1 and caspase-3 cleavage

Directory of Open Access Journals (Sweden)

Justin Y.D. Lu

2017-10-01

Full Text Available Clinical evidence points to neuroprotective effects of smoking in Parkinson’s disease (PD, but the molecular mechanisms remain unclear. We investigated the pharmacological pathways involved in these neuroprotective effects, which could provide novel ideas for developing targeted neuroprotective treatments for PD. We used the ETC complex I inhibitor methylpyridinium ion (MPP+ to induce cell death in SH-SY5Y cells as a cellular model for PD and found that nicotine inhibits cell death. Using choline as a nicotinic acetylcholine receptor (nAChR agonist, we found that nAChR stimulation was sufficient to protect SH-SY5Y cells against cell death from MPP+. Blocking α7 nAChR with methyllycaconitine (MLA prevented the protective effects of nicotine, demonstrating that these receptors are necessary for the neuroprotective effects of nicotine. The neuroprotective effect of nicotine involves other pathways relevant to PD. Cleaved Poly (ADP-ribose polymerase-1 (PARP-1 and cleaved caspase-3 were decreased by nicotine in 6-hydroxydopamine (6-OHDA lesioned mice and in MPP+-treated SH-SY5Y cells. In conclusion, our data indicate that nicotine likely exerts neuroprotective effects in PD through the α7 nAChR and downstream pathways including PARP-1 and caspase-3. This knowledge could be pursued in future research to develop neuroprotective treatments for PD.

[Research progress of dipeptidyl peptidase 4 inhibitors on healing of chronic diabetic foot ulcers].

Science.gov (United States)

Gao, Yunyi; Liang, Yujie; Ran, Xingwu

2018-05-01

To review the effect of dipeptidyl peptidase 4 (DPP-4) inhibitors on the wound healing and its mechanisms in chronic diabetic foot ulcers. The latest literature concerning DPP-4 inhibitors for chronic diabetic foot ulcers was extensively reviewed, as well as the potential benefit and mechanism of DPP-4 inhibitors on wound healing of diabetic foot ulcers was analyzed thoroughly. DPP-4 inhibitors can accelerated the ulcer healing. The mechanisms probably include inhibiting the expression of the matrix metalloproteinase (MMP) and restoring the balance of the wound MMP and the tissue inhibitors of MMP; promoting recruitment of endothelial progenitor cells and augmenting angiogenesis; optimizing extracellular matrix construction and the immune response to persistent hypoxia in chronic diabetes wounds, and so on. At present, clinical researches show that DPP-4 inhibitors may be considered as an adjuvant treatment for chronic diabetic foot ulcers. DPP-4 inhibitors show promise in the local wound healing of chronic diabetic foot ulcers. However, more strictly designed, adequately powered, long-term follow-up, and high-quality randomized control trials are needed to further verify their efficacy and safety for chronic diabetic foot ulcers.

Comparing the therapeutic efficiency of aminoguanidine and 3-aminobenzamide in lung and intestine toxicity caused by nitrogen mustard in rats

International Nuclear Information System (INIS)

Yaren, H.; Korkmaz, A.; Kunak, Z. I.; Uysal, B.; Topal, T.; Kurt, B; Kenar, L.

2009-01-01

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) and peroxynitrite are responsible for sulfur mustard (SM) induced toxicity. Since endogenous production of peroxynitrite is known to lead to poly(ADP-ribose) polymerase (PARP) activation and sometimes ultimately cell death, in this study, it was aimed to compare the therapeutic efficiencies of aminoguanidine (iNOS inhibitor) and 3 aminobenzamide (PARP inhibitor) in lung and intestine toxicity caused by nitrogen mustard in rats. A total of 40 male Sprague-Dawley rats were divided into 4 groups. Group 1 served as control and given 2 ml saline, three groups received single dose of mechlorethamine (MEC) (3.5 mg/kg subcutaneously) with the same time intervals. Group 2 received MEC only, group 3 received selective iNOS inhibitor aminoguanidine (AG) (100 mg/kg i.p.) and, group 4 received PARP inhibitor 3 aminobenzamide (3-AB) (20 mg/kg i.p.). MEC injection resulted in severe lung toxicity with strong interstitial and alveolar edema, hemorrhage, emphysematous changes, Mild inflammatory cell infiltration and septal thickening. MEC injection also caused mucosal thinning, mild inflammatory cell infiltration, ischemic changes and multifocal, superficial ulcerations (erosions) in small intestine. In AG group, interstitial and alveolar edema, hemorrhage slightly reduced in lung comparing to MEC group. Inflammatory cell infiltration was minimal, septal thickening was similar to MEC group at densely edematous and hemorrhagical areas. In 3 AB group, edematous and hemorrhagic areas were very small, inflammatory cell infiltration was minimal and there were no densly densely edematous and hemorrhagical areas in lung. The results were better than AB group. In intestine, results of AG group were better than MEC group but worse than 3 AB group. These results suggest that both iNOS and PARP inhibitors are effective but PARP inhibitors may be more promising for treatment of SM induced early lung and intestinal toxicity.(author)

Rev1 contributes to proper mitochondrial function via the PARP-NAD(+)-SIRT1-PGC1 alpha axis

DEFF Research Database (Denmark)

Fakouri, Nima Borhan; Durhuus, Jon Ambaek; Regnell, Christine Elisabeth

2017-01-01

(ADP) ribose polymerase 1 (PARP1) activity, low endogenous NAD+, low expression of SIRT1 and PGC1α and low adenosine monophosphate (AMP)-activated kinase (AMPK) activity. We conclude that replication stress via Rev1-deficiency contributes to metabolic stress caused by compromized mitochondrial function via...... the PARP-NAD+-SIRT1-PGC1α axis....

Recurrent hypoinsulinemic hyperglycemia in neonatal rats increases PARP-1 and NF-κB expression and leads to microglial activation in the cerebral cortex.

Science.gov (United States)

Gisslen, Tate; Ennis, Kathleen; Bhandari, Vineet; Rao, Raghavendra

2015-11-01

Hyperglycemia is a common metabolic problem in extremely low-birth-weight preterm infants. Neonatal hyperglycemia is associated with increased mortality and brain injury. Glucose-mediated oxidative injury may be responsible. Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in DNA repair and cell survival. However, PARP-1 overactivation leads to cell death. NF-κB is coactivated with PARP-1 and regulates microglial activation. The effects of recurrent hyperglycemia on PARP-1/NF-κB expression and microglial activation are not well understood. Rat pups were subjected to recurrent hypoinsulinemic hyperglycemia of 2 h duration twice daily from postnatal (P) day 3-P12 and killed on P13. mRNA and protein expression of PARP-1/NF-κB and their downstream effectors were determined in the cerebral cortex. Microgliosis was determined using CD11 immunohistochemistry. Recurrent hyperglycemia increased PARP-1 expression confined to the nucleus and without causing PARP-1 overactivation and cell death. NF-κB mRNA expression was increased, while IκB mRNA expression was decreased. inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) mRNA expressions were decreased. Hyperglycemia significantly increased the number of microglia. Recurrent hyperglycemia in neonatal rats is associated with upregulation of PARP-1 and NF-κB expression and subsequent microgliosis but not neuronal cell death in the cerebral cortex.

The HRD Decision-Which PARP Inhibitor to Use for Whom and When.

Science.gov (United States)

Kohn, Elise C; Lee, Jung-Min; Ivy, S Percy

2017-12-01

Rucaparib, a polyADPribose polymerase inhibitor (PARPi), was approved recently for use in women with high-grade serous ovarian cancer (HGSOC). It is now one of three approved PARPi for use in recurrent ovarian cancer, a family of agents that has changed the HGSOC treatment landscape and outcome. Clin Cancer Res; 23(23); 7155-7. ©2017 AACR See related article by Balasubramaniam et al., p. 7165 . ©2017 American Association for Cancer Research.

RECOVERY OF amiRNA3-PARP1 TRANSGENIC MAIZE PLANTS ...

African Journals Online (AJOL)

ACSS

Positive plant selectable marker genes are commonly used in plant transformation because they not only enhance the frequency of generation transgenic tissues but are considered biosafe, unlike antibiotic or herbicide resistance genes. In this study, the binary vector pNOV2819-ubiamiRNA3PARP1, harbouring the ...

Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization

Energy Technology Data Exchange (ETDEWEB)

La Ferla, Marco; Mercatanti, Alberto; Rocchi, Giulia; Lodovichi, Samuele; Cervelli, Tiziana; Pignata, Luca [Yeast Genetics and Genomics, Institute of Clinical Physiology, National Council of Research (CNR), via Moruzzi 1, 56122 Pisa (Italy); Caligo, Maria Adelaide [Section of Genetic Oncology, University Hospital and University of Pisa, via Roma 57, 56125 Pisa (Italy); Galli, Alvaro, E-mail: alvaro.galli@ifc.cnr.it [Yeast Genetics and Genomics, Institute of Clinical Physiology, National Council of Research (CNR), via Moruzzi 1, 56122 Pisa (Italy)

2015-04-15

Highlights: • The human poly (ADP-ribose) polymerase 1 (PARP-1) gene affects growth and UV-induced homologous recombination in yeast. • PARP-1 chemical inhibition impacts yeast growth and UV-induced recombination. • A genome-wide screen identifies 99 yeast genes that suppress the growth defect inferred by PARP-1. • Bioinformatics analysis identifies 41 human orthologues that may have a role in PARP-1 intracellular localization. • The findings suggest that PARP-1 nuclear localization may affect the response to PARP inhibitors in cancer therapy. - Abstract: The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the

Pan-cancer analysis of genomic scar signatures associated with homologous recombination deficiency suggests novel indications for existing cancer drugs

DEFF Research Database (Denmark)

Marquard, Andrea Marion; Eklund, Aron Charles; Joshi, Tejal

2015-01-01

Ovarian and triple-negative breast cancers with BRCA1 or BRCA2 loss are highly sensitive to treatment with PARP inhibitors and platinum-based cytotoxic agents and show an accumulation of genomic scars in the form of gross DNA copy number aberrations. Cancers without BRCA1 or BRCA2 loss...... but with accumulation of similar genomic scars also show increased sensitivity to platinum-based chemotherapy. Therefore, reliable biomarkers to identify DNA repair-deficient cancers prior to treatment may be useful for directing patients to platinum chemotherapy and possibly PARP inhibitors. Recently, three SNP array...... may be strong candidates for clinical trials with PARP inhibitors or platinum-based chemotherapeutic regimens....

The effects of GLP-1 analogues, DPP-4 inhibitors and SGLT2 inhibitors on the renal system.

Science.gov (United States)

Schernthaner, Guntram; Mogensen, Carl Erik; Schernthaner, Gerit-Holger

2014-09-01

Diabetic nephropathy (DN) affects an estimated 20%-40% of patients with type 2 diabetes mellitus (T2DM). Key modifiable risk factors for DN are albuminuria, anaemia, dyslipidaemia, hyperglycaemia and hypertension, together with lifestyle factors, such as smoking and obesity. Early detection and treatment of these risk factors can prevent DN or slow its progression, and may even induce remission in some patients. DN is generally preceded by albuminuria, which frequently remains elevated despite treatment in patients with T2DM. Optimal treatment and prevention of DN may require an early, intensive, multifactorial approach, tailored to simultaneously target all modifiable risk factors. Regular monitoring of renal function, including urinary albumin excretion, creatinine clearance and glomerular filtration rate, is critical for following any disease progression and making treatment adjustments. Dipeptidyl peptidase (DPP)-4 inhibitors and sodium-glucose cotransporter 2 (SGLT2) inhibitors lower blood glucose levels without additional risk of hypoglycaemia, and may also reduce albuminuria. Further investigation of the potential renal benefits of DPP-4 and SGLT2 inhibitors is underway. © The Author(s) 2014.

Xeroderma Pigmentosum Group A Promotes Autophagy to Facilitate Cisplatin Resistance in Melanoma Cells through the Activation of PARP1.

Science.gov (United States)

Ge, Rui; Liu, Lin; Dai, Wei; Zhang, Weigang; Yang, Yuqi; Wang, Huina; Shi, Qiong; Guo, Sen; Yi, Xiuli; Wang, Gang; Gao, Tianwen; Luan, Qi; Li, Chunying

2016-06-01

Xeroderma pigmentosum group A (XPA), a key protein in the nucleotide excision repair pathway, has been shown to promote the resistance of tumor cells to chemotherapeutic drugs by facilitating the DNA repair process. However, the role of XPA in the resistance of melanoma to platinum-based drugs like cisplatin is largely unknown. In this study, we initially found that XPA was expressed at higher levels in cisplatin-resistant melanoma cells than in cisplatin-sensitive ones. Furthermore, the knockdown of XPA not only increased cellular apoptosis but also inhibited cisplatin-induced autophagy, which rendered the melanoma cells more sensitive to cisplatin. Moreover, we discovered that the increased XPA in resistant melanoma cells promoted poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) activation and that the inhibition of PARP1 could attenuate the cisplatin-induced autophagy. Finally, we proved that the inhibition of PARP1 and the autophagy process made resistant melanoma cells more susceptible to cisplatin treatment. Our study shows that XPA can promote cell-protective autophagy in a DNA repair-independent manner by enhancing the activation of PARP1 in melanoma cells resistant to cisplatin and that the XPA-PARP1-mediated autophagy process can be targeted to overcome cisplatin resistance in melanoma chemotherapy. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Angiotensin converting enzyme inhibitors and Alzheimer disease in the presence of the apolipoprotein E4 allele.

Science.gov (United States)

Qiu, Wendy Wei Qiao; Lai, Angela; Mon, Timothy; Mwamburi, Mkaya; Taylor, Warren; Rosenzweig, James; Kowall, Neil; Stern, Robert; Zhu, Haihao; Steffens, David C

2014-02-01

The effect of angiotensin converting enzyme (ACE) inhibitors on Alzheimer disease (AD) remains unclear, with conflicting results reported. We studied the interaction of the Apolipoprotein E (ApoE) genotype and ACE inhibitors on AD. This was a cross-sectional study of homebound elderly with an AD diagnosis and documentation of medications taken. ApoE genotype was determined. A total of 355 subjects with status on ApoE alleles and cognitive diagnoses were studied. The average age (mean ± SD) of this population was 73.3 ± 8.3 years old, and 73% were female. Cross-sectionally, there was no difference in the number of AD cases between ApoE4 carriers and ApoE4 non-carriers or between ACE inhibitor users and non-users in the homebound elderly. ApoE4 carriers treated with ACE inhibitors, however, had more diagnoses of AD compared with those who did not have the treatment (28% versus 6%, p = 0.01) or ApoE4 non-carriers treated with an ACE inhibitor (28% versus 10%, p = 0.03). ACE inhibitor use was associated with AD diagnosis only in the presence of an E4 allele. Using multivariate logistic regression analysis, we found that in diagnosed AD cases there was a significant interaction between ApoE4 and ACE inhibitor use (odds ratio: 20.85; 95% confidence interval: 3.08-140.95; p = 0.002) after adjusting for age, sex, ethnicity, and education. The effects of ACE inhibitors on AD may be different depending on ApoE genotype. A prospective study is needed to determine whether ACE inhibitor use accelerates or poorly delays AD development in ApoE4 carriers compared with ApoE4 non-carriers. Copyright © 2014. Published by Elsevier Inc.

Alogliptin: a new addition to the class of DPP-4 inhibitors

Directory of Open Access Journals (Sweden)

Radha Andukuri

2009-07-01

Full Text Available Radha Andukuri, Andjela Drincic, Marc RendellDivision of Endocrinology, Department of Medicine, Creighton University School of Medicine, Omaha, Nebraska, USABackground: Alogliptin is an oral antihyperglycemic agent that is a selective inhibitor of the enzyme dipeptidyl peptidase-4 (DPP-4. Inhibition of DPP-4 elevates levels of the incretin hormones glucagon-like peptide (GLP-1 and glucose-dependent insulinotropic polypeptide (GIP by preventing their degradation.Objective: To review the evolution of alogliptin and its pharmacokinetics, pharmacodynamics, clinical efficacy and adverse effects. In addition, we compared alogliptin to other DPP-4 inhibitors.Methods: A comprehensive literature search was performed using the term ‘alogliptin’. Original research articles and review articles as well as scientific abstracts were included. Results: Alogliptin raises postprandial levels of GLP-1. It has excellent bioavailability exhibiting a median Tmax ranging from 1 to 2 hours and a mean half-life of 12.4 to 21.4 hours across all doses. When given as monotherapy, mean hemoglobin A1c (HbA1c reductions achieved were 0.5% to 0.6%. Combination therapy yielded similar reductions (−0.5% with metformin, −0.6% with glyburide, −0.8% with pioglitazone and –0.6% with insulin. Administration of alogliptin does not promote weight loss but has not resulted in weight gain. The agent is relatively well tolerated with few adverse effects, the major finding being a marginally higher rate of skin events, primarily pruritus.Conclusions: Alogliptin causes significant reductions in HbA1c when used alone or in combination with other oral agents in patients with type 2 diabetes similar to other DPP-4 inhibitors in current clinical use. The side effect profile also does not differ from that of other DPP-4 inhibitors. However, long-term studies are necessary before the place of alogliptin in the management of type 2 diabetes can be established.Keywords: alogliptin, DPP

A novel HDAC inhibitor, CG200745, inhibits pancreatic cancer cell growth and overcomes gemcitabine resistance.

Science.gov (United States)

Lee, Hee Seung; Park, Soo Been; Kim, Sun A; Kwon, Sool Ki; Cha, Hyunju; Lee, Do Young; Ro, Seonggu; Cho, Joong Myung; Song, Si Young

2017-01-30

Pancreatic cancer is predominantly lethal, and is primarily treated using gemcitabine, with increasing resistance. Therefore, novel agents that increase tumor sensitivity to gemcitabine are needed. Histone deacetylase (HDAC) inhibitors are emerging therapeutic agents, since HDAC plays an important role in cancer initiation and progression. We evaluated the antitumor effect of a novel HDAC inhibitor, CG200745, combined with gemcitabine/erlotinib on pancreatic cancer cells and gemcitabine-resistant pancreatic cancer cells. Three pancreatic cancer-cell lines were used to evaluate the antitumor effect of CG200745 combined with gemcitabine/erlotinib. CG200745 induced the expression of apoptotic proteins (PARP and caspase-3) and increased the levels of acetylated histone H3. CG200745 with gemcitabine/erlotinib showed significant growth inhibition and synergistic antitumor effects in vitro. In vivo, gemcitabine/erlotinib and CG200745 reduced tumor size up to 50%. CG200745 enhanced the sensitivity of gemcitabine-resistant pancreatic cancer cells to gemcitabine, and decreased the level of ATP-binding cassette-transporter genes, especially multidrug resistance protein 3 (MRP3) and MRP4. The novel HDAC inhibitor, CG200745, with gemcitabine/erlotinib had a synergistic anti-tumor effect on pancreatic cancer cells. CG200745 significantly improved pancreatic cancer sensitivity to gemcitabine, with a prominent antitumor effect on gemcitabine-resistant pancreatic cancer cells. Therefore, improved clinical outcome is expected in the future.

Dipeptidyl peptidase-4 (DPP-4) inhibitors are favourable to glucagon-like peptide-1 (GLP-1) agonists

DEFF Research Database (Denmark)

Madsbad, Sten

2012-01-01

Incretin-based therapies, which include the GLP-1 receptor agonists and DPP-4 inhibitors, use the antidiabetic properties of potentiating the GLP-1 receptor signalling via the regulation of insulin and glucagon secretion, inhibition of gastric emptying and suppression of appetite. Most physicians...... will start antidiabetic treatment with metformin, but adding a GLP-1 receptor agonist as the second drug seems to be optimal since more patients will reach an HbA1c below 7% than with a DPP-4 inhibitor or another oral antidiabetic agents and with minimal risk of hypoglycaemia. The GLP-1 receptor agonists...

Fluorescent Inhibitors as Tools To Characterize Enzymes: Case Study of the Lipid Kinase Phosphatidylinositol 4-Kinase IIIβ (PI4KB).

Science.gov (United States)

Humpolickova, Jana; Mejdrová, Ivana; Matousova, Marika; Nencka, Radim; Boura, Evzen

2017-01-12

The lipid kinase phosphatidylinositol 4-kinase IIIβ (PI4KB) is an essential host factor for many positive-sense single-stranded RNA (+RNA) viruses including human pathogens hepatitis C virus (HCV), Severe acute respiratory syndrome (SARS), coxsackie viruses, and rhinoviruses. Inhibitors of PI4KB are considered to be potential broad-spectrum virostatics, and it is therefore critical to develop a biochemical understanding of the kinase. Here, we present highly potent and selective fluorescent inhibitors that we show to be useful chemical biology tools especially in determination of dissociation constants. Moreover, we show that the coumarin-labeled inhibitor can be used to image PI4KB in cells using fluorescence-lifetime imaging microscopy (FLIM) microscopy.

High-throughput screening identifies Ceefourin 1 and Ceefourin 2 as highly selective inhibitors of multidrug resistance protein 4 (MRP4).

Science.gov (United States)

Cheung, Leanna; Flemming, Claudia L; Watt, Fujiko; Masada, Nanako; Yu, Denise M T; Huynh, Tony; Conseil, Gwenaëlle; Tivnan, Amanda; Polinsky, Alexander; Gudkov, Andrei V; Munoz, Marcia A; Vishvanath, Anasuya; Cooper, Dermot M F; Henderson, Michelle J; Cole, Susan P C; Fletcher, Jamie I; Haber, Michelle; Norris, Murray D

2014-09-01

Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transporter superfamily, is an organic anion transporter capable of effluxing a wide range of physiologically important signalling molecules and drugs. MRP4 has been proposed to contribute to numerous functions in both health and disease; however, in most cases these links remain to be unequivocally established. A major limitation to understanding the physiological and pharmacological roles of MRP4 has been the absence of specific small molecule inhibitors, with the majority of established inhibitors also targeting other ABC transporter family members, or inhibiting the production, function or degradation of important MRP4 substrates. We therefore set out to identify more selective and well tolerated inhibitors of MRP4 that might be used to study the many proposed functions of this transporter. Using high-throughput screening, we identified two chemically distinct small molecules, Ceefourin 1 and Ceefourin 2, that inhibit transport of a broad range of MRP4 substrates, yet are highly selective for MRP4 over other ABC transporters, including P-glycoprotein (P-gp), ABCG2 (Breast Cancer Resistance Protein; BCRP) and MRP1 (multidrug resistance protein 1; ABCC1). Both compounds are more potent MRP4 inhibitors in cellular assays than the most widely used inhibitor, MK-571, requiring lower concentrations to effect a comparable level of inhibition. Furthermore, Ceefourin 1 and Ceefourin 2 have low cellular toxicity, and high microsomal and acid stability. These newly identified inhibitors should be of great value for efforts to better understand the biological roles of MRP4, and may represent classes of compounds with therapeutic application. Copyright © 2014 Elsevier Inc. All rights reserved.

Efficacy and safety of dipeptidyl peptidase-4 inhibitors as an add-on to insulin treatment in patients with Type 2 diabetes

DEFF Research Database (Denmark)

Frandsen, Christian S.; Madsbad, S

2014-01-01

diabetes. METHODS: We searched the MEDLINE and PubMed databases to identify all randomized controlled clinical trials evaluating dipeptidyl peptidase-4 inhibitors as an add-on to insulin in patients with Type 2 diabetes, which were selected for review. The abstracts and posters of the recent annual...... and placebo treatment. CONCLUSION: Adding a dipeptidyl peptidase-4 inhibitor treatment to insulin has a moderate effect on HbA1c , a weight-neutral effect and a good safety profile. The risk of hypoglycaemia is not increased despite a significant improvement in HbA1c ....

Neer Award 2016: reduced muscle degeneration and decreased fatty infiltration after rotator cuff tear in a poly(ADP-ribose) polymerase 1 (PARP-1) knock-out mouse model.

Science.gov (United States)

Kuenzler, Michael B; Nuss, Katja; Karol, Agnieszka; Schär, Michael O; Hottiger, Michael; Raniga, Sumit; Kenkel, David; von Rechenberg, Brigitte; Zumstein, Matthias A

2017-05-01

Disturbed muscular architecture, atrophy, and fatty infiltration remain irreversible in chronic rotator cuff tears even after repair. Poly (adenosine 5'-diphosphate-ribose) polymerase 1 (PARP-1) is a key regulator of inflammation, apoptosis, muscle atrophy, muscle regeneration, and adipocyte development. We hypothesized that the absence of PARP-1 would lead to a reduction in damage to the muscle subsequent to combined tenotomy and neurectomy in a PARP-1 knockout (KO) mouse model. PARP-1 KO and wild-type C57BL/6 (WT group) mice were analyzed at 1, 6, and 12 weeks (total n = 84). In all mice, the supraspinatus and infraspinatus muscles of the left shoulder were detached and denervated. Macroscopic analysis, magnetic resonance imaging, gene expression analysis, immunohistochemistry, and histology were used to assess the differences in PARP-1 KO and WT mice. The muscles in the PARP-1 KO group had significantly less retraction, atrophy, and fatty infiltration after 12 weeks than in the WT group. Gene expression of inflammatory, apoptotic, adipogenic, and muscular atrophy genes was significantly decreased in PARP-1 KO mice in the first 6 weeks. Absence of PARP-1 leads to a reduction in muscular architectural damage, early inflammation, apoptosis, atrophy, and fatty infiltration after combined tenotomy and neurectomy of the rotator cuff muscle. Although the macroscopic reaction to injury is similar in the first 6 weeks, the ability of the muscles to regenerate was much greater in the PARP-1 KO group, leading to a near-normalization of the muscle after 12 weeks. Copyright © 2017 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

DPP4 inhibitors promote biological functions of human endothelial progenitor cells by targeting the SDF-1/CXCR4 signaling pathway

Directory of Open Access Journals (Sweden)

Liu Feng

2016-01-01

Full Text Available Dipeptidyl peptidase 4 (DPP4 inhibitors(oral hypoglycemic agentshave beneficial effects during the early stages of diabetes. In this study, we evaluated the role of DPP4inhibitorsonthe biological functions of cultured human endothelial progenitor cells (EPCs. After treating EPCs with the DPP4 inhibitors sitagliptin and vildagliptin, we examined the mRNA expression of DPP4, vascular endothelial growth factor (VEGF,VEGF receptor 2 (VEGFR-2,endothelial nitric oxide synthase (eNOS, caspase-3,stromal cell-derived factor-1 (SDF-1, chemokine (C-X-C motif receptor 4 (CXCR4 were measured by RT-PCR. The protein expression of SDF-1 and CXCR4 was determined by Western blot; cell proliferation was tested by the MTT method, and DPP4 activity was determined by a DPP4 assay. Our results revealed that DPP4 expression and activity were inhibited following the treatment with various doses of DPP4 inhibitors. Cell proliferation and the expression of VEGF, VEGFR-2andeNOS were up regulated, while cell apoptosis was inhibited by DPP4 inhibitors in a dose-dependent manner. DPP4 inhibitors activated the SDF-1/CXCR4 signaling pathway, shown by the elevated expression of SDF-1/CXCR4. This further proved that after the SDF-1/CXCR4 signaling pathway was blocked by its inhibitor ADM3100, the effects of DPP4 inhibitors on the proliferation and apoptosis, and the expression of VEGF, VEGFR-2and eNOS of EPCs were significantly reduced. These findings suggest that DPP4 inhibitors promote the biological functions of human EPCs by up regulating the SDF-1/CXCR4 signaling pathway.

SVMDLF: A novel R-based Web application for prediction of dipeptidyl peptidase 4 inhibitors.

Science.gov (United States)

Chandra, Sharat; Pandey, Jyotsana; Tamrakar, Akhilesh K; Siddiqi, Mohammad Imran

2017-12-01

Dipeptidyl peptidase 4 (DPP4) is a well-known target for the antidiabetic drugs. However, currently available DPP4 inhibitor screening assays are costly and labor-intensive. It is important to create a robust in silico method to predict the activity of DPP4 inhibitor for the new lead finding. Here, we introduce an R-based Web application SVMDLF (SVM-based DPP4 Lead Finder) to predict the inhibitor of DPP4, based on support vector machine (SVM) model, predictions of which are confirmed by in vitro biological evaluation. The best model generated by MACCS structure fingerprint gave the Matthews correlation coefficient of 0.87 for the test set and 0.883 for the external test set. We screened Maybridge database consisting approximately 53,000 compounds. For further bioactivity assay, six compounds were shortlisted, and of six hits, three compounds showed significant DPP4 inhibitory activities with IC 50 values ranging from 8.01 to 10.73 μm. This application is an OpenCPU server app which is a novel single-page R-based Web application for the DPP4 inhibitor prediction. The SVMDLF is freely available and open to all users at http://svmdlf.net/ocpu/library/dlfsvm/www/ and http://www.cdri.res.in/svmdlf/. © 2017 John Wiley & Sons A/S.

Reduced Airway Hyperresponsiveness by Phosphodiesterase 3 and 4 Inhibitors in Guinea-Pigs

Directory of Open Access Journals (Sweden)

Nöella Germain

1999-01-01

Full Text Available The aim of the present study was to compare the effects of selective phosphodiesterase (PDE 3, 4 and 5 inhibitors on antigen-induced airway hyperresponsiveness in sensitized guinea-pigs. When the sensitized guinea-pigs were orally pre-treated with the selective PDE4 inhibitor, Ro 20-1724 (30 mg/kg, and studied 48 h after OA, a significant reduction (p<0.01 of the leftward shift of the dose-response curve to ACh was noted, whereas it was ineffective at the lower dose (10 mg/kg. Administration of the selective PDE3 inhibitor, milrinone (30 mg/kg also elicited a significant reduction (p<0.01 of the airway hyperresponsiveness, whereas the PDE5 inhibitor zaprinast (30 mg/kg was ineffective. These results show that both PDE3 and PDE4 inhibitors are able to inhibit the antigen-induced airway hyperresponsiveness in sensitized guinea-pigs and support the potential utility of selective PDE inhibitors in the treatment of asthma.

Protective Effects of Chlorella-Derived Peptide Against UVC-Induced Cytotoxicity through Inhibition of Caspase-3 Activity and Reduction of the Expression of Phosphorylated FADD and Cleaved PARP-1 in Skin Fibroblasts

Directory of Open Access Journals (Sweden)

Jong Yuh Cherng

2012-08-01

Full Text Available UVC irradiation induces oxidative stress and leads to cell death through an apoptotic pathway. This apoptosis is caused by activation of caspase-3 and formation of poly(ADP-ribose polymerase-1 (PARP-1. In this study, the underlying mechanisms of Chlorella derived peptide (CDP activity against UVC-induced cytotoxicity were investigated. Human skin fibroblasts were treated with CDP, vitamin C, or vitamin E after UVC irradiation for a total energy of 15 J/cm². After the UVC exposure, cell proliferation and caspase-3 activity were measured at 12, 24, 48, and 72 h later. Expression of phosphorylated FADD and cleaved PARP-1 were measured 16 h later. DNA damage (expressed as pyrimidine (6-4 pyrimidone photoproducts DNA concentration and fragmentation assay were performed 24 h after the UVC exposure. Results showed that UVC irradiation induced cytotoxicity in all groups except those treated with CDP. The caspase-3 activity in CDP-treated cells was inhibited from 12 h onward. Expression of phosphorylated FADD and cleaved PARP-1 were also reduced in CDP-treated cells. Moreover, UVC-induced DNA damage and fragmentation were also prevented by the CDP treatment. This study shows that treatment of CDP provides protective effects against UVC-induced cytotoxicity through the inhibition of caspase-3 activity and the reduction of phosphorylated FADD and cleaved PARP-1 expression.

Niraparib Maintenance Therapy in Platinum-Sensitive, Recurrent Ovarian Cancer

DEFF Research Database (Denmark)

Mirza, Mansoor R; Monk, Bradley J; Herrstedt, Jørn

2016-01-01

Background Niraparib is an oral poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) 1/2 inhibitor that has shown clinical activity in patients with ovarian cancer. We sought to evaluate the efficacy of niraparib versus placebo as maintenance treatment for patients with platinum-sensitive, ......Background Niraparib is an oral poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) 1/2 inhibitor that has shown clinical activity in patients with ovarian cancer. We sought to evaluate the efficacy of niraparib versus placebo as maintenance treatment for patients with platinum...... or 4 adverse events that were reported in the niraparib group were thrombocytopenia (in 33.8%), anemia (in 25.3%), and neutropenia (in 19.6%), which were managed with dose modifications. Conclusions Among patients with platinum-sensitive, recurrent ovarian cancer, the median duration of progression...

Evaluation of Concurrent Radiation, Temozolomide and ABT-888 Treatment Followed by Maintenance Therapy with Temozolomide and ABT-888 in a Genetically Engineered Glioblastoma Mouse Model.

Science.gov (United States)

Lemasson, Benjamin; Wang, Hanxiao; Galbán, Stefanie; Li, Yinghua; Zhu, Yuan; Heist, Kevin A; Tsein, Christina; Chenevert, Thomas L; Rehemtulla, Alnawaz; Galbán, Craig J; Holland, Eric C; Ross, Brian D

2016-02-01

Despite the use of ionizing radiation (IR) and temozolomide (TMZ), outcome for glioblastoma (GBM) patients remains dismal. Poly (ADP-ribose) polymerase (PARP) is important in repair pathways for IR-induced DNA damage and TMZ-induced alkylation at N7-methylguanine and N3-methyldenine. However, optimized protocols for administration of PARP inhibitors have not been delineated. In this study, the PARP inhibitor ABT-888 was evaluated in combination with and compared to current standard-of-care in a genetically engineered mouse GBM model. Results demonstrated that concomitant TMZ/IR/ABT-888 with adjuvant TMZ/ABT-888 was more effective in inducing apoptosis and reducing proliferation with significant tumor growth delay and improved overall survival over concomitant TMZ/IR with adjuvant TMZ. Diffusion-weighted MRI, an early translatable response biomarker detected changes in tumors reflecting response at 1 day post TMZ/IR/ABT-888 treatment. This study provides strong scientific rationale for the development of an optimized dosing regimen for a PARP inhibitor with TMZ/IR for upfront treatment of GBM. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Evaluation of Concurrent Radiation, Temozolomide and ABT-888 Treatment Followed by Maintenance Therapy with Temozolomide and ABT-888 in a Genetically Engineered Glioblastoma Mouse Model

Directory of Open Access Journals (Sweden)

Benjamin Lemasson

2016-02-01

Full Text Available Despite the use of ionizing radiation (IR and temozolomide (TMZ, outcome for glioblastoma (GBM patients remains dismal. Poly (ADP-ribose polymerase (PARP is important in repair pathways for IR-induced DNA damage and TMZ-induced alkylation at N7-methylguanine and N3-methyldenine. However, optimized protocols for administration of PARP inhibitors have not been delineated. In this study, the PARP inhibitor ABT-888 was evaluated in combination with and compared to current standard-of-care in a genetically engineered mouse GBM model. Results demonstrated that concomitant TMZ/IR/ABT-888 with adjuvant TMZ/ABT-888 was more effective in inducing apoptosis and reducing proliferation with significant tumor growth delay and improved overall survival over concomitant TMZ/IR with adjuvant TMZ. Diffusion-weighted MRI, an early translatable response biomarker detected changes in tumors reflecting response at 1 day post TMZ/IR/ABT-888 treatment. This study provides strong scientific rationale for the development of an optimized dosing regimen for a PARP inhibitor with TMZ/IR for upfront treatment of GBM.

Linagliptin, a xanthine-based dipeptidyl peptidase-4 inhibitor with an unusual profile for the treatment of type 2 diabetes

DEFF Research Database (Denmark)

Deacon, Carolyn F; Holst, Jens Juul

2010-01-01

data presented at Scientific Meetings and peer-reviewed studies published since 2007. WHAT THE READER WILL GAIN: This article reviews pharmacokinetic and pharmacodynamic characteristics of linagliptin. Linagliptin belongs to a new chemical class of dipeptidyl pepidase-4 (DPP-4) inhibitors, which...... comprise xanthine-based compounds. It is a potent, long-acting inhibitor with high selectivity for DPP-4 versus the related enzymes DPP-8 and DPP-9. The drug has modest oral availability in humans, but is absorbed rapidly to inhibit plasma DPP-4 activity by > 80% over 24 h. It is not metabolized...

Possible Increase in Serum FABP4 Level Despite Adiposity Reduction by Canagliflozin, an SGLT2 Inhibitor.

Directory of Open Access Journals (Sweden)

Masato Furuhashi

Full Text Available Fatty acid-binding protein 4 (FABP4/A-FABP/aP2 is secreted from adipocytes in association with catecholamine-induced lipolysis, and elevated serum FABP4 level is associated with obesity, insulin resistance and atherosclerosis. Secreted FABP4 as a novel adipokine leads to insulin resistance via increased hepatic glucose production (HGP. Sodium-glucose cotransporter 2 (SGLT2 inhibitors decrease blood glucose level via increased urinary glucose excretion, though HGP is enhanced. Here we investigated whether canagliflozin, an SGLT2 inhibitor, modulates serum FABP4 level.Canagliflozin (100 mg/day was administered to type 2 diabetic patients (n = 39 for 12 weeks. Serum FABP4 level was measured before and after treatment.At baseline, serum FABP4 level was correlated with adiposity, renal dysfunction and noradrenaline level. Treatment with canagliflozin significantly decreased adiposity and levels of fasting glucose and HbA1c but increased average serum FABP4 level by 10.3% (18.0 ± 1.0 vs. 19.8 ± 1.2 ng/ml, P = 0.008, though elevation of FABP4 level after treatment was observed in 26 (66.7% out of 39 patients. Change in FABP4 level was positively correlated with change in levels of fasting glucose (r = 0.329, P = 0.044, HbA1c (r = 0.329, P = 0.044 and noradrenaline (r = 0.329, P = 0.041 but was not significantly correlated with change in adiposity or other variables.Canagliflozin paradoxically increases serum FABP4 level in some diabetic patients despite amelioration of glucose metabolism and adiposity reduction, possibly via induction of catecholamine-induced lipolysis in adipocytes. Increased FABP4 level by canagliflozin may undermine the improvement of glucose metabolism and might be a possible mechanism of increased HGP by inhibition of SGLT2.UMIN-CTR Clinical Trial UMIN000018151.

Checkpoint inhibitors in cancer immunotherapy: Cross reactivity of a CTLA-4 antibody and IDO-inhibitor L-1MT in pigs

DEFF Research Database (Denmark)

Al-Shatrawi, Zina Adil; Frøsig, Thomas Mørch; Jungersen, Gregers

a non-specific activation of porcine T cells. This will be further investigated to provide the basis for in vivo studies investigating checkpoint inhibitor blockade in combination with other cancer immunotherapies. Eventually our goal is to establish pigs as an alternative large animal model......Blockade of checkpoint inhibitors has recently shown very convincing results in the treatment of cancer. One key target is CTLA-4, which has been demonstrated to be a potent negative regulator of lymphocyte activation. The treatment with the FDA-approved fully human CTLA-4 monoclonal antibody...... Ipilimumab increases anticancer T-cell reactivity and overall survival of metastatic cancer patients. Indole-amine 2,3-dioxygenase (IDO) is another checkpoint inhibitor which suppresses T-cell immunity by the depletion of tryptophan in the T-cell microenvironment, and also inhibition of IDO by L-1...

Vildagliptin: the first innovative DDP-4 inhibitor

Directory of Open Access Journals (Sweden)

Edvin Villkhauer

2010-09-01

Full Text Available A review of the main stages of investigation undertaken by Novartis Pharmaceuticals in search of a new molecule for the treatment of type 2 diabetesmellitus, dipeptidyl peptidase-4 (DPP-4 inhibitor (Vildaglyptin. The data on specificity and selectivity of the action of this molecule are presentedalong with the results of its comparison with another agent of this group (sitagliptin.

Comparative review of dipeptidyl peptidase-4 inhibitors and sulphonylureas

DEFF Research Database (Denmark)

Deacon, Carolyn F.; Lebovitz, HE

2016-01-01

compound being launched in 2006, but the class now globally encompasses at least 11 different compounds. DPP-4 inhibitors improve glycaemic control with similar efficacy to SUs, but do not usually provoke hypoglycaemia or weight gain, are relatively free from adverse side effects, and have recently been...... drugs (DPP-4 inhibitors and SUs), highlighting differences and similarities between members of the same class, as well as discussing the potential advantages and disadvantages of the two drug classes. While both classes have their merits, the choice of which to use depends on the characteristics of each...

Poly[ADP-ribose] polymerase-1 expression is related to cold ischemia, acute tubular necrosis, and delayed renal function in kidney transplantation.

Directory of Open Access Journals (Sweden)

Francisco O'Valle

Full Text Available UNLABELLED: Cold ischemia time especially impacts on outcomes of expanded-criteria donor (ECD transplantation. Ischemia-reperfusion (IR injury produces excessive poly[ADP-Ribose] Polymerase-1 (PARP-1 activation. The present study explored the hypothesis that increased tubular expression of PARP-1 contributes to delayed renal function in suboptimal ECD kidney allografts and in non-ECD allografts that develop posttransplant acute tubular necrosis (ATN. MATERIALS AND METHODS: Nuclear PARP-1 immunohistochemical expression was studied in 326 paraffin-embedded renal allograft biopsies (193 with different degrees of ATN and 133 controls and in murine Parp-1 knockout model of IR injury. RESULTS: PARP-1 expression showed a significant relationship with cold ischemia time (r coefficient = 0.603, time to effective diuresis (r = 0.770, serum creatinine levels at biopsy (r = 0.649, and degree of ATN (r = 0.810 (p = 0.001, Pearson test. In the murine IR model, western blot showed an increase in PARP-1 that was blocked by Parp-1 inhibitor. Immunohistochemical study of PARP-1 in kidney allograft biopsies would allow early detection of possible delayed renal function, and the administration of PARP-1 inhibitors may offer a therapeutic option to reduce damage from IR in donor kidneys by preventing or minimizing ATN. In summary, these results suggest a pivotal role for PARP-1 in the ATN of renal transplantation. We propose the immunohistochemical assessment of PARP-1 in kidney allograft biopsies for early detection of a possible delayed renal function.

miR-520 promotes DNA-damage-induced trophoblast cell apoptosis by targeting PARP1 in recurrent spontaneous abortion (RSA).

Science.gov (United States)

Dong, Xiujuan; Yang, Long; Wang, Hui

2017-04-01

The establishment and maintenance of successful pregnancy mainly depends on trophoblast cells. Their dysfunction has been implicated in recurrent spontaneous abortion (RSA), a major complication of pregnancy. However, the underlying mechanisms of trophoblasts dysfunction remain unclear. DNA-damage-induced cell apoptosis has been reported to play a vital role in cell death. In this study, we identified a novel microRNA (miR-520) in RSA progression via regulating trophoblast cell apoptosis. Microarray analysis showed that miR-520 was highly expressed in villus of RSA patients. By using flow cytometry analysis, we observed miR-520 expression was correlated with human trophoblast cell apoptosis in vitro, along with decreased poly (ADP-ribose) polymerase-1 (PARP1) expression. With the analysis of clinic samples, we observed that miR-520 level was negatively correlated with PARP1 level in RSA villus. In addition, overexpression of PARP1 restored the miR-520-induced trophoblast cell apoptosis in vitro. The status of chromosome in trophoblast implied that miR-520-promoted DNA-damage-induced cell apoptosis to regulate RSA progression. These results indicated that the level of miR-520 might associate with RSA by prompting trophoblast cell apoptosis via PARP1 dependent DNA-damage pathway.

Discovery of Dengue Virus NS4B Inhibitors

Science.gov (United States)

Wang, Qing-Yin; Dong, Hongping; Zou, Bin; Karuna, Ratna; Wan, Kah Fei; Zou, Jing; Susila, Agatha; Yip, Andy; Shan, Chao; Yeo, Kim Long; Xu, Haoying; Ding, Mei; Chan, Wai Ling; Gu, Feng; Seah, Peck Gee; Liu, Wei; Lakshminarayana, Suresh B.; Kang, CongBao; Lescar, Julien; Blasco, Francesca; Smith, Paul W.

2015-01-01

ABSTRACT The four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV both in vitro and in vivo. The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC50], 10 to 80 nM) but not DENV-1 and -4 (EC50, >20 μM). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial “hit” (compound 1), leading to compound 14a, which has good in vivo pharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2- and -3-infected patients. IMPORTANCE Dengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions in the world where it was not previously endemic. While there are several promising vaccine candidates in clinical trials, approved vaccines or antivirals are not yet available. Here we describe the identification and characterization of a spiropyrazolopyridone as a novel inhibitor of DENV by targeting the viral NS4B protein. The compound potently inhibits two of the four serotypes of DENV (DENV-2 and -3) both in vitro and in vivo. Our

NR4A1 is an endogenous inhibitor of vocal fold fibrosis.

Science.gov (United States)

Hiwatashi, Nao; Bing, Renjie; Kraja, Iv; Branski, Ryan C

2017-09-01

NR4A1 was recently identified as an endogenous inhibitor of transforming growth factor (TGF)-β-induced fibrosis, and the role of this nuclear receptor has not been elucidated in tissue health or the response to injury in the vocal folds. Given the clinical implications of vocal fold fibrosis, we investigated NR4A1 expression during vocal fold wound healing in vivo and the regulatory roles of NR4A1 on vocal fold fibroblasts (VFFs) in vitro with the ultimate goal of developing targeted therapies for this challenging patient population. In vivo and in vitro. In vivo, the temporal pattern of NR4A1 mRNA expression was quantified following rat vocal fold injury. In vitro, the role of NR4A1 on TGF-β1-mediated transcription of genes underlying fibrosis as well as myofibroblast differentiation and collagen gel contraction was quantified in our human VFF line. Small interfering RNA was employed to alter NR4A1 expression to further elucidate this complex system. Nr4a1 mRNA increased 1 day after injury and peaked at 7 days. Knockdown of NR4A1 resulted in upregulation of COL1A1 and TGF-β1, with TGF-β1 stimulation (both P vocal fold health or disease. Upregulation of TGF-β following vocal fold injury was concurrent with increased NR4A1 expression. These data provide a foundation for the development of therapeutic strategies given persistent TGF-β signaling in vocal fold fibrosis. N/A Laryngoscope, 127:E317-E323, 2017. © 2017 The American Laryngological, Rhinological and Otological Society, Inc.

Discovery of a novel orally active PDE-4 inhibitor effective in an ovalbumin-induced asthma murine model.

Science.gov (United States)

Kwak, Hyun Jeong; Nam, Ji Yeon; Song, Jin Sook; No, Zaesung; Yang, Sung Don; Cheon, Hyae Gyeong

2012-06-15

Phosphodiesterase-4 (PDE-4) is responsible for metabolizing adenosine 3',5'-cyclic monophosphate that reduces the activation of a wide range of inflammatory cells including eosinophils. PDE-4 inhibitors are under development for the treatment of respiratory diseases such as asthma and chronic obstructive pulmonary disease. Herein, we report a novel PDE-4 inhibitor, PDE-423 (3-[1-(3-cyclopropylmethoxy-4-difluoromethoxybenzyl)-1H-pyrazol-3-yl]-benzoic acid), which shows good in vitro and in vivo oral activities. PDE-423 exhibited in vitro IC(50)s of 140 nM and 550 nM in enzyme assay and cell-based assay, respectively. In vivo study using ovalbumin-induced asthmatic mice revealed that PDE-423 reduced methacholine-stimulated airway hyperreactivity in a dose-dependent manner by once daily oral administration (ED(50)=18.3 mg/kg), in parallel with decreased eosinophil peroxidase activity and improved lung histology. In addition, PDE-423 was effective in diminishing lipopolysaccharide-induced neutrophilia in vivo as well as in vitro. Oral administration of PDE-423 (100 mg/kg) had no effect on the duration of xylazine/ketamine-induced anesthesia and did not induce vomiting incidence in ferrets up to the dose of 1000 mg/kg. The present study indicates that a novel PDE-4 inhibitor, PDE-423, has good pharmacological profiles implicating this as a potential candidate for the development of a new anti-asthmatic drug. Copyright © 2012 Elsevier B.V. All rights reserved.

vPARP Adjusts MVP Expression in Drug-resistant Cell Lines in Conjunction with MDR Proteins.

Science.gov (United States)

Wojtowicz, Karolina; Januchowski, Radoslaw; Nowicki, Michal; Zabel, Maciej

2017-06-01

The definition of vault (ribonucleoprotein particles) function remains highly complex. Vaults may cooperate with multidrug resistance (MDR) proteins, supporting their role in drug resistance. This topic is the main theme of this publication. The cell viability was determined by an MTT assay. The protein expression was detected by western blot analysis. The proteins were knocked-down using siRNA. No major vault protein (MVP) in the LoVo/Dx and W1PR cell lines after tunicamycin treatment was shown. In W1PR cells with knocked-down MVP, a statistically significant decrease in cell viability was noted. In LoVo/Dx, W1TR and A2780TR cells were vault poly-ADP-ribose polymerase (vPARP) was knockdown, a decrease in cell viability was shown. Also, MVP silencing induced an increase in glycoprotein P (Pgp) expression in LoVo/Dx cells. MVP is important for the drug resistance of cancer cells, but it probably requires the presence of vPARP for full activation. Some correlations between MDR proteins and vaults exist. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Andrographolide Induces Apoptosis of C6 Glioma Cells via the ERK-p53-Caspase 7-PARP Pathway

Directory of Open Access Journals (Sweden)

Shih-Hung Yang

2014-01-01

Full Text Available Background. Glioma is the most malignant tumor of the central nervous system. Efforts on the development of new chemotherapy are mandatory. Andrographolide (AND, a diterpenoid lactone isolated from the Andrographis paniculata, has been shown to have antitumor activities in several types of cancer cells. Whether AND can exert its antitumor activity in glioblastoma cells remains unknown. This study examined the anticancer effects of AND, both in vitro and in vivo. Methods. Cell apoptosis was assayed by flow cytometry and nuclear staining. The signaling pathway for AND was determined by western blotting. The effects of AND on tumor growth was evaluated in a mouse model. Results and Conclusion. In vitro, with application of specific inhibitors and siRNA, AND-induced apoptosis was proven through ROS-ERK-P53-caspase 7-PARP signaling pathway. In vivo, AND significantly retarded tumor growth and caused regression of well-formed tumors in vivo. Furthermore, AND did not induce apoptosis or activate ERK and p53 in primary cultured astrocyte cells, and it may serve as a potential therapeutic candidate for the treatment of glioma.

Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization.

Science.gov (United States)

La Ferla, Marco; Mercatanti, Alberto; Rocchi, Giulia; Lodovichi, Samuele; Cervelli, Tiziana; Pignata, Luca; Caligo, Maria Adelaide; Galli, Alvaro

2015-04-01

The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the deubiquitination enzyme gene OTU1, the nuclear pore protein POM152 and the SNT1 that encodes for the Set3C subunit of the histone deacetylase complex. In these strains the PARP-1 level was roughly the same as in the wild type. PARP-1 localized in the nucleus more in the snt1Δ than in the wild type strain; after UV radiation, PARP-1 localized in the nucleus more in hho1 and pom152 deletion strains than in the wild type indicating that these functions may have a role on regulating PARP-1 level and activity in the nucleus. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of epoxybergamottin as a CYP3A4 inhibitor in grapefruit peel.

Science.gov (United States)

Wangensteen, H; Molden, E; Christensen, H; Malterud, K E

2003-02-01

The oral availability of many drugs metabolised by the enzyme cytochrome P(450) 3A4 (CYP3A4) is increased if co-administered with grapefruit juice. Extracts from grapefruit peel have also demonstrated inhibitory activity and, during commercial manufacturing of grapefruit juice, inhibitory components might be squeezed into the juice from the peel. Thus, the aim of this in vitro study was to identify CYP3A4 inhibitors in grapefruit peel. Grapefruit peel was extracted with diethyl ether, and the extract was further fractionated by normal-phase chromatography. Fractions demonstrating significant CYP3A4 inhibitory activity, as measured by the relative reduction in N-demethylation of diltiazem in transfected human liver epithelial cells, were subsequently separated by preparative thin-layer chromatography. Constituents of the fractions and isolated compounds were identified by nuclear magnetic resonance spectroscopy. Analysis of diltiazem and N-demethyl-diltiazem was performed using high-performance liquid chromatography. Of the identified components in grapefruit peel, only epoxybergamottin demonstrated a concentration-dependent inhibition of the CYP3A4-mediated N-demethylation of diltiazem. The IC(50) value was calculated to be 4.2+/-1.1 micro M. Coumarins without the furan ring and flavonoids isolated from grapefruit peel did not interfere with the metabolism of diltiazem. The results indicated the presence of other CYP3A4 inhibitors in grapefruit peel, but these agents were lost during the purification process excluding their identification. The furanocoumarin epoxybergamottin, present in grapefruit peel, is an inhibitor of CYP3A4. In commercial manufacturing of grapefruit juice, epoxybergamottin is possibly distributed into the juice. During manufacturing, however, epoxybergamottin may be hydrolysed to 6',7'-dihydroxybergamottin, which has been suggested as an important CYP3A4 inhibitor in grapefruit juice.

Discovery of a potent and orally available acyl-CoA: cholesterol acyltransferase inhibitor as an anti-atherosclerotic agent: (4-phenylcoumarin)acetanilide derivatives.

Science.gov (United States)

Ogino, Masaki; Fukui, Seiji; Nakada, Yoshihisa; Tokunoh, Ryosuke; Itokawa, Shigekazu; Kakoi, Yuichi; Nishimura, Satoshi; Sanada, Tsukasa; Fuse, Hiromitsu; Kubo, Kazuki; Wada, Takeo; Marui, Shogo

2011-01-01

Acyl-CoA: cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes cholesterol esterification. ACAT inhibitors are expected to be potent therapeutic agents for the treatment of atherosclerosis. A series of potent ACAT inhibitors based on an (4-phenylcoumarin)acetanilide scaffold was identified. Evaluation of the structure-activity relationships of a substituent on this scaffold, with an emphasis on improving the pharmacokinetic profile led to the discovery of 2-[7-chloro-4-(3-chlorophenyl)-6-methyl-2-oxo-2H-chromen-3-yl]-N-[4-chloro-2-(trifluoromethyl)phenyl]acetamide (23), which exhibited potent ACAT inhibitory activity (IC50=12 nM) and good pharmacokinetic profile in mice. Compound 23 also showed regressive effects on atherosclerotic plaques in apolipoprotein (apo)E knock out (KO) mice at a dose of 0.3 mg/kg per os (p.o.).

Inadequate Triglyceride Management Worsens the Durability of Dipeptidyl Peptidase-4 Inhibitor in Subjects with Type 2 Diabetes Mellitus

Directory of Open Access Journals (Sweden)

Masashi Shimoda

2017-01-01

Full Text Available Dipeptidyl peptidase-4 (DPP-4 inhibitors are often used all over the world and exert various beneficial effects including glucose-lowering effect in many subjects with type 2 diabetes. It is poorly understood, however, which factors are closely related with the durability of glucose-lowering effect by DPP-4 inhibitor. In this study, we examined retrospectively which factors could mainly influence the durability of DPP-4 inhibitor. We enrolled 212 participants with type 2 diabetes to whom DPP-4 inhibitor was administered for over 1 year without an addition or increase of other hypoglycemic agents. Age and baseline HbA1c level were significantly higher in the effective group than those in the ineffective group. The effective group had a tendency of smaller amounts of weight change, average total cholesterol, and average triglyceride compared with the ineffective group. Multiple logistic regression analysis showed that average triglyceride and baseline HbA1c were independent predictors associated with the durability of DPP-4 inhibitor. Moreover, an average triglyceride level contributed to the durability of DPP-4 inhibitor in the obese group (BMI ≥ 25 kg/m2 but not in the nonobese group (BMI < 25 kg/m2. These results suggest the importance of strict triglyceride management to maintain the durability of glucose-lowering effect by DPP-4 inhibitor, especially in obese subjects with type 2 diabetes.

Design and synthesis of 4-heteroaryl 1,2,3,4-tetrahydroisoquinolines as triple reuptake inhibitors.

Science.gov (United States)

Liu, Shuang; Zha, Congxiang; Nacro, Kassoum; Hu, Min; Cui, Wenge; Yang, Yuh-Lin; Bhatt, Ulhas; Sambandam, Aruna; Isherwood, Matthew; Yet, Larry; Herr, Michael T; Ebeltoft, Sarah; Hassler, Carla; Fleming, Linda; Pechulis, Anthony D; Payen-Fornicola, Anne; Holman, Nicholas; Milanowski, Dennis; Cotterill, Ian; Mozhaev, Vadim; Khmelnitsky, Yuri; Guzzo, Peter R; Sargent, Bruce J; Molino, Bruce F; Olson, Richard; King, Dalton; Lelas, Snjezana; Li, Yu-Wen; Johnson, Kim; Molski, Thaddeus; Orie, Anitra; Ng, Alicia; Haskell, Roy; Clarke, Wendy; Bertekap, Robert; O'Connell, Jonathan; Lodge, Nicholas; Sinz, Michael; Adams, Stephen; Zaczek, Robert; Macor, John E

2014-07-10

A series of 4-bicyclic heteroaryl 1,2,3,4-tetrahydroisoquinoline inhibitors of the serotonin transporter (SERT), norepinephrine transporter (NET), and dopamine transporter (DAT) was discovered. The synthesis and structure-activity relationship (SAR) of these triple reuptake inhibitors (TRIs) will be discussed. Compound 10i (AMR-2), a very potent inhibitor of SERT, NET, and DAT, showed efficacy in the rat forced-swim and mouse tail suspension models with minimum effective doses of 0.3 and 1 mg/kg (po), respectively. At efficacious doses in these assays, 10i exhibited substantial occupancy levels at the three transporters in both rat and mouse brain. The study of the metabolism of 10i revealed the formation of a significant active metabolite, compound 13.

The cardiovascular safety trials of DPP-4 inhibitors, GLP-1 agonists, and SGLT2 inhibitors.

Science.gov (United States)

Secrest, Matthew H; Udell, Jacob A; Filion, Kristian B

2017-04-01

In this paper, we review the results of large, double-blind, placebo-controlled randomized trials mandated by the US Food and Drug Administration to examine the cardiovascular safety of newly-approved antihyperglycemic agents in patients with type 2 diabetes. The cardiovascular effects of dipeptidyl peptidase-4 (DPP-4) inhibitors remain controversial: while these drugs did not reduce or increase the risk of primary, pre-specified composite cardiovascular outcomes, one DPP-4 inhibitor (saxagliptin) increased the risk of hospitalization for heart failure in the overall population; another (alogliptin) demonstrated inconsistent effects on heart failure hospitalization across subgroups of patients, and a third (sitagliptin) demonstrated no effect on heart failure. Evidence for cardiovascular benefits of glucagon-like peptide-1 (GLP-1) agonists has been similarly heterogeneous, with liraglutide and semaglutide reducing the risk of composite cardiovascular outcomes, but lixisenatide having no reduction or increase in cardiovascular risk. The effect of GLP-1 agonists on retinopathy remains a potential concern. In the only completed trial to date to assess a sodium-glucose cotransporter-2 (SGLT2) inhibitor, empagliflozin reduced the risk of composite cardiovascular endpoints, predominantly through its impact on cardiovascular mortality and heart failure hospitalization. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of APE1 T2197G (Asp148Glu Polymorphism on APE1, XRCC1, PARP1 and OGG1 Expression in Patients with Colorectal Cancer

Directory of Open Access Journals (Sweden)

Juliana C. Santos

2014-09-01

Full Text Available It has been hypothesized that genetic variation in base excision repair (BER might modify colorectal adenoma risk. Thus, we evaluated the influence of APE1 T2197G (Asp148Glu polymorphism on APE1, XRCC1, PARP1 and OGG1 expression in normal and tumor samples from patients with colorectal cancer. The results indicate a downregulation of OGG1 and an upregulation of XRCC1 expression in tumor tissue. Regarding the anatomical location of APE1, OGG1 and PARP-1, a decrease in gene expression was observed among patients with cancer in the rectum. In patients with or without some degree of tumor invasion, a significant downregulation in OGG1 was observed in tumor tissue. Interestingly, when taking into account the tumor stage, patients with more advanced grades (III and IV showed a significant repression for APE1, OGG1 and PARP-1. XRCC1 expression levels were significantly enhanced in tumor samples and were correlated with all clinical and histopathological data. Concerning the polymorphism T2197G, GG genotype carriers exhibited a significantly reduced expression of genes of the BER repair system (APE1, XRCC1 and PARP1. In summary, our data show that patients with colorectal cancer present expression changes in several BER genes, suggesting a role for APE1, XRCC1, PARP1 and OGG1 and APE1 polymorphism in colorectal carcinogenesis.

Comparison between SGLT2 inhibitors and DPP4 inhibitors added to insulin therapy in type 2 diabetes: a systematic review with indirect comparison meta-analysis.

Science.gov (United States)

Min, Se Hee; Yoon, Jeong-Hwa; Hahn, Seokyung; Cho, Young Min

2017-01-01

Both sodium glucose cotransporter 2 (SGLT2) inhibitors and dipeptidyl peptidase-4 (DPP4) inhibitors can be used to treat patients with type 2 diabetes mellitus (T2DM) that is inadequately controlled with insulin therapy, and yet there has been no direct comparison of these two inhibitors. We searched MEDLINE, EMBASE, LILACS, the Cochrane Central Register of Controlled Trials and ClinicalTrials.gov through June 2015. Randomized controlled trials published in English that compare SGLT2 inhibitor plus insulin (SGLT2i/INS) with placebo plus insulin or DPP4 inhibitor plus insulin (DPP4i/INS) with placebo plus insulin in patients with T2DM were selected. Data on the study characteristics, efficacy and safety outcomes were extracted. We compared the efficacy and safety between SGLT2i/INS and DPP4i/INS indirectly with covariates adjustment. Risk of potential bias was assessed. Fourteen eligible randomized controlled trials comprising 6980 patients were included (five SGLT2 inhibitor studies and nine DPP4 inhibitor studies). Covariate-adjusted indirect comparison using meta-regression analyses revealed that SGLT2i/INS achieved greater reduction in HbA 1c [weighted mean difference (WMD) -0.24%, 95% confidence interval (CI) -0.43 to -0.05%], fasting plasma glucose (WMD -18.0 mg/dL, 95% CI -28.5 to -7.6 mg/dL) and body weight (WMD -2.38 kg, 95% CI -3.18 to -1.58 kg) from baseline than DPP4i/INS without increasing the risk of hypoglycaemia (relative risks 1.19, 95% CI 0.78 to 1.82). Sodium glucose cotransporter 2 inhibitors achieved better glycaemic control and greater weight reduction than DPP4 inhibitors without increasing the risk of hypoglycaemia in patients with T2DM that is inadequately controlled with insulin. There has been no direct comparison of SGLT2 inhibitors and DPP4 inhibitors in patients with T2DM inadequately controlled with insulin therapy. In this study, we performed indirect meta-analysis comparing SGLT2 inhibitors and DPP4 inhibitors added to insulin

Dipeptidyl peptidase-4 inhibitors for the treatment of type 2 diabetes

DEFF Research Database (Denmark)

Deacon, Carolyn F; Holst, Jens Juul

2013-01-01

INTRODUCTION: Dipeptidyl peptidase (DPP)-4 inhibitors belong to one class of drugs that have been approved for treatment of type 2 diabetes (T2D) based on the glucose-lowering actions of the gastrointestinal hormone glucagon-like peptide (GLP)-1. Several different compounds are now available...... of the different DPP-4 inhibitors, all are small orally active compounds with broadly similar HbA1c-lowering efficacy. They improve glycaemic control in T2D, without increasing the risk of hypoglycaemia or causing weight gain. They can be used as monotherapy or in combination with other anti-diabetic therapies......, and although their mechanism of action (inhibition of the catalytic activity of DPP-4) is the same, there are fundamental differences between them. AREAS COVERED: The authors discuss the differences between different DPP-4 inhibitors and review their therapeutic efficacy and key safety data. The literature...

[Expression of PARP/NF-κB and intervention effect of 5-AIQ/PDTC in SAP rats with adrenal damage].

Science.gov (United States)

Yang, Bo; Guo, Wen-Yi; Yu, Jia; Zhao, Kai-liang; Shi, Qiao; Zuo, Teng; Wang, Wei-xing

2013-10-15

To explore the expression of poly (ADP-ribose) polymerase/nuclear factor-κB (PARP/NF-κB) and intervention effect of 5-aminoisoquinolinone/pyrrolidine dithiocarbamate (5-AIQ/PDTC) in severe acute pancreatitis (SAP) rats with adrenal damage. The primarily cultured adrenocortical cells were quantitatively divided into control group (SO), pancreatitis group (SAP), PDTC drug control group (SO+PDTC), PDTC intervention group (SAP+PDTC), 5-AIQ drug control group (SO+ 5-AIQ) and 5-AIQ intervention group (SAP+5-AIQ). The SAP and 2 intervention groups were stimulated with the sera of SAP rats. Then corresponding drugs were added and culture continued for 12 hours. The corticosterone levels and PARP/NF-κB expression were observed for each group. Adrenal cells in vitro cultured were round or oval, had secretory granules and could be stained by 3β-hydroxysteroid dehydrogenase antibody. The adherence rate was 60% after 48-hour culturing. The corticosterone level of SAP group was significantly lower than that of SO group [ (216.4 ± 15.7) vs (294.8 ± 16.3) µg/L, P SAP group (P SAP and PDTC intervention groups were higher than SO group while 5-AIQ intervention group was significantly lower than SAP and PDTC intervention groups, but higher than SO and drug control groups. The expression of NF-κB in SAP group was higher than that in SO group. Two intervention groups were lower than SAP group, but higher than SO and drug control groups. The pathway of PARP/NF-κB participates in adrenal damage of SAP rats. To a certain extent, the uses of 5-AIQ and PDTC may alleviate adrenal damage.

A Selenium Containing Inhibitor for the Treatment of Hepatocellular Cancer

Directory of Open Access Journals (Sweden)

Hephzibah Rani S. Tagaram

2016-03-01

Full Text Available Hepatocellular carcinoma (HCC is the third most deadly cancer in the world. New treatment strategies are desperately needed due to limited standard therapies. Activation of the Erk, Akt, and STAT3pathways is implicated in the prognosis of HCC. The Se,Se′-1,4-phenylenebis(1,2-ethanediyl bisisoselenourea (PBISe, is a selenium-containing MAPK and PI3 kinase inhibitor, effectively inhibit tumorigenesis in a variety of experimental models. The aim of our study is to demonstrate the potential role of PBISe in the treatment of HCC. The anti-proliferative and pro-apoptotic ability of PBISe is studied in vitro in four human HCC cell lines and in vivo in a spontaneous murine HCC model. Inhibition of cancer growth was performed by cell viability assay and apoptosis by caspase 3/7, PARP cleavage, annexin-V, and TUNEL assays. Role of PBISe on PI3 kinase, MAPK and STAT3 signaling is determined by Western blotting. In vivo effects of PBISe on tumor sizes were monitored using MRI in a spontaneous murine HCC. Liver tissues from the PBISe-treated mice are analyzed for angiogenesis, proliferation, and signaling pathway markers. Overall, PBISe activated caspase-3/7 and increased DNA fragmentation, which is positively correlated with the increased PARP cleavage. PBISe promoted apoptosis by inhibiting PI3K, MAPK, and STAT3 signaling with significant reduction in the tumor sizes (p < 0.007. PBISe-treated tumors reduced survival marker PCNA, and angiogenesis markers Vegf-A, Vegf-R3 and CD34. These results demonstrate the chemotherapeutic effects of PBISe, by inhibiting tumor growth and facilitating tumor apoptosis for HCC treatment.

Scaffold-hopping from xanthines to tricyclic guanines: A case study of dipeptidyl peptidase 4 (DPP4) inhibitors

Energy Technology Data Exchange (ETDEWEB)

Pissarnitski, Dmitri A.; Zhao, Zhiqiang; Cole, David; Wu, Wen-Lian; Domalski, Martin; Clader, John W.; Scapin, Giovanna; Voigt, Johannes; Soriano, Aileen; Kelly, Theresa; Powles, Mary Ann; Yao, Zuliang; Burnett, Duane A. (Merck)

2016-11-01

Molecular modeling of unbound tricyclic guanine scaffolds indicated that they can serve as effective bioisosteric replacements of xanthines. This notion was further confirmed by a combination of X-ray crystallography and SAR studies, indicating that tricyclic guanine DPP4 inhibitors mimic the binding mode of xanthine inhibitors, exemplified by linagliptin. Realization of the bioisosteric relationship between these scaffolds potentially will lead to a wider application of cyclic guanines as xanthine replacements in drug discovery programs for a variety of biological targets. Newly designed DPP4 inhibitors achieved sub-nanomolar potency range and demonstrated oral activity in vivo in mouse glucose tolerance test.

Isolation and Characterization of an α-Glucosidase Inhibitor from Musa spp. (Baxijiao Flowers

Directory of Open Access Journals (Sweden)

Zhanwu Sheng

2014-07-01

Full Text Available The use of α-glucosidase inhibitors is considered to be an effective strategy in the treatment of diabetes. Using a bioassay-guided fractionation technique, five Bacillus stearothermophilus α-glucosidase inhibitors were isolated from the flowers of Musa spp. (Baxijiao. Using NMR spectroscopy analysis they were identified as vanillic acid (1, ferulic acid (2, β-sitosterol (3, daucosterol (4 and 9-(4′-hydroxyphenyl-2-methoxyphenalen-1-one (5. The half maximal inhibitory concentration (IC50 values of compounds 1–5 were 2004.58, 1258.35, 283.67, 247.35 and 3.86 mg/L, respectively. Compared to a known α-glucosidase inhibitor (acarbose, IC50 = 999.31 mg/L, compounds 3, 4 and 5 showed a strong α-glucosidase inhibitory effect. A Lineweaver-Burk plot indicated that compound 5 is a mixed-competitive inhibitor, while compounds 3 and 4 are competitive inhibitors. The inhibition constants (Ki of compounds 3, 4 and 5 were 20.09, 2.34 and 4.40 mg/L, respectively. Taken together, these data show that the compounds 3, 4 and 5 are potent α-glucosidase inhibitors.

Poly(ADP-ribose) polymerase as a novel regulator of 17β-estradiol-induced cell growth through a control of the estrogen receptor/IGF-1 receptor/PDZK1 axis.

Science.gov (United States)

Kim, Hogyoung; Tarhuni, Abdelmetalab; Abd Elmageed, Zakaria Y; Boulares, A Hamid

2015-07-17

We and others have extensively investigated the role of PARP-1 in cell growth and demise in response to pathophysiological cues. Most of the clinical trials on PARP inhibitors are targeting primarily estrogen receptor (ER) negative cancers with BRCA-deficiency. It is surprising that the role of the enzyme has yet to be investigated in ER-mediated cell growth. It is noteworthy that ER is expressed in the majority of breast cancers. We recently showed that the scaffolding protein PDZK1 is critical for 17β-estradiol (E2)-induced growth of breast cancer cells. We demonstrated that E2-induced PDZK1 expression is indirectly regulated by ER and requires IGF-1 receptor (IGF-1R). The breast cancer cell lines MCF-7 and BT474 were used as ER(+) cell culture models. Thieno[2,3-c]isoquinolin-5-one (TIQ-A) and olaparib (AZD2281) were used as potent inhibitors of PARP. PARP-1 knockdown by shRNA was used to show specificity of the effects to PARP-1. In this study, we aimed to determine the effect of PARP inhibition on estrogen-induced growth of breast cancer cells and examine whether the potential effect is linked to PDZK1 and IGF-1R expression. Our results show that PARP inhibition pharmacologically by TIQ-A or olaparib or by PARP-1 knockdown blocked E2-dependent growth of MCF-7 cells. Such inhibitory effect was also observed in olaparib-treated BT474 cells. The effect of PARP inhibition on cell growth coincided with an efficient reduction in E2-induced PDZK1 expression. This effect was accompanied by a similar decrease in the cell cycle protein cyclin D1. PARP appeared to regulate E2-induced PDZK1 at the mRNA level. Such regulation may be linked to a modulation of IGF-1R as PARP inhibition pharmacologically or by PARP-1 knockdown efficiently reduced E2-induced expression of the receptor at the protein and mRNA levels. Overall, our results show for the first time that PARP regulates E2-mediated cell growth by controlling the ER/IGF-1R/PDZK1 axis. These findings suggest that the

Effect of veliparib (ABT-888) on cardiac repolarization in patients with advanced solid tumors : a randomized, placebo-controlled crossover study

NARCIS (Netherlands)

Munasinghe, Wijith; Stodtmann, Sven; Tolcher, Anthony; Calvo, Emiliano; Gordon, Michael; Jalving, Mathilde; de Vos-Geelen, Judith; Medina, Diane; Bergau, Dennis; Nuthalapati, Silpa; Hoffman, David; Shepherd, Stacie; Xiong, Hao

2016-01-01

Veliparib (ABT-888) is an orally bioavailable potent inhibitor of poly(ADP-ribose) polymerase (PARP)-1 and PARP-2. This phase 1 study evaluated the effect of veliparib on corrected QT interval using Fridericia's formula (QTcF). Eligible patients with advanced solid tumors received single-dose oral

Novel Insights into Structure-Activity Relationships of N-Terminally Modified PACE4 Inhibitors.

Science.gov (United States)

Kwiatkowska, Anna; Couture, Frédéric; Levesque, Christine; Ly, Kévin; Beauchemin, Sophie; Desjardins, Roxane; Neugebauer, Witold; Dory, Yves L; Day, Robert

2016-02-04

PACE4 plays important roles in prostate cancer cell proliferation. The inhibition of this enzyme has been shown to slow prostate cancer progression and is emerging as a promising therapeutic strategy. In previous work, we developed a highly potent and selective PACE4 inhibitor, the multi-Leu (ML) peptide, an octapeptide with the sequence Ac-LLLLRVKR-NH2 . Here, with the objective of developing a useful compound for in vivo administration, we investigate the effect of N-terminal modifications. The inhibitory activity, toxicity, stability, and cell penetration properties of the resulting analogues were studied and compared to the unmodified inhibitor. Our results show that the incorporation of a polyethylene glycol (PEG) moiety leads to a loss of antiproliferative activity, whereas the attachment of a lipid chain preserves or improves it. However, the lipidated peptides are significantly more toxic when compared with their unmodified counterparts. Therefore, the best results were achieved not by the N-terminal extension but by the protection of both ends with the d-Leu residue and 4-amidinobenzylamide, which yielded the most stable inhibitor, with an excellent activity and toxicity profile. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Single-cell and subcellular pharmacokinetic imaging allows insight into drug action in vivo.

Science.gov (United States)

Thurber, Greg M; Yang, Katy S; Reiner, Thomas; Kohler, Rainer H; Sorger, Peter; Mitchison, Tim; Weissleder, Ralph

2013-01-01

Pharmacokinetic analysis at the organ level provides insight into how drugs distribute throughout the body, but cannot explain how drugs work at the cellular level. Here we demonstrate in vivo single-cell pharmacokinetic imaging of PARP-1 inhibitors and model drug behaviour under varying conditions. We visualize intracellular kinetics of the PARP-1 inhibitor distribution in real time, showing that PARP-1 inhibitors reach their cellular target compartment, the nucleus, within minutes in vivo both in cancer and normal cells in various cancer models. We also use these data to validate predictive finite element modelling. Our theoretical and experimental data indicate that tumour cells are exposed to sufficiently high PARP-1 inhibitor concentrations in vivo and suggest that drug inefficiency is likely related to proteomic heterogeneity or insensitivity of cancer cells to DNA-repair inhibition. This suggests that single-cell pharmacokinetic imaging and derived modelling improve our understanding of drug action at single-cell resolution in vivo.

Dipeptidyl Peptidase-4 Inhibitor Anagliptin Prevents Intracranial Aneurysm Growth by Suppressing Macrophage Infiltration and Activation.

Science.gov (United States)

Ikedo, Taichi; Minami, Manabu; Kataoka, Hiroharu; Hayashi, Kosuke; Nagata, Manabu; Fujikawa, Risako; Higuchi, Sei; Yasui, Mika; Aoki, Tomohiro; Fukuda, Miyuki; Yokode, Masayuki; Miyamoto, Susumu

2017-06-19

Chronic inflammation plays a key role in the pathogenesis of intracranial aneurysms (IAs). DPP-4 (dipeptidyl peptidase-4) inhibitors have anti-inflammatory effects, including suppressing macrophage infiltration, in various inflammatory models. We examined whether a DPP-4 inhibitor, anagliptin, could suppress the growth of IAs in a rodent aneurysm model. IAs were surgically induced in 7-week-old male Sprague Dawley rats, followed by oral administration of 300 mg/kg anagliptin. We measured the morphologic parameters of aneurysms over time and their local inflammatory responses. To investigate the molecular mechanisms, we used lipopolysaccharide-treated RAW264.7 macrophages. In the anagliptin-treated group, aneurysms were significantly smaller 2 to 4 weeks after IA induction. Anagliptin inhibited the accumulation of macrophages in IAs, reduced the expression of MCP-1 (monocyte chemotactic protein 1), and suppressed the phosphorylation of p65. In lipopolysaccharide-stimulated RAW264.7 cells, anagliptin treatment significantly reduced the production of tumor necrosis factor α, MCP-1, and IL-6 (interleukin 6) independent of GLP-1 (glucagon-like peptide 1), the key mediator in the antidiabetic effects of DPP-4 inhibitors. Notably, anagliptin activated ERK5 (extracellular signal-regulated kinase 5), which mediates the anti-inflammatory effects of statins, in RAW264.7 macrophages. Preadministration with an ERK5 inhibitor blocked the inhibitory effect of anagliptin on MCP-1 and IL-6 expression. Accordingly, the ERK5 inhibitor also counteracted the suppression of p65 phosphorylation in vitro. A DPP-4 inhibitor, anagliptin, prevents the growth of IAs via its anti-inflammatory effects on macrophages. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Analysis of the effects of phosphodiesterase type 3 and 4 inhibitors in cerebral arteries

DEFF Research Database (Denmark)

Birk, Steffen; Edvinsson, Lars; Olesen, Jes

2004-01-01

Inhibitors of phosphodiesterases 3 and 4, the main cyclic AMP (cAMP) degrading enzymes in arteries, may have therapeutic potential in cerebrovascular disorders. We analysed the effects of such phosphodiesterases in guinea pig cerebral arteries with organ bath technique and cyclic nucleotide assays...... the major contributors to total cAMP hydrolysis in the arteries examined. The phosphodiesterase 3 inhibitors additionally attenuated cyclic GMP (cGMP) hydrolysis, but relaxant responses were not dependent on an intact endothelium or on the nitric oxide-cGMP pathway. Conversely, the phosphodiesterase 4...

Caspase-1 Deficiency Alleviates Dopaminergic Neuronal Death via Inhibiting Caspase-7/AIF Pathway in MPTP/p Mouse Model of Parkinson's Disease.

Science.gov (United States)

Qiao, Chen; Zhang, Lin-Xia; Sun, Xi-Yang; Ding, Jian-Hua; Lu, Ming; Hu, Gang

2017-08-01

Caspase family has been recognized to be involved in dopaminergic (DA) neuronal death and to exert an unfavorable role in Parkinson's disease (PD) pathology. Our previous study has revealed that caspase-1, as an important component of NLRP3 inflammasome, induces microglia-mediated neuroinflammation in the pathogenesis of PD. However, the role of caspase-1 in DA neuronal degeneration in the onset of PD remains unclear. Here, we showed that caspase-1 knockout ameliorated DA neuronal loss and dyskinesia in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine/probenecid (MPTP/p)-induced PD model mice. We further found that caspase-1 knockout decreased MPTP/p-induced caspase-7 cleavage, subsequently inhibited nuclear translocation of poly (ADP-ribose) polymerase 1 (PARP1), and reduced the release of apoptosis-inducing factor (AIF). Consistently, we demonstrated that caspase-1 inhibitor suppressed caspase-7/PARP1/AIF-mediated apoptosis pathway by 1-methyl-4-phenylpyridinium ion (MPP + ) stimulation in SH-SY5Y cells. Caspase-7 overexpression reduced the protective effects of caspase-1 inhibitor on SH-SY5Y cell apoptosis. Collectively, our results have revealed that caspase-1 regulates DA neuronal death in the pathogenesis of PD in mice via caspase-7/PARP1/AIF pathway. These findings will shed new insight into the potential of caspase-1 as a target for PD therapy.

Cassava starch graft copolymers an eco-friendly corrosion inhibitor for steel in H{sub 2}SO{sub 4} solution

Energy Technology Data Exchange (ETDEWEB)

Li, Xianghong; Deng, Shuduan [Southwest Forestry University, Kunming (China)

2015-11-15

Cassava starch graft copolymer (CSGC) was prepared by grafting acryl amide (AA) onto cassava starch (CS). The inhibition effect of CSGC on the corrosion of cold rolled steel (CRS) in 1.0M H{sub 2}SO{sub 4} solution was first studied by weight loss, potentiodynamic polarization curves, electrochemical impedance spectroscopy (EIS) and scanning electron microscope (SEM) methods. The results show that CSGC is a good inhibitor, and inhibition efficiency of CSGC is higher than that of CS or AA. The adsorption of CSGC on steel surface obeys Langmuir adsorption isotherm. CSGC is a mixed-type inhibitor at 20 .deg. C, while mainly a cathodic inhibitor at 50 .deg. C.

Crystal structures of HIV-1 nonnucleoside reverse transcriptase inhibitors: N-benzyl-4-methyl-benzimidazoles

Science.gov (United States)

Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

2009-07-01

HIV-1 nonnucleoside reverse transcriptase inhibitors are potentially specific and effective drugs in AIDS therapy. The presence of two aromatic systems with an angled orientation in the molecule of the inhibitor is crucial for interactions with HIV-1 RT. The inhibitor drives like a wedge into the cluster of aromatic residues of RT HIV-1 and restrains the enzyme in a conformation that blocks the chemical step of nucleotide incorporation. Structural studies provide useful information for designing new, more active inhibitors. The crystal structures of four NNRTIs are presented here. The investigated compounds are derivatives of N-benzyl-4-methyl-benzimidazole with various aliphatic and aromatic substituents at carbon 2 positions and a 2,6-dihalogeno-substituted N-benzyl moiety. Structural data reported here show that the conformation of the investigated compounds is relatively rigid. Such feature is important for the nonnucleoside inhibitor binding to HIV-1 reverse transcriptase.

Structural Biology and Molecular Modeling in the Design of Novel DPP-4 Inhibitors

Science.gov (United States)

Scapin, Giovanna

Inhibition of dipeptidyl peptidase IV (DPP-4) is a promising new approach for the treatment of type 2 diabetes. DPP-4 is the enzyme responsible for inactivating the incretin hormones glucagon-like peptide 1 (GLP-1) and glucose dependent insulinotropic polypeptide (GIP), two hormones that play important roles in glucose homeostasis. The potent, orally bioavailable and highly selective small molecule DPP-4 inhibitor sitagliptin has been approved by the FDA as novel drug for the treatment of type 2 diabetes. The comparison between the binding mode of sitagliptin (a β-amino acid) and that of a second class of inhibitors (α-amino acid-based) initially led to the successful identification and design of structurally diverse and highly potent DPP-4 inhibitors. Further analysis of the crystal structure of sitagliptin bound to DPP-4 suggested that the central β-amino butanoyl moiety could be replaced by a rigid group. This was confirmed by molecular modeling, and the resulting cyclohexylamine analogs were synthesized and found to be potent DPP-4 inhibitors. However, the triazolopyrazine was predicted to be distorted in order to fit in the binding pocket, and the crystal structure showed that multiple conformations exist for this moiety. Additional molecular modeling studies were then used to improve potency of the cyclohexylamine series. In addition, a 3-D QSAR method was used to gain insight for reducing off-target DPP-8/9 activities. Novel compounds were thus synthesized and found to be potent DPP-4 inhibitors. Two compounds in particular were designed to be highly selective against off-target "DPP-4 Activity- and/or Structure Homologues" (DASH) enzymes while maintaining potency against DPP-4.

Changes in glucose-induced plasma active glucagon-like peptide-1 levels by co-administration of sodium–glucose cotransporter inhibitors with dipeptidyl peptidase-4 inhibitors in rodents

Directory of Open Access Journals (Sweden)

Takahiro Oguma

2016-12-01

Full Text Available We investigated whether structurally different sodium–glucose cotransporter (SGLT 2 inhibitors, when co-administered with dipeptidyl peptidase-4 (DPP4 inhibitors, could enhance glucagon-like peptide-1 (GLP-1 secretion during oral glucose tolerance tests (OGTTs in rodents. Three different SGLT inhibitors—1-(β-d-Glucopyranosyl-4-chloro-3-[5-(6-fluoro-2-pyridyl-2-thienylmethyl]benzene (GTB, TA-1887, and canagliflozin—were examined to assess the effect of chemical structure. Oral treatment with GTB plus a DPP4 inhibitor enhanced glucose-induced plasma active GLP-1 (aGLP-1 elevation and suppressed glucose excursions in both normal and diabetic rodents. In DPP4-deficient rats, GTB enhanced glucose-induced aGLP-1 elevation without affecting the basal level, whereas metformin, previously reported to enhance GLP-1 secretion, increased both the basal level and glucose-induced elevation. Oral treatment with canagliflozin and TA-1887 also enhanced glucose-induced aGLP-1 elevation when co-administered with either teneligliptin or sitagliptin. These data suggest that structurally different SGLT2 inhibitors enhance plasma aGLP-1 elevation and suppress glucose excursions during OGTT when co-administered with DPP4 inhibitors, regardless of the difference in chemical structure. Combination treatment with DPP4 inhibitors and SGLT2 inhibitors having moderate SGLT1 inhibitory activity may be a promising therapeutic option for improving glycemic control in patients with type 2 diabetes mellitus.

Treatment progression in sulfonylurea and dipeptidyl peptidase-4-inhibitor cohorts of type 2 diabetes patients on metformin

Directory of Open Access Journals (Sweden)

Peng X

2016-08-01

Full Text Available Xiaomei Peng, Dingfeng Jiang, Dongju Liu, Oralee J Varnado, Jay P Bae Eli Lilly and Company, Global Patient Outcomes and Real World Evidence, Indianapolis, IN, USA Background: Metformin is an oral antidiabetic drug (OAD widely used as first-line therapy in type 2 diabetes (T2D treatments. Numerous treatment pathways after metformin failure exist. It is important to understand how treatment choices influence subsequent therapy progressions. This retrospective study compares adherence to, persistence with, and treatment progression in sulfonylurea (SU and dipeptidyl peptidase-4 (DPP-4 inhibitor patient cohorts with T2D on metformin. Methods: Using health insurance claims data, matched patient cohorts were created and OAD use was compared in patients with T2D initiating SU or DPP-4 inhibitors (index drugs since January 1, 2010, to December 31, 2010, with background metformin therapy. Propensity score matching adjusted for possible selection bias. Persistence was measured via Cox regression as days to a ≥60-day gap in index drug possession; adherence was defined as proportion of days covered (PDC ≥80%. Evolving treatment patterns were traced at 6-month intervals for 24 months following index drug discontinuation. Results: From among 19,621 and 7,484 patients in the SU and DPP-4 inhibitor cohorts, respectively, 6,758 patient pairs were matched. Persistence at 12 months in the SU cohort was 48.0% compared to 52.5% for the DPP-4 inhibitor cohort. PDC adherence (mean [SD] during the 12-month follow-up period was 63.3 (29.7 for the SU cohort and 65.5 (28.7 for the DPP-4 inhibitor cohort. PDC ≥80% was 40.5% and 43.4% in the SU and DPP-4 inhibitor cohorts, respectively. A higher percentage of patients in the SU cohort remained untreated. Following index drug discontinuation, monotherapy was more common in the SU cohort, while use of two or three OADs was more common in the DPP-4 inhibitor cohort. Insulin therapy initiation was higher in the SU

V3-independent competitive resistance of a dual-X4 HIV-1 to the CXCR4 inhibitor AMD3100.

Directory of Open Access Journals (Sweden)

Yosuke Maeda

Full Text Available A CXCR4 inhibitor-resistant HIV-1 was isolated from a dual-X4 HIV-1 in vitro. The resistant variant displayed competitive resistance to the CXCR4 inhibitor AMD3100, indicating that the resistant variant had a higher affinity for CXCR4 than that of the wild-type HIV-1. Amino acid sequence analyses revealed that the resistant variant harbored amino acid substitutions in the V2, C2, and C4 regions, but no remarkable changes in the V3 loop. Site-directed mutagenesis confirmed that the changes in the C2 and C4 regions were principally involved in the reduced sensitivity to AMD3100. Furthermore, the change in the C4 region was associated with increased sensitivity to soluble CD4, and profoundly enhanced the entry efficiency of the virus. Therefore, it is likely that the resistant variant acquired the higher affinity for CD4/CXCR4 by the changes in non-V3 regions. Taken together, a CXCR4 inhibitor-resistant HIV-1 can evolve using a non-V3 pathway.

Exogenous fatty acid binding protein 4 promotes human prostate cancer cell progression.

Science.gov (United States)

Uehara, Hisanori; Takahashi, Tetsuyuki; Oha, Mina; Ogawa, Hirohisa; Izumi, Keisuke

2014-12-01

Epidemiologic studies have found that obesity is associated with malignant grade and mortality in prostate cancer. Several adipokines have been implicated as putative mediating factors between obesity and prostate cancer. Fatty acid binding protein 4 (FABP4), a member of the cytoplasmic fatty acid binding protein multigene family, was recently identified as a novel adipokine. Although FABP4 is released from adipocytes and mean circulating concentrations of FABP4 are linked with obesity, effects of exogenous FABP4 on prostate cancer progression are unclear. In this study, we examined the effects of exogenous FABP4 on human prostate cancer cell progression. FABP4 treatment promoted serum-induced prostate cancer cell invasion in vitro. Furthermore, oleic acid promoted prostate cancer cell invasion only if FABP4 was present in the medium. These promoting effects were reduced by FABP4 inhibitor, which inhibits FABP4 binding to fatty acids. Immunostaining for FABP4 showed that exogenous FABP4 was taken up into DU145 cells in three-dimensional culture. In mice, treatment with FABP4 inhibitor reduced the subcutaneous growth and lung metastasis of prostate cancer cells. Immunohistochemical analysis showed that the number of apoptotic cells, positive for cleaved caspase-3 and cleaved PARP, was increased in subcutaneous tumors of FABP4 inhibitor-treated mice, as compared with control mice. These results suggest that exogenous FABP4 might promote human prostate cancer cell progression by binding with fatty acids. Additionally, exogenous FABP4 activated the PI3K/Akt pathway, independently of binding to fatty acids. Thus, FABP4 might be a key molecule to understand the mechanisms underlying the obesity-prostate cancer progression link. © 2014 UICC.

A possible usage of a CDK4 inhibitor for breast cancer stem cell-targeted therapy

International Nuclear Information System (INIS)

Han, Yu Kyeong; Lee, Jae Ho; Park, Ga-Young; Chun, Sung Hak; Han, Jeong Yun; Kim, Sung Dae; Lee, Janet; Lee, Chang-Woo; Yang, Kwangmo; Lee, Chang Geun

2013-01-01

Highlights: ► A CDK4 inhibitor may be used for breast cancer stem cell-targeted therapy. ► The CDK4 inhibitor differentiated the cancer stem cell population (CD24 − /CD44 + ) of MDA-MB-231. ► The differentiation of the cancer stem cells by the CDK4 inhibitor radiosensitized MDA-MB-231. -- Abstract: Cancer stem cells (CSCs) are one of the main reasons behind cancer recurrence due to their resistance to conventional anti-cancer therapies. Thus, many efforts are being devoted to developing CSC-targeted therapies to overcome the resistance of CSCs to conventional anti-cancer therapies and decrease cancer recurrence. Differentiation therapy is one potential approach to achieve CSC-targeted therapies. This method involves inducing immature cancer cells with stem cell characteristics into more mature or differentiated cancer cells. In this study, we found that a CDK4 inhibitor sensitized MDA-MB-231 cells but not MCF7 cells to irradiation. This difference appeared to be associated with the relative percentage of CSC-population between the two breast cancer cells. The CDK4 inhibitor induced differentiation and reduced the cancer stem cell activity of MDA-MB-231 cells, which are shown by multiple marker or phenotypes of CSCs. Thus, these results suggest that radiosensitization effects may be caused by reducing the CSC-population of MDA-MB-231 through the use of the CDK4 inhibitor. Thus, further investigations into the possible application of the CDK4 inhibitor for CSC-targeted therapy should be performed to enhance the efficacy of radiotherapy for breast cancer

Differential Effects of Phosphatidylinositol 4-Kinase (PI4K and 3-Kinase (PI3K Inhibitors on Stomatal Responses to Environmental Signals

Directory of Open Access Journals (Sweden)

Koh Iba

2017-05-01

Full Text Available Specific cellular components including products of phosphatidylinositol (PI metabolism play an important role as signaling molecules in stomatal responses to environmental signals. In this study, pharmacological inhibitors of a set of cellular components, including PI4-kinase (PI4K and PI3K, were used to investigate stomatal closure in response to CO2, darkness, and abscisic acid (ABA. Treatment with PAO, a specific inhibitor of PI4K, specifically inhibited the stomatal response to CO2 compared with that to darkness and ABA. In contrast, treatment with LY294002, a PI3K-specific inhibitor, specifically inhibited the stomatal response to darkness compared with that to CO2 and ABA. The specific inhibitory effects of PAO and LY294002 were also observed as changes in the spatial density of dot-like structures labeled by green fluorescent protein-tagged PATROL1, a protein that controls stomatal aperture possibly via regulation of H+-ATPase amount in guard cell plasma membranes. Our results suggest an important role for PI4K and PI3K in the CO2 and darkness signal transduction pathways, respectively, that mediate PATROL1 dynamics.

The heat shock protein 90 inhibitor 17-AAG suppresses growth and induces apoptosis in human cholangiocarcinoma cells.

Science.gov (United States)

Zhang, Jianjun; Zheng, Zhichao; Zhao, Yan; Zhang, Tao; Gu, Xiaohu; Yang, Wei

2013-11-01

The aim of this study was to investigate the effects of 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (HSP90) inhibitor, on the proliferation, cell cycle, and apoptosis of human cholangiocarcinoma (CCA) cells. Cell proliferation and cell cycle distribution were measured by the MTT assay and flow cytometry analysis, respectively. Induction of apoptosis was determined by flow cytometry and Hoechst staining. The expressions of cleaved poly ADP-ribose polymerase (PARP), Bcl-2, Survivin, and Cyclin B1 were detected by Western blot analysis. The activity of caspase-3 was also examined. We found that 17-AAG inhibited cell growth and induced G2/M cell cycle arrest and apoptosis in CCA cells together with the down-regulation of Bcl-2, Survivin and Cyclin B1, and the up-regulation of cleaved PARP. Moreover, increased caspase-3 activity was also observed in CCA cells treated with 17-AAG. In conclusion, our data suggest that the inhibition of HSP90 function by 17-AAG may provide a promising therapeutic strategy for the treatment of human CCA.

17-beta estradiol inhibits oxidative stress-induced accumulation of AIF into nucleolus and PARP1-dependent cell death via estrogen receptor alpha.

Science.gov (United States)

Batnasan, Enkhzaya; Wang, Ruoxi; Wen, Jitao; Ke, Yueshuang; Li, Xiaoxue; Bohio, Ameer Ali; Zeng, Xianlu; Huo, Hongliang; Han, Liping; Boldogh, Istvan; Ba, Xueqing

2015-01-05

Oxidative stress-induced DNA damage results in over-activation of poly(ADP-ribose) polymerase 1 (PARP1), leading to parthanatos, a newly discovered cell elimination pathway. Inhibition of PARP1-dependent cell death has shown to improve the outcome of diseases, including stroke, heart ischemia, and neurodegenerative diseases. In the present study we aimed to detect whether estrogen plays a protective role in inhibiting parthanatos. We utilized human mammary adenocarcinoma cells (MCF7) that abundantly express the estrogen receptor alpha and beta (ERα and ERβ). Parthanatos was induced by challenging the cells with hydrogen peroxide (H2O2). Microscopic imaging and molecular biological techniques, such as Western blot analysis and RNA interference, were performed. The results showed 17β estradiol (E2) protected MCF7 cells from PARP1-dependent cell death by decreasing protein PARylation, and AIF translocation into nuclei/nucleoli. Down-regulation of ERα expression by siRNA before E2 addition resulted in the failure of the E2-mediated inhibition of H2O2-induced protein PARylation and AIF nucleolar translocation. Together these data suggest that estrogen via its alpha-type receptor inhibits oxidative stress-induced, PARP1-dependent cell death. The present study provided us insight into how to apply hormone therapy in intervention of parthanatos-implicated ischemic and degenerative diseases. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Structural exploration for the refinement of anticancer matrix metalloproteinase-2 inhibitor designing approaches through robust validated multi-QSARs

Science.gov (United States)

Adhikari, Nilanjan; Amin, Sk. Abdul; Saha, Achintya; Jha, Tarun

2018-03-01

Matrix metalloproteinase-2 (MMP-2) is a promising pharmacological target for designing potential anticancer drugs. MMP-2 plays critical functions in apoptosis by cleaving the DNA repair enzyme namely poly (ADP-ribose) polymerase (PARP). Moreover, MMP-2 expression triggers the vascular endothelial growth factor (VEGF) having a positive influence on tumor size, invasion, and angiogenesis. Therefore, it is an urgent need to develop potential MMP-2 inhibitors without any toxicity but better pharmacokinetic property. In this article, robust validated multi-quantitative structure-activity relationship (QSAR) modeling approaches were attempted on a dataset of 222 MMP-2 inhibitors to explore the important structural and pharmacophoric requirements for higher MMP-2 inhibition. Different validated regression and classification-based QSARs, pharmacophore mapping and 3D-QSAR techniques were performed. These results were challenged and subjected to further validation to explain 24 in house MMP-2 inhibitors to judge the reliability of these models further. All these models were individually validated internally as well as externally and were supported and validated by each other. These results were further justified by molecular docking analysis. Modeling techniques adopted here not only helps to explore the necessary structural and pharmacophoric requirements but also for the overall validation and refinement techniques for designing potential MMP-2 inhibitors.

Acetyl-CoA Carboxylase-α Inhibitor TOFA Induces Human Cancer Cell Apoptosis

Science.gov (United States)

Wang, Chun; Xu, Canxin; Sun, Mingwei; Luo, Dixian; Liao, Duan-fang; Cao, Deliang

2009-01-01

Acetyl-CoA carboxylase-α (ACCA) is a rate-limiting enzyme in long chain fatty acid synthesis, playing a critical role in cellular energy storage and lipid synthesis. ACCA is upregulated in multiple types of human cancers and small interfering RNA-mediated ACCA silencing in human breast and prostate cancer cells results in oxidative stress and apoptosis. This study reports for the first time that TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, is cytotoxic to lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15, with an IC50 at approximately 5.0, 5.0, and 4.5 μg/ml, respectively. TOFA at 1.0–20.0 μg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Supplementing simultaneously the cells with palmitic acids (100 μM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA. Taken together, these data suggest that TOFA is a potent cytotoxic agent to lung and colon cancer cells, inducing apoptosis through disturbing their fatty acid synthesis. PMID:19450551

Acetyl-CoA carboxylase-alpha inhibitor TOFA induces human cancer cell apoptosis.

Science.gov (United States)

Wang, Chun; Xu, Canxin; Sun, Mingwei; Luo, Dixian; Liao, Duan-Fang; Cao, Deliang

2009-07-31

Acetyl-CoA carboxylase-alpha (ACCA) is a rate-limiting enzyme in long chain fatty acid synthesis, playing a critical role in cellular energy storage and lipid synthesis. ACCA is upregulated in multiple types of human cancers and small interfering RNA-mediated ACCA silencing in human breast and prostate cancer cells results in oxidative stress and apoptosis. This study reports for the first time that TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, is cytotoxic to lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15, with an IC(50) at approximately 5.0, 5.0, and 4.5 microg/ml, respectively. TOFA at 1.0-20.0 microg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Supplementing simultaneously the cells with palmitic acids (100 microM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA. Taken together, these data suggest that TOFA is a potent cytotoxic agent to lung and colon cancer cells, inducing apoptosis through disturbing their fatty acid synthesis.

Synthesis, pharmacological evaluation and molecular docking studies of pyrimidinedione based DPP-4 inhibitors as antidiabetic agents

Science.gov (United States)

Jha, Vibhu; Bhadoriya, Kamlendra Singh

2018-04-01

Dipeptidyl peptidase-4 (DPP-4) inhibitors are a class of newly developed antidiabetic drugs that bock DPP-4. DPP-4 is responsible for degradation of incretins harmones such as GLP-1 (Glucagon like Peptide) and GIP (Gastric inhibitory polypeptide) that maintain blood-glucose level. Pyrimidinedione based compounds were designed and synthesized for DPP-4 inhibitory activity. These heterocycles were designed by taking Alogliptin as a reference DPP-4 inhibitors and synthesized as N-methylated and N-benzylated pyrimidinediones. These compounds were subjected to DPP-4 assay, five out of nine synthesized compounds have shown in vitro DPP-4 inhibitory activity in significant range. Further, molecular docking studies of these compounds were performed on DPP-4 subunit and compared with natural DPP-4 inhibitors like Flavone, Resveratrol, Quercetin, Diprotin A. Docking studies have led to the conclusion that there are some identical amino acid interactions as Tyr 666 and Tyr 662, seen in both synthesized compounds and natural DPP-4 inhibitors. This study completely gives a good scope for further derivatisation and optimization of synthesized compounds to get clinical candidate as DPP-4 inhibitor for antidiabetic activity.

Inhibitor of differentiation 4 (Id4) is a potential tumor suppressor in prostate cancer

International Nuclear Information System (INIS)

Carey, Jason PW; Asirvatham, Ananthi J; Galm, Oliver; Ghogomu, Tandeih A; Chaudhary, Jaideep

2009-01-01

Inhibitor of differentiation 4 (Id4), a member of the Id gene family is also a dominant negative regulator of basic helix loop helix (bHLH) transcription factors. Some of the functions of Id4 appear to be unique as compared to its other family members Id1, Id2 and Id3. Loss of Id4 gene expression in many cancers in association with promoter hypermethylation has led to the proposal that Id4 may act as a tumor suppressor. In this study we provide functional evidence that Id4 indeed acts as a tumor suppressor and is part of a cancer associated epigenetic re-programming. Data mining was used to demonstrate Id4 expression in prostate cancer. Methylation specific polymerase chain reaction (MSP) analysis was performed to understand molecular mechanisms associated with Id4 expression in prostate cancer cell lines. The effect of ectopic Id4 expression in DU145 cells was determined by cell cycle analysis (3H thymidine incorporation and FACS), expression of androgen receptor, p53 and cyclin dependent kinase inhibitors p27 and p21 by a combination of RT-PCR, real time-PCR, western blot and immuno-cytochemical analysis. Id4 expression was down-regulated in prostate cancer. Id4 expression was also down-regulated in prostate cancer line DU145 due to promoter hyper-methylation. Ectopic Id4 expression in DU145 prostate cancer cell line led to increased apoptosis and decreased cell proliferation due in part by an S-phase arrest. In addition to S-phase arrest, ectopic Id4 expression in PC3 cells also resulted in prolonged G2/M phase. At the molecular level these changes were associated with increased androgen receptor (AR), p21, p27 and p53 expression in DU145 cells. The results suggest that Id4 acts directly as a tumor suppressor by influencing a hierarchy of cellular processes at multiple levels that leads to a decreased cell proliferation and change in morphology that is possibly mediated through induction of previously silenced tumor suppressors

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation*

Science.gov (United States)

Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch; Sinning, Steffen; Kristensen, Anders Skov; Strømgaard, Kristian; Andersen, Jacob

2015-01-01

The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu406 is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT. PMID:25903124

Proteasome inhibitor carfilzomib interacts synergistically with histone deacetylase inhibitor vorinostat in Jurkat T-leukemia cells.

Science.gov (United States)

Gao, Minjie; Gao, Lu; Tao, Yi; Hou, Jun; Yang, Guang; Wu, Xiaosong; Xu, Hongwei; Tompkins, Van S; Han, Ying; Wu, Huiqun; Zhan, Fenghuang; Shi, Jumei

2014-06-01

In the present study, we investigated the interactions between proteasome inhibitor carfilzomib (CFZ) and histone deacetylase inhibitor vorinostat in Jurkat T-leukemia cells. Coexposure of cells to minimally lethal concentrations of CFZ with very low concentration of vorinostat resulted in synergistic antiproliferative effects and enhanced apoptosis in Jurkat T-leukemia cells, accompanied with the sharply increased reactive oxygen species (ROS), the striking decrease in the mitochondrial membrane potential (MMP), the increased release of cytochrome c, the enhanced activation of caspase-9 and -3, and the cleavage of PARP. The combined treatment of Jurkat cells pre-treated with ROS scavengers N-acetylcysteine (NAC) significantly blocked the loss of mitochondrial membrane potential, suggesting that ROS generation was a former event of the loss of mitochondrial membrane potential. Furthermore, NAC also resulted in a marked reduction in apoptotic cells, indicating a critical role for increased ROS generation by combined treatment. In addition, combined treatment arrested the cell cycle in G2-M phase. These results imply that CFZ interacted synergistically with vorinostat in Jurkat T-leukemia cells, which raised the possibility that the combination of carfilzomib with vorinostat may represent a novel strategy in treating T-cell Leukemia. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Design and synthesis of N-(4-aminopyridin-2-yl)amides as B-Raf(V600E) inhibitors.

Science.gov (United States)

Li, Xiaokai; Shen, Jiayi; Tan, Li; Zhang, Zhang; Gao, Donglin; Luo, Jinfeng; Cheng, Huimin; Zhou, Xiaoping; Ma, Jie; Ding, Ke; Lu, Xiaoyun

2016-06-15

B-Raf(V600E) was an effective target for the treatment of human cancers. Based on a pan-Raf inhibitor TAK-632, a series of N-(4-aminopyridin-2-yl)amide derivatives were designed as novel B-Raf(V600E) inhibitors. Detailed structure-activity studies of the compounds revealed that most of the compounds displayed potent enzymatic activity against B-Raf(V600E), and good selectivity over B-Raf(WT). One of the most promising compound 4l exhibited potent inhibitory activity with an IC50 value of 38nM for B-raf(V600E), and displayed antiproliferative activities against colo205 and HT29 cells with IC50 values of 0.136 and 0.094μM, respectively. It also displayed good selectivity on both enzymatic and cellular assays over B-Raf(WT). These inhibitors may serve as lead compounds for further developing novel B-Raf(V600E) inhibitors as anticancer drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fragment-based lead discovery of small molecule inhibitors for the EPHA4 receptor tyrosine kinase

NARCIS (Netherlands)

van Linden, O.P.J.; Farenc, C; Zoutman, W.H.; Hameetman, L; Wijtmans, M.; Leurs, R.; Tensen, C.P.; Siegal, G.; de Esch, I.J.P.

2011-01-01

The in silico identification, optimization and crystallographic characterization of a 6,7,8,9-tetrahydro-3H-pyrazolo[3,4-c]isoquinolin-1-amine scaffold as an inhibitor for the EPHA4 receptor tyrosine kinase is described. A database containing commercially available compounds was subjected to an in

Discovery of Novel Tricyclic Heterocycles as Potent and Selective DPP-4 Inhibitors for the Treatment of Type 2 Diabetes

Energy Technology Data Exchange (ETDEWEB)

Wu, Wen-Lian; Hao, Jinsong; Domalski, Martin; Burnett, Duane A.; Pissarnitski, Dmitri; Zhao, Zhiqiang; Stamford, Andrew; Scapin, Giovanna; Gao, Ying-Duo; Soriano, Aileen; Kelly, Terri M.; Yao, Zuliang; Powles, Mary Ann; Chen, Shiying; Mei, Hong; Hwa, Joyce (Merck)

2016-05-12

In our efforts to develop second generation DPP-4 inhibitors, we endeavored to identify distinct structures with long-acting (once weekly) potential. Taking advantage of X-ray cocrystal structures of sitagliptin and other DPP-4 inhibitors, such as alogliptin and linagliptin bound to DPP-4, and aided by molecular modeling, we designed several series of heterocyclic compounds as initial targets. During their synthesis, an unexpected chemical transformation provided a novel tricyclic scaffold that was beyond our original design. Capitalizing on this serendipitous discovery, we have elaborated this scaffold into a very potent and selective DPP-4 inhibitor lead series, as highlighted by compound 17c.

Tetomilast: new promise for phosphodiesterase-4 inhibitors?

Science.gov (United States)

Bickston, Stephen J; Snider, Kenneth R; Kappus, Matthew R

2012-12-01

Tetomilast is a novel thiazole phosphodiesterase-4 (PDE-4) inhibitor, which may prove useful in both the treatment of inflammatory bowel disease (IBD) and chronic obstructive pulmonary disease (COPD). Here, the authors review the pharmacology of the drug, and offer critical review of the available data for use of tetomilast in the treatment of IBD. Peer-reviewed publications, including Phase I and II clinical trials, all other formats included. Tetomilast may be beneficial in IBD. Small differences in molecules and in recombinant proteins can translate into substantial differences in clinical effects and toxicity in IBD. This is a reasonable approach when exploring new options like tetomilast.

Indeno-1-one [2,3-b]quinoxaline as an effective inhibitor for the corrosion of mild steel in 0.5 M H2SO4 solution

International Nuclear Information System (INIS)

Obot, I.B.; Obi-Egbedi, N.O.

2010-01-01

Indeno-1-one [2,3-b] quinoxaline (INQUI), synthesized in our laboratory, was tested as inhibitor for the corrosion of mild steel in 0.5 M H 2 SO 4 using gravimetric method at 30 o C. The inhibitor (INQUI) showed about 81% inhibition efficiency (E (%)) at an optimum concentration of 10 x 10 -6 M. The inhibition efficiency increases with increase in inhibitor concentration but decreases with increase in immersion time. The adsorption of the inhibitor on the mild steel in the acid solution was found to accord with Temkin's adsorption isotherm. The calculated value of the free energy for the adsorption process, ΔG ads , reveals a strong chemisorbed bond between the inhibitor and mild steel surface and a spontaneous adsorption of the inhibitor on the mild steel surface. Density functional theory (DFT) proves that INQUI molecule is adsorbed on the mild steel surface by the most negatively charged nitrogen and oxygen atoms.

The PDE4 inhibitor CHF-6001 and LAMAs inhibit bronchoconstriction-induced remodeling in lung slices

NARCIS (Netherlands)

Kistemaker, Loes E M; Oenema, Tjitske A; Baarsma, Hoeke A; Bos, I. Sophie T.; Schmidt, Martina; Facchinetti, Fabrizio; Civelli, Maurizio; Villetti, Gino; Gosens, Reinoud

2017-01-01

Combination therapy of PDE4 inhibitors and anticholinergics induces bronchoprotection in COPD. Mechanical forces that arise during bronchoconstriction may contribute to airway remodeling. Therefore, we investigated the impact of PDE4 inhibitors and anticholinergics on bronchoconstriction-induced

Predictive Factors for Efficacy of Dipeptidyl Peptidase-4 Inhibitors in Patients with Type 2 Diabetes Mellitus

Directory of Open Access Journals (Sweden)

Shusuke Yagi

2015-08-01

Full Text Available BackgroundPredictive factors for the efficacy of dipeptidyl peptidase-4 (DPP-4 inhibitors for lowering glycosylated hemoglobin (HbA1c remain unclear in patients with type 2 diabetes mellitus. The aim of this study is therefore to clarify predictive factors of the efficacy of DPP-4 inhibitors for lowering HbA1c after 12 months of treatment.MethodsA total of 191 consecutive type 2 diabetic patients (male sex 55%, mean age, 68.3±35.8 years, who had been treated with DPP-4 inhibitors for 12 months, were enrolled in this study and evaluated retrospectively.ResultsAfter 12 months of DPP-4 inhibitor treatment, random blood glucose level, and HbA1c level, decreased from 167±63 to 151±49 mg/dL (P<0.01, and from 7.5%±1.3% to 6.9%±0.9% (P<0.01 respectively, without severe side effects. Multiple regression analysis showed that predictors of DPP-4 inhibitor treatment efficacy in lowering HbA1c level after 12 months were a decrease in HbA1c level after 3 months of treatment, a high baseline HbA1c level, a low baseline body mass index, and the absence of coronary artery disease.ConclusionMost suitable candidates for treatment with DPP-4 inhibitors are diabetics who are not obese and do not have coronary artery disease. In addition, long-term efficacy of DPP-4 inhibitors can be predicted by decrement of HbA1c after 3 months of treatment.

Discovery of Potent and Selective Inhibitors for ADAMTS-4 through DNA-Encoded Library Technology (ELT).

Science.gov (United States)

Ding, Yun; O'Keefe, Heather; DeLorey, Jennifer L; Israel, David I; Messer, Jeffrey A; Chiu, Cynthia H; Skinner, Steven R; Matico, Rosalie E; Murray-Thompson, Monique F; Li, Fan; Clark, Matthew A; Cuozzo, John W; Arico-Muendel, Christopher; Morgan, Barry A

2015-08-13

The aggrecan degrading metalloprotease ADAMTS-4 has been identified as a novel therapeutic target for osteoarthritis. Here, we use DNA-encoded Library Technology (ELT) to identify novel ADAMTS-4 inhibitors from a DNA-encoded triazine library by affinity selection. Structure-activity relationship studies based on the selection information led to the identification of potent and highly selective inhibitors. For example, 4-(((4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-6-(((4-methylpiperazin-1-yl)methyl)amino)-1,3,5-triazin-2-yl)amino)methyl)-N-ethyl-N-(m-tolyl)benzamide has IC50 of 10 nM against ADAMTS-4, with >1000-fold selectivity over ADAMT-5, MMP-13, TACE, and ADAMTS-13. These inhibitors have no obvious zinc ligand functionality.

Computational study on the inhibitor binding mode and allosteric regulation mechanism in hepatitis C virus NS3/4A protein.

Directory of Open Access Journals (Sweden)

Weiwei Xue

Full Text Available HCV NS3/4A protein is an attractive therapeutic target responsible for harboring serine protease and RNA helicase activities during the viral replication. Small molecules binding at the interface between the protease and helicase domains can stabilize the closed conformation of the protein and thus block the catalytic function of HCV NS3/4A protein via an allosteric regulation mechanism. But the detailed mechanism remains elusive. Here, we aimed to provide some insight into the inhibitor binding mode and allosteric regulation mechanism of HCV NS3/4A protein by using computational methods. Four simulation systems were investigated. They include: apo state of HCV NS3/4A protein, HCV NS3/4A protein in complex with an allosteric inhibitor and the truncated form of the above two systems. The molecular dynamics simulation results indicate HCV NS3/4A protein in complex with the allosteric inhibitor 4VA adopts a closed conformation (inactive state, while the truncated apo protein adopts an open conformation (active state. Further residue interaction network analysis suggests the communication of the domain-domain interface play an important role in the transition from closed to open conformation of HCV NS3/4A protein. However, the inhibitor stabilizes the closed conformation through interaction with several key residues from both the protease and helicase domains, including His57, Asp79, Asp81, Asp168, Met485, Cys525 and Asp527, which blocks the information communication between the functional domains interface. Finally, a dynamic model about the allosteric regulation and conformational changes of HCV NS3/4A protein was proposed and could provide fundamental insights into the allosteric mechanism of HCV NS3/4A protein function regulation and design of new potent inhibitors.

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation.

Science.gov (United States)

Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch; Sinning, Steffen; Kristensen, Anders Skov; Strømgaard, Kristian; Andersen, Jacob

2015-06-05

The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu(406) is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Minocycline Blocks Asthma-associated Inflammation in Part by Interfering with the T Cell Receptor-Nuclear Factor κB-GATA-3-IL-4 Axis without a Prominent Effect on Poly(ADP-ribose) Polymerase*

Science.gov (United States)

Naura, Amarjit S.; Kim, Hogyoung; Ju, Jihang; Rodriguez, Paulo C.; Jordan, Joaquin; Catling, Andrew D.; Rezk, Bashir M.; Elmageed, Zakaria Y. Abd; Pyakurel, Kusma; Tarhuni, Abdelmetalab F.; Abughazleh, Mohammad Q.; Errami, Youssef; Zerfaoui, Mourad; Ochoa, Augusto C.; Boulares, A. Hamid

2013-01-01

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N′-nitro-N-nitroso-guanidine-treated mice or H2O2-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production. PMID:23184953

Minocycline blocks asthma-associated inflammation in part by interfering with the T cell receptor-nuclear factor κB-GATA-3-IL-4 axis without a prominent effect on poly(ADP-ribose) polymerase.

Science.gov (United States)

Naura, Amarjit S; Kim, Hogyoung; Ju, Jihang; Rodriguez, Paulo C; Jordan, Joaquin; Catling, Andrew D; Rezk, Bashir M; Abd Elmageed, Zakaria Y; Pyakurel, Kusma; Tarhuni, Abdelmetalab F; Abughazleh, Mohammad Q; Errami, Youssef; Zerfaoui, Mourad; Ochoa, Augusto C; Boulares, A Hamid

2013-01-18

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.

Evaluation of EML4-ALK Fusion Proteins in Non–Small Cell Lung Cancer Using Small Molecule Inhibitors

Directory of Open Access Journals (Sweden)

Yongjun Li

2011-01-01

Full Text Available The echinoderm microtubule–associated protein-like 4–anaplastic lymphoma kinase (EML4-ALK fusion gene resulting from an inversion within chromosome 2p occurs in approximately 5% of non–small cell lung cancer and is mu-tually exclusive with Ras and EGFR mutations. In this study, we have used a potent and selective ALK small molecule inhibitor, NPV-TAE684, to assess the oncogenic role of EML4-ALK in non–small cell lung cancer (NSCLC. We show here that TAE684 inhibits proliferation and induces cell cycle arrest, apoptosis, and tumor regression in two NSCLC models that harbor EML4-ALK fusions. TAE684 inhibits EML4-ALK activation and its downstream signaling including ERK, AKT, and STAT3. We used microarray analysis to carry out targeted pathway studies of gene expression changes in H2228 NSCLC xenograft model after TAE684 treatment and identified a gene signature of EML4-ALK inhibition. The gene signature represents 1210 known human genes, and the top biologic processes represented by these genes are cell cycle, DNA synthesis, cell proliferation, and cell death. We also compared the effect of TAE684 with PF2341066, a c-Met and ALK small molecule inhibitor currently in clinical trial in cancers harboring ALK fusions, and demonstrated that TAE684 is a much more potent inhibitor of EML4-ALK. Our data demonstrate that EML4-ALK plays an important role in the pathogenesis of a subset of NSCLC and provides insight into the mech-anism of EML4-ALK inhibition by a small molecule inhibitor.

Identification of [4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-2-pyrimidinyl] amines and ethers as potent and selective cyclooxygenase-2 inhibitors.

Science.gov (United States)

Swarbrick, Martin E; Beswick, Paul J; Gleave, Robert J; Green, Richard H; Bingham, Sharon; Bountra, Chas; Carter, Malcolm C; Chambers, Laura J; Chessell, Iain P; Clayton, Nick M; Collins, Sue D; Corfield, John A; Hartley, C David; Kleanthous, Savvas; Lambeth, Paul F; Lucas, Fiona S; Mathews, Neil; Naylor, Alan; Page, Lee W; Payne, Jeremy J; Pegg, Neil A; Price, Helen S; Skidmore, John; Stevens, Alexander J; Stocker, Richard; Stratton, Sharon C; Stuart, Alastair J; Wiseman, Joanne O

2009-08-01

A novel series of [4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-2-pyrimidine-based cyclooxygenase-2 (COX-2) inhibitors, which have a different arrangement of substituents compared to the more common 1,2-diarylheterocycle based molecules, have been discovered. For example, 2-(butyloxy)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)pyrimidine (47), a member of the 2-pyrimidinyl ether series, has been shown to be a potent and selective inhibitor with a favourable pharmacokinetic profile, high brain penetration and good efficacy in rat models of hypersensitivity.

Distinct spatio temporal patterns and PARP dependence of XRCC1 recruitment to single-strand break and base excision repair

International Nuclear Information System (INIS)

Campalans, Anna; Kortulewski, Thierry; Amouroux, Rachel; Radicella, J. Pablo; Menoni, Herve; Vermeulen, Wim

2013-01-01

Single-strand break repair (SSBR) and base excision repair (BER) of modified bases and abasic sites share several players. Among them is XRCC1, an essential scaffold protein with no enzymatic activity, required for the coordination of both pathways. XRCC1 is recruited to SSBR by PARP-1, responsible for the initial recognition of the break. The recruitment of XRCC1 to BER is still poorly understood. Here we show by using both local and global induction of oxidative DNA base damage that XRCC1 participation in BER complexes can be distinguished from that in SSBR by several criteria. We show first that XRCC1 recruitment to BER is independent of PARP. Second, unlike SSBR complexes that are assembled within minutes after global damage induction, XRCC1 is detected later in BER patches, with kinetics consistent with the repair of oxidized bases. Third, while XRCC1-containing foci associated with SSBR are formed both in eu- and heterochromatin domains, BER complexes are assembled in patches that are essentially excluded from heterochromatin and where the oxidized bases are detected. (authors)

Inhibitor of differentiation 4 (Id4 is a potential tumor suppressor in prostate cancer

Directory of Open Access Journals (Sweden)

Carey Jason PW

2009-06-01

Full Text Available Abstract Background Inhibitor of differentiation 4 (Id4, a member of the Id gene family is also a dominant negative regulator of basic helix loop helix (bHLH transcription factors. Some of the functions of Id4 appear to be unique as compared to its other family members Id1, Id2 and Id3. Loss of Id4 gene expression in many cancers in association with promoter hypermethylation has led to the proposal that Id4 may act as a tumor suppressor. In this study we provide functional evidence that Id4 indeed acts as a tumor suppressor and is part of a cancer associated epigenetic re-programming. Methods Data mining was used to demonstrate Id4 expression in prostate cancer. Methylation specific polymerase chain reaction (MSP analysis was performed to understand molecular mechanisms associated with Id4 expression in prostate cancer cell lines. The effect of ectopic Id4 expression in DU145 cells was determined by cell cycle analysis (3H thymidine incorporation and FACS, expression of androgen receptor, p53 and cyclin dependent kinase inhibitors p27 and p21 by a combination of RT-PCR, real time-PCR, western blot and immuno-cytochemical analysis. Results Id4 expression was down-regulated in prostate cancer. Id4 expression was also down-regulated in prostate cancer line DU145 due to promoter hyper-methylation. Ectopic Id4 expression in DU145 prostate cancer cell line led to increased apoptosis and decreased cell proliferation due in part by an S-phase arrest. In addition to S-phase arrest, ectopic Id4 expression in PC3 cells also resulted in prolonged G2/M phase. At the molecular level these changes were associated with increased androgen receptor (AR, p21, p27 and p53 expression in DU145 cells. Conclusion The results suggest that Id4 acts directly as a tumor suppressor by influencing a hierarchy of cellular processes at multiple levels that leads to a decreased cell proliferation and change in morphology that is possibly mediated through induction of previously

Emerging role of dipeptidyl peptidase-4 inhibitors in the management of type 2 diabetes

Directory of Open Access Journals (Sweden)

Bernd Richter

2008-08-01

Full Text Available Bernd Richter, Elizabeth Bandeira-Echtler, Karla Bergerhoff, Christian LerchCochrane Metabolic and Endocrine Disorders Group, Department of General Practice, Heinrich-Heine University Duesseldorf, Duesseldorf, GermanyBackground: In type 2 diabetes mellitus (T2DM there is a progressive loss of β-cell function. One new approach yielding promising results is the use of the orally active dipeptidyl peptidase-4 (DPP-4 inhibitors. However, every new compound for T2DM has to prove long-term safety especially on cardiovascular outcomes.Objectives: Systematic review and meta-analysis of the effects of sitagliptin and vildagliptin therapy on main efficacy parameters and safety.Selection criteria, data collection, and analysis: Randomized controlled clinical studies of at least 12 weeks’ duration in T2DM.Results: DPP-4 inhibitors versus placebo showed glycosylated hemoglobin A1c (A1c improvements of 0.7% versus placebo but not compared to monotherapy with other hypoglycemic agents (0.3% in favor of controls. The overall risk profile of DPP-4 inhibitors was low, however a 34% relative risk increase (95% confidence interval 10% to 64%, P = 0.004 was noted for all-cause infection associated with sitagliptin use. No data on immune function, health-related quality of life and diabetic complications could be extracted.Conclusions: DPP-4 inhibitors have some theoretical advantages over existing therapies with oral antidiabetic compounds but should currently be restricted to individual patients. Long-term data on cardiovascular outcomes and safety are needed before widespread use of these new agents.Keywords: DPP-4 inhibitors, sitagliptin, vildagliptin, systematic review, meta-analysis

Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti-inflammatory effects in mdx mice with Duchenne muscular dystrophy.

Science.gov (United States)

Nio, Yasunori; Tanaka, Masayuki; Hirozane, Yoshihiko; Muraki, Yo; Okawara, Mitsugi; Hazama, Masatoshi; Matsuo, Takanori

2017-12-01

Duchenne muscular dystrophy (DMD) is the most common inherited muscular dystrophy. Patients experience DMD in their 20s from cardiac or respiratory failure related to progressive muscle wasting. Currently, the only treatments for the symptoms of DMD are available. Muscle fibrosis, a DMD feature, leads to reduced muscle function and muscle mass, and hampers pharmaceutical therapeutic efficacy. Although antifibrotic agents may be useful, none is currently approved. Phosphodiesterase 4 (PDE4) inhibitors have exhibited antifibrotic effects in human and animal models. In this study, we showed beneficial effects of the PDE4 inhibitor piclamilast in the DMD mdx mouse. Piclamilast reduced the mRNA level of profibrotic genes, including collagen 1A1, in the gastrocnemius and diaphragm, in the mdx mouse, and significantly reduced the Sirius red staining area. The PDE5 inhibitors sildenafil and tadalafil ameliorated functional muscle ischemia in boys with DMD, and sildenafil reversed cardiac dysfunction in the mdx mouse. Single-treatment piclamilast or sildenafil showed similar antifibrotic effects on the gastrocnemius; combination therapy showed a potent antifibrotic effect, and piclamilast and combination therapy increased peroxisome proliferator-activated receptor γ coactivator-1α mRNA in mouse gastrocnemius. In summary, we confirmed that piclamilast has significant antifibrotic effects in mdx mouse muscle and is a potential treatment for muscle fibrosis in DMD.-Nio, Y., Tanaka, M., Hirozane, Y., Muraki, Y., Okawara, M., Hazama, M., Matsuo, T. Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti-inflammatory effects in mdx mice with Duchenne muscular dystrophy. © FASEB.

DNA repair in plants studied by comet assay

Directory of Open Access Journals (Sweden)

Karel J Angelis

2015-06-01

Fig. 2B. Effect of mutation and of inhibitors of PARP1 on SSB repair kinetics. SSBs induced by 1 hr treatment with 2 mM MMS in atparp1 (red and in Arabidopsis wt in presence of 3 mM 3-aminobenzamide (3-ABA, turquoise and 10 μM HsPARP1 specific AG14361 (green inhibitors. (Angelis and Kozák, unpublished data

High resolution crystal structure of rat long chain hydroxy acid oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1, 2, 3-thiadiazole. Implications for inhibitor specificity and drug design

Energy Technology Data Exchange (ETDEWEB)

Chen, Zhi-wei; Vignaud, Caroline; Jaafar, Adil; Lévy, Bernard; Guéritte, Françoise; Guénard, Daniel; Lederer, Florence; Mathews, F. Scott (CNRS-UMR); (WU-MED)

2012-05-24

Long chain hydroxy acid oxidase (LCHAO) is responsible for the formation of methylguanidine, a toxic compound with elevated serum levels in patients with chronic renal failure. Its isozyme glycolate oxidase (GOX), has a role in the formation of oxalate, which can lead to pathological deposits of calcium oxalate, in particular in the disease primary hyperoxaluria. Inhibitors of these two enzymes may have therapeutic value. These enzymes are the only human members of the family of FMN-dependent L-2-hydroxy acid-oxidizing enzymes, with yeast flavocytochrome b{sub 2} (Fcb2) among its well studied members. We screened a chemical library for inhibitors, using in parallel rat LCHAO, human GOX and the Fcb2 flavodehydrogenase domain (FDH). Among the hits was an inhibitor, CCPST, with an IC{sub 50} in the micromolar range for all three enzymes. We report here the crystal structure of a complex between this compound and LCHAO at 1.3 {angstrom} resolution. In comparison with a lower resolution structure of this enzyme, binding of the inhibitor induces a conformational change in part of the TIM barrel loop 4, as well as protonation of the active site histidine. The CCPST interactions are compared with those it forms with human GOX and those formed by two other inhibitors with human GOX and spinach GOX. These compounds differ from CCPST in having the sulfur replaced with a nitrogen in the five-membered ring as well as different hydrophobic substituents. The possible reason for the {approx}100-fold difference in affinity between these two series of inhibitors is discussed. The present results indicate that specificity is an issue in the quest for therapeutic inhibitors of either LCHAO or GOX, but they may give leads for this quest.

Synergistic in-vitro effects of combining an antiglycolytic, 3-bromopyruvate, and a bromodomain-4 inhibitor on U937 myeloid leukemia cells.

Science.gov (United States)

Kapp, Nicolette; Stander, Xiao X; Stander, Barend A

2018-06-01

This project investigated the in-vitro effects of a glycolytic inhibitor, 3-bromopyruvate (3-BrP), in combination with and a new in silico-designed inhibitor of the bromodomain-4 (BRD-4) protein, ITH-47, on the U937 acute myeloid leukemia cell line. 3-BrP is an agent that targets the altered metabolism of cancer cells by interfering with glucose metabolism in the glycolytic pathway. ITH-47 is an acetyl-lysine inhibitor that displaces bromdomain 4 proteins from chromatin by competitively binding to the acetyl-lysine recognition pocket of this bromodomain and extraterminal (BET) BRD protein, thereby preventing transcription of cancer-associated genes and further cell growth. Cell growth studies determined the IC50 after 48 h exposure for 3-BrP and ITH-47 to be 6 and 2 μmol/l, respectively. When combined, 2.4 and 1 μmol/l of 3-BrP and ITH-47, respectively, inhibited 50% of the cell population, yielding a synergistic combination index of 0.9. Subsequent mechanistic studies showed that the IC50 concentrations of ITH-47 and 3-BrP and the combination increased observable apoptotic bodies and cell shrinkage in U937 cells treated for 48 h. Cell cycle analysis showed an increase in the sub-G1 fraction in all treated cells, suggesting that cell death was increased in the treated samples. Annexin-V-FITC apoptosis analysis showed a statistically significant increase in the number of cells in early and late apoptosis, indicating that cell death occurred through apoptosis and not necrosis. Only U937 cells exposed to ITH-47 showed a decrease in mitochondrial membrane potential compared with the vehicle control. Reactive oxygen species production was decreased in all treated samples. ITH-47-exposed cells showed a decrease in c-Myc, Bcl-2, and p53 gene expressions. 3-BrP-treated cells showed an increase in c-myc and p53 gene expressions. The combination of ITH-47 and 3-BrP lead to downregulation of c-myc and Bcl-2 genes. ITH-47 exposure conditions yielded a marked decrease

Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

International Nuclear Information System (INIS)

Kizhakkayil, Jaleel; Thayyullathil, Faisal; Chathoth, Shahanas; Hago, Abdulkader; Patel, Mahendra; Galadari, Sehamuddin

2010-01-01

Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3β (GSK3β), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3β. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.

Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

Energy Technology Data Exchange (ETDEWEB)

Kizhakkayil, Jaleel; Thayyullathil, Faisal; Chathoth, Shahanas; Hago, Abdulkader; Patel, Mahendra [Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain (United Arab Emirates); Galadari, Sehamuddin, E-mail: sehamuddin@uaeu.ac.ae [Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain (United Arab Emirates)

2010-04-09

Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3{beta} (GSK3{beta}), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3{beta}. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.

Poly(ADP-ribose polymerase 1 is indispensable for transforming growth factor-β Induced Smad3 activation in vascular smooth muscle cell.

Directory of Open Access Journals (Sweden)

Dan Huang

Full Text Available BACKGROUND: Transforming growth factor type-β (TGF-β/Smad pathway plays an essential role in vascular fibrosis. Reactive oxygen species (ROS generation also mediates TGF-β signaling-induced vascular fibrosis, suggesting that some sort of interaction exists between Smad and redox pathways. However, the underlying molecular mechanism is largely unknown. This study aims to investigate the influence of poly(ADP-ribose polymerase 1 (PARP1, a downstream effector of ROS, on TGF-β signaling transduction through Smad3 pathway in rat vascular smooth muscle cells (VSMCs. METHODS AND RESULTS: TGF-β1 treatment promoted PARP1 activation through induction of ROS generation in rat VSMCs. TGF-β1-induced phosphorylation and nuclear accumulation of Smad3 was prevented by treatment of cells with PARP inhibitor, 3-aminobenzamide (3AB or N-(6-oxo-5,6-dihydrophenanthridin-2-yl-2-(N,N-dimethylaminoacetami (PJ34, or PARP1 siRNA. TGF-β1 treatment promoted poly(ADP-ribosylation of Smad3 via activation of PARP1 in the nucleus. Poly(ADP-ribosylation enhanced Smad-Smad binding element (SBE complex formation in nuclear extracts and increased DNA binding activity of Smad3. Pretreatment with 3AB, PJ34, or PARP1 siRNA prevented TGF-β1-induced Smad3 transactivation and expression of Smad3 target genes, including collagen Iα1, collagen IIIα1 and tissue inhibitor of metalloproteinase 1, in rat VSMCs. CONCLUSIONS: PARP1 is indispensable for TGF-β1 induced Smad3 activation in rat VSMCs. Targeting PARP1 may be a promising therapeutic approach against vascular diseases induced by dysregulation of TGF-β/Smad3 pathway.

Treating ER+ Breast Cancer with CDK4/6 Inhibitors.

Science.gov (United States)

2017-08-01

Data from the MONARCH2, PALOMA-1, and TREnd trials strongly support using CDK4/6 inhibitors alongside standard endocrine therapy for advanced ER-positive breast cancer. Including these targeted agents not only improves progression-free survival but may reverse acquired resistance to hormone treatment. ©2017 American Association for Cancer Research.

Arsenite-induced ROS/RNS generation causes zinc loss and inhibits the activity of poly(ADP-ribose) polymerase-1.

Science.gov (United States)

Wang, Feng; Zhou, Xixi; Liu, Wenlan; Sun, Xi; Chen, Chen; Hudson, Laurie G; Jian Liu, Ke

2013-08-01

Arsenic enhances the genotoxicity of other carcinogenic agents such as ultraviolet radiation and benzo[a]pyrene. Recent reports suggest that inhibition of DNA repair is an important aspect of arsenic cocarcinogenesis, and DNA repair proteins such as poly(ADP ribose) polymerase (PARP)-1 are direct molecular targets of arsenic. Although arsenic has been shown to generate reactive oxygen/nitrogen species (ROS/RNS), little is known about the role of arsenic-induced ROS/RNS in the mechanism underlying arsenic inhibition of DNA repair. We report herein that arsenite-generated ROS/RNS inhibits PARP-1 activity in cells. Cellular exposure to arsenite, as well as hydrogen peroxide and NONOate (nitric oxide donor), decreased PARP-1 zinc content, enzymatic activity, and PARP-1 DNA binding. Furthermore, the effects of arsenite on PARP-1 activity, DNA binding, and zinc content were partially reversed by the antioxidant ascorbic acid, catalase, and the NOS inhibitor, aminoguanidine. Most importantly, arsenite incubation with purified PARP-1 protein in vitro did not alter PARP-1 activity or DNA-binding ability, whereas hydrogen peroxide or NONOate retained PARP-1 inhibitory activity. These results strongly suggest that cellular generation of ROS/RNS plays an important role in arsenite inhibition of PARP-1 activity, leading to the loss of PARP-1 DNA-binding ability and enzymatic activity. Copyright © 2013 Elsevier Inc. All rights reserved.

Dipeptidyl peptidase-4 inhibitors in type 2 diabetes therapy – focus on alogliptin

Directory of Open Access Journals (Sweden)

Capuano A

2013-09-01

Full Text Available Annalisa Capuano,1 Liberata Sportiello,1 Maria Ida Maiorino,2 Francesco Rossi,1 Dario Giugliano,2 Katherine Esposito3 1Department of Experimental Medicine, 2Department of Medical, Surgical, Neurological, Metabolic Sciences, and Geriatrics, 3Department of Clinical and Experimental Medicine and Surgery, Second University of Naples, Naples, Italy Abstract: Type 2 diabetes mellitus is a complex and progressive disease that is showing an apparently unstoppable increase worldwide. Although there is general agreement on the first-line use of metformin in most patients with type 2 diabetes, the ideal drug sequence after metformin failure is an area of increasing uncertainty. New treatment strategies target pancreatic islet dysfunction, in particular gut-derived incretin hormones. Inhibition of the enzyme dipeptidyl peptidase-4 (DPP-4 slows degradation of endogenous glucagon-like peptide-1 (GLP-1 and thereby enhances and prolongs the action of the endogenous incretin hormones. The five available DPP-4 inhibitors, also known as 'gliptins' (sitagliptin, vildagliptin, saxagliptin, linagliptin, alogliptin, are small molecules used orally with similar overall clinical efficacy and safety profiles in patients with type 2 diabetes. The main differences between the five gliptins on the market include: potency, target selectivity, oral bioavailability, long or short half-life, high or low binding to plasma proteins, metabolism, presence of active or inactive metabolites, excretion routes, dosage adjustment for renal and liver insufficiency, and potential drug–drug interactions. On average, treatment with gliptins is expected to produce a mean glycated hemoglobin (HbA1c decrease of 0.5%–0.8%, with about 40% of diabetic subjects at target for the HbA1c goal <7%. There are very few studies comparing DPP-4 inhibitors. Alogliptin as monotherapy or added to metformin, pioglitazone, glibenclamide, voglibose, or insulin therapy significantly improves glycemic control

Clinical trial simulation methods for estimating the impact of DPP-4 inhibitors on cardiovascular disease

Directory of Open Access Journals (Sweden)

Schuetz CA

2015-06-01

Full Text Available Charles Andy Schuetz,1 Siew Hwa Ong,2 Matthias Blüher3 1Evidera Inc., Bethesda, MD, USA; 2Novartis Pharma AG, Basel, Switzerland; 3Department of Medicine, University of Leipzig, Leipzig, Germany Introduction: Dipeptidyl peptidase-4 (DPP-4 inhibitors are a class of oral antidiabetic agents for the treatment of type 2 diabetes mellitus, which lower blood glucose without causing severe hypoglycemia. However, the first cardiovascular (CV safety trials have only recently reported their results, and our understanding of these therapies remains incomplete. Using clinical trial simulations, we estimated the effectiveness of DPP-4 inhibitors in preventing major adverse cardiovascular events (MACE in a population like that enrolled in the SAVOR-TIMI (the Saxagliptin Assessment of Vascular Outcomes Recorded in Patients with Diabetes Mellitus – Thrombolysis in Myocardial Infarction 53 trial. Methods: We used the Archimedes Model to simulate a clinical trial of individuals (N=11,000 with diagnosed type 2 diabetes and elevated CV risk, based on established disease or multiple risk factors. The DPP-4 class was modeled with a meta-analysis of HbA1c and weight change, pooling results from published trials of alogliptin, linagliptin, saxagliptin, sitagliptin, and vildagliptin. The study treatments were added-on to standard care, and outcomes were tracked for 20 years. Results: The DPP-4 class was associated with an HbA1c drop of 0.66% (0.71%, 0.62% and a weight drop of 0.14 (-0.07, 0.36 kg. These biomarker improvements produced a relative risk (RR for MACE at 5 years of 0.977 (0.968, 0.986. The number needed to treat to prevent one occurrence of MACE at 5 years was 327 (233, 550 in the elevated CV risk population. Conclusion: Consistent with recent trial publications, our analysis indicates that DPP-4 inhibitors do not increase the risk of MACE relative to the standard of care. This study provides insights about the long-term benefits of DPP-4 inhibitors and

Gastrointestinal Adverse Events of Dipeptidyl Peptidase 4 Inhibitors in Type 2 Diabetes: A Systematic Review and Network Meta-analysis.

Science.gov (United States)

Wu, Shanshan; Chai, Sanbao; Yang, Jun; Cai, Ting; Xu, Yang; Yang, Zhirong; Zhang, Yuan; Ji, Linong; Sun, Feng; Zhan, Siyan

2017-09-01

The purpose of this study was to systematically evaluate the effect of dipeptidyl peptidase 4 inhibitors on gastrointestinal adverse events in patients with type 2 diabetes. MEDLINE, Embase, the Cochrane Library, and ClinicalTrials.gov were searched from inception through April 28, 2016. Randomized controlled trials that compared dipeptidyl peptidase 4 inhibitor-based therapies with placebo and other hypoglycemic agents in type 2 diabetes were included. The duration of studies was at least 4 weeks. A total of 165 randomized controlled trials and 122,072 patients were included in the study. Dipeptidyl peptidase 4 inhibitors did not increase the incidence of gastrointestinal adverse events after the treatment with alogliptin (odds ratio [OR] = 0.83; 95% CI, 0.59-1.15), linagliptin (OR = 1.11; 95% CI, 0.92-1.35), saxagliptin (OR = 0.96; 95% CI, 0.80-1.15), sitagliptin (OR = 0.95; 95% CI, 0.64-1.14), teneligliptin (OR = 1.50; 95% CI, 0.81-2.77), and vildagliptin (OR = 0.80; 95% CI, 0.63-1.01) compared with placebo. Compared with glucagon-like peptide 1 receptor agonists, dipeptidyl peptidase 4 inhibitors significantly decreased the incidence of gastrointestinal adverse events with alogliptin (OR = 0.26; 95% CI, 0.15-0.44), linagliptin (OR = 0.43; 95% CI, 0.25-0.74), saxagliptin (OR = 0.28; 95% CI, 0.17-0.46), sitagliptin (OR = 0.24; 95% CI, 0.17-0.35), and vildagliptin (OR = 0.27; 95% CI, 0.18-0.41). Dipeptidyl peptidase 4 inhibitors were not associated with an increased risk of gastrointestinal adverse events relative to metformin and α-glucosidase inhibitors, respectively. The network meta-analysis found that compared with glucagon-like peptide 1 receptor agonists, metformin, and α-glucosidase inhibitor, dipeptidyl peptidase 4 inhibitors are associated with a lower incidence of gastrointestinal adverse events. Copyright © 2017 Elsevier HS Journals, Inc. All rights reserved.

Effects of intracellular iron overload on cell death and identification of potent cell death inhibitors.

Science.gov (United States)

Fang, Shenglin; Yu, Xiaonan; Ding, Haoxuan; Han, Jianan; Feng, Jie

2018-06-11

Iron overload causes many diseases, while the underlying etiologies of these diseases are unclear. Cell death processes including apoptosis, necroptosis, cyclophilin D-(CypD)-dependent necrosis and a recently described additional form of regulated cell death called ferroptosis, are dependent on iron or iron-dependent reactive oxygen species (ROS). However, whether the accumulation of intracellular iron itself induces ferroptosis or other forms of cell death is largely elusive. In present study, we study the role of intracellular iron overload itself-induced cell death mechanisms by using ferric ammonium citrate (FAC) and a membrane-permeable Ferric 8-hydroxyquinoline complex (Fe-8HQ) respectively. We show that FAC-induced intracellular iron overload causes ferroptosis. We also identify 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor GSK2334470 as a potent ferroptosis inhibitor. Whereas, Fe-8HQ-induced intracellular iron overload causes unregulated necrosis, but partially activates PARP-1 dependent parthanatos. Interestingly, we identify many phenolic compounds as potent inhibitors of Fe-8HQ-induced cell death. In conclusion, intracellular iron overload-induced cell death form might be dependent on the intracellular iron accumulation rate, newly identified cell death inhibitors in our study that target ferroptosis and unregulated oxidative cell death represent potential therapeutic strategies against iron overload related diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

Factors Associated with Utilization of Dipeptidyl-4 Inhibitors in Patients with Type 2 Diabetes Mellitus: A Cross-Sectional Retrospective Study

Directory of Open Access Journals (Sweden)

Hasniza Zaman Huri

2014-01-01

Full Text Available Dipeptidyl-4 (DPP-4 inhibitors are oral antidiabetic agents recently introduced to Malaysia. Thus, limited data is available on their utilization patterns and factors associated with their use. This study aims to analyse the utilization patterns of DPP-4 inhibitors, factors that influenced the choice of agent, and the rationale for treatment with DPP-4 inhibitors in patients with type 2 diabetes mellitus. This retrospective study was conducted to address the utilization pattern of DPP-4 inhibitors and factors that influence choice in type 2 diabetes mellitus patients. 299 subjects taking either sitagliptin or vildagliptin from September 2008 to September 2012 were included in the study. Sitagliptin was more frequently prescribed than vildagliptin. Of the patients prescribed DPP-4 inhibitors, 95% received combinations of these and other agents, whereas only 5% were prescribed DPP-4 inhibitors as monotherapy. Factors affecting the utilization of DPP-4 inhibitors included age (P=0.049 and concomitant use of beta blockers (P=0.045 and aspirin (P=0.008. Early identification of factors associated with DPP-4 inhibitors is essential to enhance quality use of the drugs.

Antiviral Activity and Resistance Analysis of NS3/4A Protease Inhibitor Grazoprevir and NS5A Inhibitor Elbasvir in Hepatitis C Virus GT4 Replicons.

Science.gov (United States)

Asante-Appiah, Ernest; Curry, Stephanie; McMonagle, Patricia; Ingravallo, Paul; Chase, Robert; Nickle, David; Qiu, Ping; Howe, Anita; Lahser, Frederick C

2017-07-01

Although genotype 4 (GT4)-infected patients represent a minor overall percentage of the global hepatitis C virus (HCV)-infected population, the high prevalence of the genotype in specific geographic regions coupled with substantial sequence diversity makes it an important genotype to study for antiviral drug discovery and development. We evaluated two direct-acting antiviral agents-grazoprevir, an HCV NS3/4A protease inhibitor, and elbasvir, an HCV NS5A inhibitor-in GT4 replicons prior to clinical studies in this genotype. Following a bioinformatics analysis of available GT4 sequences, a set of replicons bearing representative GT4 clinical isolates was generated. For grazoprevir, the 50% effective concentration (EC 50 ) against the replicon bearing the reference GT4a (ED43) NS3 protease and NS4A was 0.7 nM. The median EC 50 for grazoprevir against chimeric replicons encoding NS3/4A sequences from GT4 clinical isolates was 0.2 nM (range, 0.11 to 0.33 nM; n = 5). The difficulty in establishing replicons bearing NS3/4A resistance-associated substitutions was substantially overcome with the identification of a G162R adaptive substitution in NS3. Single NS3 substitutions D168A/V identified from de novo resistance selection studies reduced grazoprevir antiviral activity by 137- and 47-fold, respectively, in the background of the G162R replicon. For elbasvir, the EC 50 against the replicon bearing the reference full-length GT4a (ED43) NS5A gene was 0.0002 nM. The median EC 50 for elbasvir against chimeric replicons bearing clinical isolates from GT4 was 0.0007 nM (range, 0.0002 to 34 nM; n = 14). De novo resistance selection studies in GT4 demonstrated a high propensity to suppress the emergence of amino acid substitutions that confer high-potency reductions to elbasvir. Phenotypic characterization of the NS5A amino acid substitutions identified (L30F, L30S, M31V, and Y93H) indicated that they conferred 15-, 4-, 2.5-, and 7.5-fold potency losses, respectively, to elbasvir

Veliparib in combination with radiotherapy for the treatment of MGMT unmethylated glioblastoma

OpenAIRE

Jue, Toni Rose; Nozue, Kyoko; Lester, Ashleigh J.; Joshi, Swapna; Schroder, Lisette B. W.; Whittaker, Shane P.; Nixdorf, Sheri; Rapkins, Robert W.; Khasraw, Mustafa; McDonald, Kerrie L.

2017-01-01

Background The O 6 -methylguanine methyltransferase (MGMT) gene is frequently unmethylated in patients with glioblastoma (GBM), rendering them non-responsive to the standard treatment regime of surgery followed by concurrent radiotherapy (RT) and temozolomide. Here, we investigate the efficacy of adding a PARP inhibitor, veliparib, to radiotherapy to treat MGMT unmethylated GBM. Methods The inhibition of PARP with veliparib (ABT-888), a potent and orally bioavailable inhibitor in combination ...

Teneligliptin: a DPP-4 inhibitor for the treatment of type 2 diabetes

Directory of Open Access Journals (Sweden)

Kishimoto M

2013-05-01

Full Text Available Miyako KishimotoDepartment of Diabetes and Metabolic Medicine, Center Hospital, Tokyo, Japan; Diabetes and Metabolism Information Center, Diabetes Research Center, National Center for Global Health and Medicine, Tokyo, JapanAbstract: Dipeptidyl peptidase-4 (DPP-4 inhibitors have recently emerged as a new class of antidiabetic that show favorable results in improving glycemic control with a minimal risk of hypoglycemia and weight gain. Teneligliptin, a novel DPP-4 inhibitor, exhibits a unique structure characterized by five consecutive rings, which produce a potent and long-lasting effect. Teneligliptin is currently used in cases showing insufficient improvement in glycemic control even after diet control and exercise or a combination of diet control, exercise, and sulfonylurea- or thiazolidine-class drugs. In adults, teneligliptin is orally administered at a dosage of 20 mg once daily, which can be increased up to 40 mg per day. Because the metabolites of this drug are eliminated via renal and hepatic excretion, no dose adjustment is necessary in patients with renal impairment. The safety profile of teneligliptin is similar to those of other available DPP-4 inhibitors. However, caution needs to be exercised when administering teneligliptin to patients who are prone to QT prolongation. One study has reported that the postprandial blood glucose-lowering effects of teneligliptin administered prior to breakfast were sustained throughout the day, and the effects observed after dinner were similar to those observed after breakfast or lunch. Thus, although clinical data for this new drug are limited, this drug shows promise in stabilizing glycemic fluctuations throughout the day and consequently suppressing the progression of diabetic complications. However, continued evaluation in long-term studies and clinical trials is required to evaluate the efficacy and safety of the drug as well as to identify additional indications for its clinical use

Effects of an inhibitor of the γ-secretase complex on proliferation and apoptotic parameters in a FOXL2-mutated granulosa tumor cell line (KGN).

Science.gov (United States)

Irusta, Griselda; Pazos, Maria Camila; Maidana, Camila Pazos; Abramovich, Dalhia; De Zúñiga, Ignacio; Parborell, Fernanda; Tesone, Marta

2013-07-01

Ovarian granulosa cell tumors (GCTs) represent 3%-5% of all ovarian malignancies. Treatments have limited proven efficacy and biologically targeted treatment is lacking. The aim of this study was to investigate the role of Notch signaling in the proliferation, steroidogenesis, apoptosis, and phosphatidylinositol 3-kinase (PI3K)/AKT pathway in a FOXL2-mutated granulosa tumor cell line (KGN) representative of the adult form of GCTs. When Notch signaling is initiated, the receptors expose a cleavage site in the extracellular domain to the metalloproteinase TACE and, following this cleavage, Notch undergoes another cleavage mediated by the presenilin-gamma-secretase complex. To achieve our goal, DAPT, an inhibitor of the gamma-secretase complex, was used to investigate the role of the Notch system in parameters associated with cell growth and death, using a human granulosa cell tumor line (KGN) as an experimental model. We observed that JAGGED1, DLL4, NOTCH1, and NOTCH4 were highly expressed in KGN cells as compared to granulosa-lutein cells obtained from assisted reproductive techniques patients. The proliferation and viability of KGN cells, as well as progesterone and estradiol production, decreased in the presence of 20 μM DAPT. Apoptotic parameters like PARP and caspase 8 cleavages, BAX, and BCLXs increased in KGN cells cultured with DAPT, whereas others such as BCL2, BCLXl, FAS, and FAS ligand did not change. AKT phosphorylation decreased and PTEN protein increased when Notch signaling was inhibited in KGN cells. We conclude that the Notch system acts as a survival pathway in KGN cells, and might be interacting with the PI3K/AKT pathway.

A comparison of the inhibitory activity of selective PDE4 inhibitors on eosinophil recruitment in guinea pig skin

Directory of Open Access Journals (Sweden)

Mauro M Teixeira

1997-12-01

Full Text Available The elevation of intracellular cyclic AMP by phosphodiesterase (PDE4 inhibitors in eosinophils is associated with inhibition of the activation and recruitment of these cells. We have previously shown that systemic treatment with the PDE4 inhibitor rolipram effectively inhibt eosinophil migration in guinea pig skin. In the present study we compare the oral potency and efficacy of the PDE4 inhibitors rolipram, RP 73401 and CDP 840 on allergic and PAF-induced eosinophil recruitment. Rolipram and RP 73401 were equally effective and potent when given by the oral route and much more active than the PDE4 inhibitor CDP 840. We suggest that this guinea pig model of allergic and mediator-induced eosinophil recruitment is both a sensitive and simple tool to test the efficacy and potency of PDE4 inhibitors in vivo.

A natural product-like JAK2/STAT3 inhibitor induces apoptosis of malignant melanoma cells.

Directory of Open Access Journals (Sweden)

Ke-Jia Wu

Full Text Available The JAK2/STAT3 signaling pathway plays a critical role in tumorigenesis, and has been suggested as a potential molecular target for anti-melanoma therapeutics. However, few JAK2 inhibitors were being tested for melanoma therapy. In this study, eight amentoflavone analogues were evaluated for their activity against human malignant melanoma cells. The most potent analogue, compound 1, inhibited the phosphorylation of JAK2 and STAT3 in human melanoma cells, but had no discernible effect on total JAK2 and STAT3 levels. A cellular thermal shift assay was performed to identify that JAK2 is engaged by 1 in cell lysates. Moreover, compound 1 showed higher antiproliferative activity against human melanoma A375 cells compared to a panel of cancer and normal cell lines. Compound 1 also activated caspase-3 and cleaved PARP, which are markers of apoptosis, and suppressed the anti-apoptotic Bcl-2 level. Finally, compound 1 induced apoptosis in 80% of treated melanoma cells. To our knowledge, compound 1 is the first amentoflavone-based JAK2 inhibitor to be investigated for use as an anti-melanoma agent.

Small-Molecule Inhibitors Targeting DNA Repair and DNA Repair Deficiency in Research and Cancer Therapy.

Science.gov (United States)

Hengel, Sarah R; Spies, M Ashley; Spies, Maria

2017-09-21

To maintain stable genomes and to avoid cancer and aging, cells need to repair a multitude of deleterious DNA lesions, which arise constantly in every cell. Processes that support genome integrity in normal cells, however, allow cancer cells to develop resistance to radiation and DNA-damaging chemotherapeutics. Chemical inhibition of the key DNA repair proteins and pharmacologically induced synthetic lethality have become instrumental in both dissecting the complex DNA repair networks and as promising anticancer agents. The difficulty in capitalizing on synthetically lethal interactions in cancer cells is that many potential targets do not possess well-defined small-molecule binding determinates. In this review, we discuss several successful campaigns to identify and leverage small-molecule inhibitors of the DNA repair proteins, from PARP1, a paradigm case for clinically successful small-molecule inhibitors, to coveted new targets, such as RAD51 recombinase, RAD52 DNA repair protein, MRE11 nuclease, and WRN DNA helicase. Copyright © 2017 Elsevier Ltd. All rights reserved.

First Russian DPP-4 inhibitor Gosogliptin comparing to Vildagliptin in type 2 diabetes mellitus patients

Directory of Open Access Journals (Sweden)

Karina Oganesovna Galstyan

2015-01-01

The results showed that gosogliptin is an effective hypoglycaemic agent from the group of DPP-4 inhibitors and can be recommended for use in patients with T2DM both as monotherapy and in combination with metformin.

Investigating CH4 production in an oxic plant-soil system -a new approach combining isotopic labelling (13C) and inhibitors

Science.gov (United States)

Lenhart, Katharina; Keppler, Frank

2017-04-01

Typically, aerated soil are net sinks of atmospheric methane (CH4), being highest in native ecosystems (pristine forests > managed forests > grasslands > crop fields). However, this does not exclude a simultaneous endogenic CH4 production in the plant-soil system, which cannot be detected simply via CH4 flux measurements. Methanogenic archaea producing CH4 under anoxic conditions were thought to be the only biotic source of CH4 in the soil. However, until recently a non-archaeal pathway of CH4 formation is known where CH4 is produced under oxic conditions in plants (Keppler et al. 2006) and fungi (Lenhart et al. 2012). Additionally, abiotic formation of CH4 from soil organic matter was reported (Jugold et al. 2012) and may be ubiquitous in terrestrial ecosystems. The major goal of this project was to determine soil endogenic CH4 sources and to estimate their contribution to the endogenic CH4 production. Especially the effect of plants and fungi on soil CH4 production was investigated. Therefore, a series of experiments was carried out on field fresh soil collected in a grassland and a forest ecosystem under controlled laboratory conditions. By combining selective inhibitors and 13C labelling, CH4 production rates of several CH4 sources were quantified. The major difficulty was to detect the comparatively small flux of CH4 production against the background of the high CH4 consumption rates due to methanotrophic bacteria. Therefore, we supplemented bare soil and soil with vegetation with selective inhibitors and 13C labelled substrates in a closed chamber system. In a first step, CH4 production was determined by the inhibition of CH4 oxidizing bacteria with Difluoromethane (DFM, 2ml l-1). In the following, a 13C labelled substrate (either CO2, Acetate, or Methionine -S-CH3 labelled) was added in combination with a specific inhibitor -either for archaeal methanogenesis (Bromoethanesulfonate), bacteria (Streptomycin), or fungi (Captan, Cycloheximide). Gas samples were

The Effect of Adding Corrosion Inhibitors into an Electroless Nickel Plating Bath for Magnesium Alloys

Science.gov (United States)

Hu, Rong; Su, Yongyao; Liu, Hongdong; Cheng, Jiang; Yang, Xin; Shao, Zhongcai

2016-10-01

In this work, corrosion inhibitors were added into an electroless nickel plating bath to realize nickel-phosphorus (Ni-P) coating deposition on magnesium alloy directly. The performance of five corrosion inhibitors was evaluated by inhibition efficiency. The results showed that only ammonium hydrogen fluoride (NH4HF2) and ammonium molybdate ((NH4)2MoO4) could be used as corrosion inhibitors for magnesium alloy in the bath. Moreover, compounding NH4HF2 and (NH4)2MoO4, the optimal concentrations were both at 1.5 ~ 2%. The deposition process of Ni-P coating was observed by using a scanning electron microscope (SEM). It showed corrosion inhibitors inhibited undesired dissolution of magnesium substrate during the electroless plating process. In addition, SEM observation indicated that the corrosion inhibition reaction and the Ni2+ replacement reaction were competitive at the initial deposition time. Both electrochemical analysis and thermal shock test revealed that the Ni-P coating exhibited excellent corrosion resistance and adhesion properties in protecting the magnesium alloy.

Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

International Nuclear Information System (INIS)

Fahrer, Joerg; Wagner, Silvia; Buerkle, Alexander; Koenigsrainer, Alfred

2009-01-01

Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

Energy Technology Data Exchange (ETDEWEB)

Fahrer, Joerg, E-mail: joerg.fahrer@uni-ulm.de [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Wagner, Silvia [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany); Buerkle, Alexander [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Koenigsrainer, Alfred [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany)

2009-08-14

Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

Yttrium 3-(4-nitrophenyl)-2-propenoate used as inhibitor against copper alloy corrosion in 0.1 M NaCl solution

International Nuclear Information System (INIS)

Nam, Nguyen Dang; Thang, Vo Quoc; Hoai, Nguyen To; Hien, Pham Van

2016-01-01

Highlights: • Yttrium 3-(4-nitrophenyl)-2-propenoate has been studied as an effective corrosion inhibitor for copper. • A high inhibition performance is attributed to the forming protective inhibiting deposits. • Yttrium 3-(4-nitrophenyl)-2-propenoate mitigates corrosion by promoting random distribution of minor anodes. - Abstract: Yttrium 3-(4-nitrophenyl)-2-propenoate has been studied as an effective corrosion inhibitor for copper alloy in 0.1 M chloride solution. The results show that the surface of copper alloy coupons exposed to solutions containing 0.45 mM yttrium 3-(4-nitrophenyl)-2-propenoate had no signs of corrosion attack due to protective film formation, whereas the surface of copper alloy coupons exposed to non-inhibitor and lower concentrations of yttrium 3-(4-nitrophenyl)-2-propenoate containing solutions were severely corroded. A high inhibition performance is attributed to the forming protective inhibiting deposits that slow down the electrochemical corrosion reactions and mitigate corrosion by promoting random distribution of minor anodes.

Pyrogallol, ROS generator inhibits As4.1 juxtaglomerular cells via cell cycle arrest of G2 phase and apoptosis

International Nuclear Information System (INIS)

Park, Woo Hyun; Han, Yong Hwan; Kim, Suhn Hee; Kim, Sung Zoo

2007-01-01

Pyrogallol as a catechin compound has been employed as an O 2 · - generator and often used to investigate the role of ROS in the biological system. Here, we investigated the in vitro effect of pyrogallol on cell growth, cell cycle and apoptosis in As4.1 juxtaglomerular cells. Dose-dependent inhibition of cell growth was observed with IC 50 of about 60 μM for 48 h using MTT assay. Pyrogallol (100 μM) did not alter intracellular H 2 O 2 level and catalase activity, but increased the intracellular O 2 · - level and decreased SOD activity in As4.1 cells. DNA flow cytometric analysis indicated that 50 and 100 μM pyrogallol significantly increased G2 phase cells as compared with those of pyrogallol-untreated cells. Also, pyrogallol induced apoptosis as evidenced by flow cytometric detection of sub-G1 DNA content, annexin V binding assay and DAPI staining. This apoptosis process was accompanied with the loss of mitochondrial transmembrane potential (ΔΨ m ), Bcl-2 decrease, caspase-3 activation and PARP cleavage. Pan caspase inhibitor (Z-VAD) could significantly rescue As4.1 cells from pyrogallol-induced cell death. But, the inhibitors of caspase-3, caspase-8, and caspase-9 did not prevent apoptotic events in pyrogallol-treated As4.1 cells. Taken together, we have demonstrated that an ROS inducer, pyrogallol inhibits the growth of As4.1 JG cells via cell cycle arrest and apoptosis, and suggest that the compound exhibits an anti-proliferative efficacy on these cells

CDK4/6 inhibitor PD0332991 in glioblastoma treatment: does it have a future?

Directory of Open Access Journals (Sweden)

Lisette eSchroder

2015-11-01

Full Text Available Glioblastoma is aggressive, highly infiltrating, and the most frequent malignant form of brain cancer. With a median survival time of only 14.6 months, when treated with the standard of care, it is essential to find new therapeutic options. A specific CDK4/6 inhibitor, PD0332991, obtained accelerated approval from the Food and Drug Administration for the treatment of patients with advanced estrogen receptor-positive and HER2-negative breast cancer. Common alterations in the cyclin D1-Cyclin Dependent Kinase 4/6-Retinoblastoma 1 pathway in glioblastoma make PD0332991 also an interesting drug for the treatment of glioblastoma. Promising results in in vitro studies, where patient derived glioblastoma cell lines showed sensitivity to PD0332991, gave motive to start in vivo studies. Outcomes of these studies have been contrasting in terms of PD0332991 efficacy within the brain: more research is necessary to conclude whether CDK4/6 inhibitor can be beneficial in the treatment of glioblastoma.

Identification of quinazoline based inhibitors of IRAK4 for the treatment of inflammation

Energy Technology Data Exchange (ETDEWEB)

Smith, Graham F.; Altman, Michael D.; Andresen, Brian; Baker, James; Brubaker, Jason D.; Chen, Hongmin; Chen, Yiping; Childers, Matthew; Donofrio, Anthony; Ferguson, Heidi; Fischer, Christian; Fischmann, Thierry O.; Gibeau, Craig; Hicks, Alexander; Jin, Sue; Kattar, Sam; Kleinschek, Melanie A.; Leccese, Erica; Lesburg, Charles; Li, Chaomin; Lim, Jongwon; Liu, Duan; Maclean, John K.F.; Mansoor, Faruk; Moy, Lilly Y.; Mulrooney, Erin F.; Necheva, Antoaneta S.; Presland, Jeremy; Rakhilina, Larissa; Yang, Ruojing; Torres, Luis; Zhang-Hoover, Jie; Northrup, Alan (Merck); (Oncorus); (Theravance Biopharma); (AstraZeneca); (Blueprint Medicines)

2017-06-01

Interleukin-1 receptor associated kinase 4 (IRAK4) has been implicated in IL-1R and TLR based signaling. Therefore selective inhibition of the kinase activity of this protein represents an attractive target for the treatment of inflammatory diseases. Medicinal chemistry optimization of high throughput screening (HTS) hits with the help of structure based drug design led to the identification of orally-bioavailable quinazoline based IRAK4 inhibitors with excellent pharmacokinetic profile and kinase selectivity. These highly selective IRAK4 compounds show activity in vivo via oral dosing in a TLR7 driven model of inflammation.

Increase of Intracellular Cyclic AMP by PDE4 Inhibitors Affects HepG2 Cell Cycle Progression and Survival.

Science.gov (United States)

Massimi, Mara; Cardarelli, Silvia; Galli, Francesca; Giardi, Maria Federica; Ragusa, Federica; Panera, Nadia; Cinque, Benedetta; Cifone, Maria Grazia; Biagioni, Stefano; Giorgi, Mauro

2017-06-01

Type 4 cyclic nucleotide phosphodiesterases (PDE4) are major members of a superfamily of enzymes (PDE) involved in modulation of intracellular signaling mediated by cAMP. Broadly expressed in most human tissues and present in large amounts in the liver, PDEs have in the last decade been key therapeutic targets for several inflammatory diseases. Recently, a significant body of work has underscored their involvement in different kinds of cancer, but with no attention paid to liver cancer. The present study investigated the effects of two PDE4 inhibitors, rolipram and DC-TA-46, on the growth of human hepatoma HepG2 cells. Treatment with these inhibitors caused a marked increase of intracellular cAMP level and a dose- and time-dependent effect on cell growth. The concentrations of inhibitors that halved cell proliferation to about 50% were used for cell cycle experiments. Rolipram (10 μM) and DC-TA-46 (0.5 μM) produced a decrease of cyclin expression, in particular of cyclin A, as well as an increase in p21, p27 and p53, as evaluated by Western blot analysis. Changes in the intracellular localization of cyclin D1 were also observed after treatments. In addition, both inhibitors caused apoptosis, as demonstrated by an Annexin-V cytofluorimetric assay and analysis of caspase-3/7 activity. Results demonstrated that treatment with PDE4 inhibitors affected HepG2 cell cycle and survival, suggesting that they might be useful as potential adjuvant, chemotherapeutic or chemopreventive agents in hepatocellular carcinoma. J. Cell. Biochem. 118: 1401-1411, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Enhanced effects by 4-phenylbutyrate in combination with RTK inhibitors on proliferation in brain tumor cell models

International Nuclear Information System (INIS)

Marino, Ana-Maria; Sofiadis, Anastasios; Baryawno, Ninib; Johnsen, John Inge; Larsson, Catharina; Vukojevic, Vladana; Ekstroem, Tomas J.

2011-01-01

Highlights: → The histone deacetylase inhibitor 4-phenylbutyrate substantially enhance efficacy of the receptor tyrosine kinase inhibitors gefitinib or vandetanib in glioma and medulloblastoma cell lines. → Cell death increases and clonogenic survival is reduced in the combination treatments, over mono-therapy. → Combination treatments with these drugs may improve clinical outcome for cancer therapy. -- Abstract: We have investigated in vitro effects of anticancer therapy with the histone deacetylase inhibitor (HDACi) 4-phenylbutyrate (4-PB) combined with receptor tyrosine kinase inhibitors (RTKi) gefitinib or vandetanib on the survival of glioblastoma (U343MGa) and medulloblastoma (D324Med) cells. In comparison with individual effects of these drugs, combined treatment with gefitinib/4-PB or vandetanib/4-PB resulted in enhanced cell killing and reduced clonogenic survival in both cell lines. Our results suggest that combined treatment using HDACi and RTKi may beneficially affect the outcome of cancer therapy.

New 5-benzylidenethiazolidin-4-one inhibitors of bacterial MurD ligase: design, synthesis, crystal structures, and biological evaluation.

Science.gov (United States)

Zidar, Nace; Tomašić, Tihomir; Šink, Roman; Kovač, Andreja; Patin, Delphine; Blanot, Didier; Contreras-Martel, Carlos; Dessen, Andréa; Premru, Manica Müller; Zega, Anamarija; Gobec, Stanislav; Mašič, Lucija Peterlin; Kikelj, Danijel

2011-11-01

Mur ligases (MurC-MurF), a group of bacterial enzymes that catalyze four consecutive steps in the formation of cytoplasmic peptidoglycan precursor, are becoming increasingly adopted as targets in antibacterial drug design. Based on the crystal structure of MurD cocrystallized with thiazolidine-2,4-dione inhibitor I, we have designed, synthesized, and evaluated a series of improved glutamic acid containing 5-benzylidenerhodanine and 5-benzylidenethiazolidine-2,4-dione inhibitors of MurD with IC(50) values up to 28 μM. Inhibitor 37, with an IC(50) of 34 μM, displays a weak antibacterial activity against S. aureus ATCC 29213 and E. faecalis ATCC 29212 with minimal inhibitory concentrations of 128 μg/mL. High-resolution crystal structures of MurD in complex with two new inhibitors (compounds 23 and 51) reveal details of their binding modes within the active site and provide valuable information for further structure-based optimization. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Catecholic amides as potential selective phosphodiesterase 4D inhibitors: Design, synthesis, pharmacological evaluation and structure-activity relationships.

Science.gov (United States)

Zhou, Zhong-Zhen; Ge, Bing-Chen; Chen, Yu-Fang; Shi, Xiu-Dong; Yang, Xue-Mei; Xu, Jiang-Ping

2015-11-15

In this study, a series of catechol-based amides (8a-n) with different amide linkers linking the catecholic moiety to the terminal phenyl ring was designed and synthesized as potent phosphodiesterase (PDE) 4D inhibitors. The inhibitory activities of these compounds were evaluated against the core catalytic domains of human PDE4 (PDE4CAT), full-length PDE4B1 and PDE4D7 enzymes, and other PDE family members. The results indicated the majority of compounds 8a-n displayed moderate to good inhibitory activities against PDE4CAT. Among these compounds, compound 8 j with a short amide linker (-CONHCH2-) displayed comparable PDE4CAT inhibitory activity (IC50=410 nM) with rolipram. More interestingly, compound 8 g, a potent and selective PDE4D inhibitor (IC50=94 nM), exhibited a 10-fold selectivity over the PDE4B subtypes and an over 1000-fold selectivity against other PDE family members. Docking simulations suggested that 8 g forms three extra H-bonds with the N-H of residue Asn487 and two water molecules. Copyright © 2015 Elsevier Ltd. All rights reserved.

Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis.

Science.gov (United States)

Loor, Gabriel; Kondapalli, Jyothisri; Schriewer, Jacqueline M; Chandel, Navdeep S; Vanden Hoek, Terry L; Schumacker, Paul T

2010-12-15

Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, whereas other studies implicate the activation of the mitochondrial permeability transition pore as the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, whereas it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetylcysteine and exogenous glutathione or by overexpression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells overexpressing Cu,Zn-SOD or Mn-SOD. Overexpression of antiapoptotic Bcl-X(L) protected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D, or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochrome c, Bax/Bak, caspase-9, and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them. Copyright © 2010 Elsevier Inc. All rights reserved.

Inhibitor for the Corrosion of Mild Steel in H 2 SO 4 | Patel | South ...

African Journals Online (AJOL)

An extract of Terminalia chebula fruits was investigated as a corrosion inhibitor of mild steel in 0.5 M H2SO4 by means of conventional mass loss, electrochemical polarization, electrochemical impedance spectroscopy and scanning electron microscopy. The mass loss results showed that the extract of Terminalia chebula is ...

Poly(ADP-ribose polymerase inhibition reveals a potential mechanism to promote neuroprotection and treat neuropathic pain

Directory of Open Access Journals (Sweden)

Prashanth Komirishetty

2016-01-01

Full Text Available Neuropathic pain is triggered by the lesions to peripheral nerves which alter their structure and function. Neuroprotective approaches that limit the pathological changes and improve the behavioral outcome have been well explained in different experimental models of neuropathy but translation of such strategies to clinics has been disappointing. Experimental evidences revealed the role of free radicals, especially peroxynitrite after the nerve injury. They provoke oxidative DNA damage and consequent over-activation of the poly(ADP-ribose polymerase (PARP upregulates pro-inflammatory pathways, causing bioenergetic crisis and neuronal death. Along with these changes, it causes mitochondrial dysfunction leading to neuronal apoptosis. In related preclinical studies agents that neutralize the free radicals and pharmacological inhibitors of PARP have shown benefits in treating experimental neuropathy. This article reviews the involvement of PARP over-activation in trauma induced neuropathy and therapeutic significance of PARP inhibitors in the experimental neuropathy and neuropathic pain.

Poly(ADP-ribose) polymerase inhibition reveals a potential mechanism to promote neuroprotection and treat neuropathic pain.

Science.gov (United States)

Komirishetty, Prashanth; Areti, Aparna; Gogoi, Ranadeep; Sistla, Ramakrishna; Kumar, Ashutosh

2016-10-01

Neuropathic pain is triggered by the lesions to peripheral nerves which alter their structure and function. Neuroprotective approaches that limit the pathological changes and improve the behavioral outcome have been well explained in different experimental models of neuropathy but translation of such strategies to clinics has been disappointing. Experimental evidences revealed the role of free radicals, especially peroxynitrite after the nerve injury. They provoke oxidative DNA damage and consequent over-activation of the poly(ADP-ribose) polymerase (PARP) upregulates pro-inflammatory pathways, causing bioenergetic crisis and neuronal death. Along with these changes, it causes mitochondrial dysfunction leading to neuronal apoptosis. In related preclinical studies agents that neutralize the free radicals and pharmacological inhibitors of PARP have shown benefits in treating experimental neuropathy. This article reviews the involvement of PARP over-activation in trauma induced neuropathy and therapeutic significance of PARP inhibitors in the experimental neuropathy and neuropathic pain.

DPP-4 inhibitor des-F-sitagliptin treatment increased insulin exocytosis from db/db mice {beta} cells

Energy Technology Data Exchange (ETDEWEB)

Nagamatsu, Shinya, E-mail: shinya@ks.kyorin-u.ac.jp [Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611 (Japan); Ohara-Imaizumi, Mica; Nakamichi, Yoko; Aoyagi, Kyota; Nishiwaki, Chiyono [Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611 (Japan)

2011-09-09

Highlights: {yields} Anti-diabetic new drug, DPP-4 inhibitor, can affect the insulin exocytosis. {yields} DPP-4 inhibitor treatment altered syntaxin 1 expression. {yields} Treatment of db/db mice with DPP-4 inhibitor increased insulin release. -- Abstract: Incretin promotes insulin secretion acutely. Recently, orally-administered DPP-4 inhibitors represent a new class of anti-hyperglycemic agents. Indeed, inhibitors of dipeptidyl peptidase-IV (DPP-4), sitagliptin, has just begun to be widely used as therapeutics for type 2 diabetes. However, the effects of sitagliptin-treatment on insulin exocytosis from single {beta}-cells are yet unknown. We therefore investigated how sitagliptin-treatment in db/db mice affects insulin exocytosis by treating db/db mice with des-F-sitagliptin for 2 weeks. Perfusion studies showed that 2 weeks-sitagliptin treatment potentiated insulin secretion. We then analyzed insulin granule motion and SNARE protein, syntaxin 1, by TIRF imaging system. TIRF imaging of insulin exocytosis showed the increased number of docked insulin granules and increased fusion events from them during first-phase release. In accord with insulin exocytosis data, des-F-sitagliptin-treatment increased the number of syntaxin 1 clusters on the plasma membrane. Thus, our data demonstrated that 2-weeks des-F-sitagliptin-treatment increased the fusion events of insulin granules, probably via increased number of docked insulin granules and that of syntaxin 1 clusters.

Effects of selective phosphodiesterases-4 inhibitors on learning and memory: a review of recent research.

Science.gov (United States)

Peng, Sheng; Sun, Haiyan; Zhang, Xiaoqing; Liu, Gongjian; Wang, Guanglei

2014-09-01

Phosphodiesterase-4 (PDE-4) regulates the intracellular level of cyclic adenosine monophosphate. Recent studies demonstrated that PDE-4 inhibitors can counteract deficits in long-term memory caused by aging or increased expression of mutant forms of human amyloid precursor proteins, and can influence the process of memory function and cognitive enhancement. Therapeutics, such as ketamine, a drug used in clinical anesthesia, can also cause memory deficits as adverse effects. Targeting PDE-4 with selective inhibitors may offer a novel therapeutic strategy to prevent, slow the progress, and, eventually, treat memory deficits.

Efficacy of cyclin dependent kinase 4 inhibitors as potent neuroprotective agents against insults relevant to Alzheimer's disease.

Directory of Open Access Journals (Sweden)

Priyankar Sanphui

Full Text Available Alzheimer's disease (AD is a progressive neurodegenerative disease with no cure till today. Aberrant activation of cell cycle regulatory proteins is implicated in neurodegenerative diseases including AD. We and others have shown that Cyclin dependent kinase 4 (Cdk4 is activated in AD brain and is required for neuron death. In this study, we tested the efficiency of commercially available Cdk4 specific inhibitors as well as a small library of synthetic molecule inhibitors targeting Cdk4 as neuroprotective agents in cellular models of neuron death. We found that several of these inhibitors significantly protected neuronal cells against death induced by nerve growth factor (NGF deprivation and oligomeric beta amyloid (Aβ that are implicated in AD. These neuroprotective agents inhibit specifically Cdk4 kinase activity, loss of mitochondrial integrity, induction of pro-apoptotic protein Bim and caspase3 activation in response to NGF deprivation. The efficacies of commercial and synthesized inhibitors are comparable. The synthesized molecules are either phenanthrene based or naphthalene based and they are synthesized by using Pschorr reaction and Buchwald coupling respectively as one of the key steps. A number of molecules of both kinds block neurodegeneration effectively. Therefore, we propose that Cdk4 inhibition would be a therapeutic choice for ameliorating neurodegeneration in AD and these synthetic Cdk4 inhibitors could lead to development of effective drugs for AD.

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation

DEFF Research Database (Denmark)

Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch

2015-01-01

) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced...... to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4...

Proof of activity with AMD11070, an orally bioavailable inhibitor of CXCR4-tropic HIV type 1.

Science.gov (United States)

Moyle, Graeme; DeJesus, Edwin; Boffito, Marta; Wong, Rebecca S; Gibney, Colleen; Badel, Karin; MacFarland, Ron; Calandra, Gary; Bridger, Gary; Becker, Stephen

2009-03-15

The X4 Antagonist Concept Trial investigates the safety and antiviral activity of AMD11070, a potent inhibitor of X4-tropic human immunodeficiency virus (HIV) in vitro in HIV-infected patients harboring X4-tropic virus. Patients enrolled in the study had an X4 virus population 2000 relative luminescence units (rlu; by the Monogram Trofile Assay) and an HIV-1 RNA level 5000 copies/mL. Patients received AMD11070 monotherapy for 10 days. Coreceptor tropism, plasma HIV-1 RNA level, and CD4 cell count were measured at study entry, on day 5, and on day 10. Daily predose and serial samples on the last day of treatment were obtained for determination of plasma AMD11070 concentration. Ten patients were given AMD11070 monotherapy (200 mg to 8 patients and 100 mg to 2 patients) twice daily for 10 days. The median baseline CD4 cell count was 160 cells/mm(3), and the median HIV-1 RNA level was 91,447 copies/mL. Four of 9 evaluable patients achieved a reduction in X4 virus population of >or= rlu. The median change in X4 virus population at the end of treatment was -0.22 log(10) rlu (range, -1.90 to 0.23 log(10) rlu). Three of 4 patients who responded to therapy showed a tropism shift from dual- or mixed-tropic viruses to exclusively R5 virus by day 10. There were no drug-related serious adverse events, adverse events of greater than grade 2, or laboratory abnormalities. These results demonstrate the activity of AMD11070, the first oral CXCR4 antagonist, against X4-tropic HIV-1. The drug was well tolerated, with no serious safety concerns. AMD11070 is on clinical hold because of histologic changes to the liver observed in long-term animal studies; additional preclinical safety assessments are pending.

Expression profiles of vault components MVP, TEP1 and vPARP and their correlation to other multidrug resistance proteins in ovarian cancer.

Science.gov (United States)

Szaflarski, Witold; Sujka-Kordowska, Patrycja; Pula, Bartosz; Jaszczyńska-Nowinka, Karolina; Andrzejewska, Małgorzata; Zawierucha, Piotr; Dziegiel, Piotr; Nowicki, Michał; Ivanov, Pavel; Zabel, Maciej

2013-08-01

Vaults are cytoplasmic ribonucleoprotein particles composed of three proteins (MVP, TEP1, vPARP) and vault‑associated RNAs (vRNAs). Although the cellular functions of vaults remain unclear, vaults are strongly linked to the development of multidrug resistance (MDR), the major obstacle to the efficient treatment of cancers. Available published data suggest that vaults and their components are frequently upregulated in broad variety of multidrug-resistant cancer cell lines and tumors of different histological origin. Here, we provide detailed analysis of vault protein expression in post-surgery ovarian cancer samples from patients that were not exposed to chemotherapy. Our analysis suggests that vault proteins are expressed in the ovaries of healthy individuals but their expression in cancer patients is changed. Specifically, MVP, TEP1 and vPARP mRNA levels are significantly decreased in cancer samples with tendency of lower expression in higher-grade tumors. The pattern of vault protein mRNA expression is strongly correlated with the expression of other MDR-associated proteins such as MDR1, MRP1 and BCRP. Surprisingly, the protein levels of MVP, TEP1 and vPARP are actually increased in the higher‑grade tumors suggesting existence of post-transcriptional regulation of vault component production.

Pharmacological Inhibition of poly(ADP-ribose) polymerases improves fitness and mitochondrial function in skeletal muscle.

NARCIS (Netherlands)

Pirinen, E.; Canto, C.; Jo, Y.S.; Morato, L.; Zhang, H.; Menzies, K.J.; Williams, E.G.; Mouchiroud, L.; Moullan, N.; Hagberg, C.; Li, W.; Timmers, S.; Imhof, R.; Verbeek, J.; Pujol, A.; Loon, B. van; Viscomi, C.; Zeviani, M.; Schrauwen, P.; Sauve, A.A.; Schoonjans, K.; Auwerx, J.

2014-01-01

We previously demonstrated that the deletion of the poly(ADP-ribose)polymerase (Parp)-1 gene in mice enhances oxidative metabolism, thereby protecting against diet-induced obesity. However, the therapeutic use of PARP inhibitors to enhance mitochondrial function remains to be explored. Here, we show

Activity of second-generation ALK inhibitors against crizotinib-resistant mutants in an NPM-ALK model compared to EML4-ALK

International Nuclear Information System (INIS)

Fontana, Diletta; Ceccon, Monica; Gambacorti-Passerini, Carlo; Mologni, Luca

2015-01-01

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor involved in both solid and hematological tumors. About 80% of ALK-positive anaplastic large-cell lymphoma (ALCL) cases are characterized by the t(2;5)(p23;q35) translocation, encoding for the aberrant fusion protein nucleophosmin (NPM)-ALK, whereas 5% of non-small-cell lung cancer (NSCLC) patients carry the inv(2)(p21;p23) rearrangement, encoding for the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion. The ALK/c-MET/ROS inhibitor crizotinib successfully improved the treatment of ALK-driven diseases. However, several cases of resistance appeared in NSCLC patients, and ALK amino acid substitutions were identified as a leading cause of resistance to crizotinib. Second-generation ALK inhibitors have been developed in order to overcome crizotinib resistance. In this work, we profiled in vitro the activity of crizotinib, AP26113, ASP3026, alectinib, and ceritinib against six mutated forms of ALK associated with clinical resistance to crizotinib (C1156Y, L1196M, L1152R, G1202R, G1269A, and S1206Y) and provide a classification of mutants according to their level of sensitivity/resistance to the drugs. Since the biological activity of ALK mutations extends beyond the specific type of fusion, both NPM-ALK- and EML4-ALK-positive cellular models were used. Our data revealed that most mutants may be targeted by using different inhibitors. One relevant exception is represented by the G1202R substitution, which was highly resistant to all drugs (>10-fold increased IC 50 compared to wild type) and may represent the most challenging mutation to overcome. These results provide a prediction of cross-resistance of known crizotinib-resistant mutations against all second-generation tyrosine kinase inhibitors (TKIs) clinically available, and therefore could be a useful tool to help clinicians in the management of crizotinib-resistance cases

HDAC inhibitors enhance neratinib activity and when combined enhance the actions of an anti-PD-1 immunomodulatory antibody in vivo.

Science.gov (United States)

Booth, Laurence; Roberts, Jane L; Poklepovic, Andrew; Avogadri-Connors, Francesca; Cutler, Richard E; Lalani, Alshad S; Dent, Paul

2017-10-27

Patients whose NSCLC tumors become afatinib resistant presently have few effective therapeutic options to extend their survival. Afatinib resistant NSCLC cells were sensitive to clinically relevant concentrations of the irreversible pan-HER inhibitor neratinib, but not by the first generation ERBB1/2/4 inhibitor lapatinib. In multiple afatinib resistant NSCLC clones, HDAC inhibitors reduced the expression of ERBB1/3/4, but activated c-SRC, which resulted in higher total levels of ERBB1/3 phosphorylation. Neratinib also rapidly reduced the expression of ERBB1/2/3/4, c-MET and of mutant K-/N-RAS; K-RAS co-localized with phosphorylated ATG13 and with cathepsin B in vesicles. Combined exposure of cells to [neratinib + HDAC inhibitors] caused inactivation of mTORC1 and mTORC2, enhanced autophagosome and subsequently autolysosome formation, and caused an additive to greater than additive induction of cell death. Knock down of Beclin1 or ATG5 prevented HDAC inhibitors or neratinib from reducing ERBB1/3/4 and K-/N-RAS expression and reduced [neratinib + HDAC inhibitor] lethality. Neratinib and HDAC inhibitors reduced the expression of multiple HDAC proteins via autophagy that was causal in the reduced expression of PD-L1, PD-L2 and ornithine decarboxylase, and increased expression of Class I MHCA. In vivo , neratinib and HDAC inhibitors interacted to suppress the growth of 4T1 mammary tumors, an effect that was enhanced by an anti-PD-1 antibody. Our data support the premises that neratinib lethality can be enhanced by HDAC inhibitors, that neratinib may be a useful therapeutic tool in afatinib resistant NSCLC, and that [neratinib + HDAC inhibitor] exposure facilitates anti-tumor immune responses.

Hepatitis C Virus NS3/4A Protease Inhibitors: A Light at the End of the Tunnel

Science.gov (United States)

Chatel-Chaix, Laurent; Baril, Martin; Lamarre, Daniel

2010-01-01

Hepatitis C virus (HCV) infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease) that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection. PMID:21994705

Hepatitis C Virus NS3/4A Protease Inhibitors: A Light at the End of the Tunnel

Directory of Open Access Journals (Sweden)

Laurent Chatel-Chaix

2010-08-01

Full Text Available Hepatitis C virus (HCV infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection.

Replication fork stability confers chemoresistance in BRCA-deficient cells

DEFF Research Database (Denmark)

Chaudhuri, Arnab Ray; Callen, Elsa; Ding, Xia

2016-01-01

/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11......Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3...... nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations...

A review of dipeptidyl peptidase-4 inhibitors. Hot topics from randomized controlled trials

DEFF Research Database (Denmark)

Deacon, Carolyn F

2018-01-01

The first clinical study to investigate effects of dipeptidyl peptidase-4 (DPP-4) inhibition was published in 2002, and since then, numerous randomized controlled trials (RCTs) have shown that DPP-4 inhibitors are efficacious, safe and well-tolerated. This review will focus upon RCTs which have i...

The PDE4 inhibitor CHF-6001 and LAMAs inhibit bronchoconstriction-induced remodeling in lung slices.

Science.gov (United States)

Kistemaker, Loes E M; Oenema, Tjitske A; Baarsma, Hoeke A; Bos, I Sophie T; Schmidt, Martina; Facchinetti, Fabrizio; Civelli, Maurizio; Villetti, Gino; Gosens, Reinoud

2017-09-01

Combination therapy of PDE4 inhibitors and anticholinergics induces bronchoprotection in COPD. Mechanical forces that arise during bronchoconstriction may contribute to airway remodeling. Therefore, we investigated the impact of PDE4 inhibitors and anticholinergics on bronchoconstriction-induced remodeling. Because of the different mechanism of action of PDE4 inhibitors and anticholinergics, we hypothesized functional interactions of these two drug classes. Guinea pig precision-cut lung slices were preincubated with the PDE4 inhibitors CHF-6001 or roflumilast and/or the anticholinergics tiotropium or glycopyorrolate, followed by stimulation with methacholine (10 μM) or TGF-β 1 (2 ng/ml) for 48 h. The inhibitory effects on airway smooth muscle remodeling, airway contraction, and TGF-β release were investigated. Methacholine-induced protein expression of smooth muscle-myosin was fully inhibited by CHF-6001 (0.3-100 nM), whereas roflumilast (1 µM) had smaller effects. Tiotropium and glycopyrrolate fully inhibited methacholine-induced airway remodeling (0.1-30 nM). The combination of CHF-6001 and tiotropium or glycopyrrolate, in concentrations partially effective by themselves, fully inhibited methacholine-induced remodeling in combination. CHF-6001 did not affect airway closure and had limited effects on TGF-β 1 -induced remodeling, but rather, it inhibited methacholine-induced TGF-β release. The PDE4 inhibitor CHF-6001, and to a lesser extent roflumilast, and the LAMAs tiotropium and glycopyrrolate inhibit bronchoconstriction-induced remodeling. The combination of CHF-6001 and anticholinergics was more effective than the individual compounds. This cooperativity might be explained by the distinct mechanisms of action inhibiting TGF-β release and bronchoconstriction. Copyright © 2017 the American Physiological Society.

Inhibitor scaffold for the histone lysine demethylase KDM4C (JMJD2C)

DEFF Research Database (Denmark)

Leurs, Ulrike; Clausen, Rasmus P; Kristensen, Jesper L

2012-01-01

The human histone demethylases of the KDM4 (JMJD2) family have been associated to diseases such as prostate and breast cancer, as well as X-linked mental retardation. Therefore, these enzymes are considered oncogenes and their selective inhibition might be a possible therapeutic approach to treat...... cancer. Here we describe a heterocyclic ring system library screened against the histone demethylase KDM4C (JMJD2C) in the search for novel inhibitory scaffolds. A 4-hydroxypyrazole scaffold was identified as an inhibitor of KDM4C; this scaffold could be employed in the further development of novel...... therapeutics, as well as for the elucidation of the biological roles of KDM4C on epigenetic regulation....

Activity of EGFR-tyrosine kinase and ALK inhibitors for EML4–ALK-rearranged non–small–cell lung cancer harbored coexisting EGFR mutation

International Nuclear Information System (INIS)

Miyanaga, Akihiko; Kawamoto, Masashi; Tsuchiya, Shinichi; Hagiwara, Koichi; Soda, Manabu; Takeuchi, Kengo; Yamamoto, Nobuyuki; Mano, Hiroyuki; Ishikawa, Yuichi; Gemma, Akihiko; Shimizu, Kumi; Noro, Rintaro; Seike, Masahiro; Kitamura, Kazuhiro; Kosaihira, Seiji; Minegishi, Yuji; Shukuya, Takehito; Yoshimura, Akinobu

2013-01-01

The EML4–ALK (echinoderm microtubule-associated protein-like 4 gene and the anaplastic lymphoma kinase gene) fusion oncogene represents a novel molecular target in a small subset of non–small–cell lung cancers (NSCLCs). The EML4–ALK fusion gene occurs generally in NSCLC without mutations in epidermal growth factor receptor (EGFR) and KRAS. We report that a case of EML4–ALK-positive NSCLC with EGFR mutation had a response of stable disease to both an EGFR tyrosine kinase inhibitor (EGFR-TKI) and ALK inhibitor. We described the first clinical report of a patient with EML4–ALK-positive NSCLC with EGFR mutation that had a response of stable disease to both single-agent EGFR-TKI and ALK inhibitor. EML4–ALK translocation may be associated with resistance to EGFR-TKI, and EGFR signaling may contribute to resistance to ALK inhibitor in EML4–ALK-positive NSCLC

Dipeptidyl peptidase-4 inhibitor ameliorates early renal injury through its anti-inflammatory action in a rat model of type 1 diabetes

Energy Technology Data Exchange (ETDEWEB)

Kodera, Ryo, E-mail: kodera@cc.okayama-u.ac.jp [Center for Innovative Clinical Medicine, Okayama University Hospital, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Shikata, Kenichi [Center for Innovative Clinical Medicine, Okayama University Hospital, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Takatsuka, Tetsuharu; Oda, Kaori; Miyamoto, Satoshi; Kajitani, Nobuo; Hirota, Daisho; Ono, Tetsuichiro [Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Usui, Hitomi Kataoka [Department of Primary Care and Medical Education, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Makino, Hirofumi [Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan)

2014-01-17

Highlights: •DPP-4 inhibitor decreased urinary albumin excretion in a rat of type 1 diabetes. •DPP-4 inhibitor ameliorated histlogical changes of diabetic nephropathy. •DPP-4 inhibitor has reno-protective effects through anti-inflammatory action. •DPP-4 inhibitor is beneficial on diabetic nephropathy besides lowering blood glucose. -- Abstract: Introduction: Dipeptidyl peptidase-4 (DPP-4) inhibitors are incretin-based drugs in patients with type 2 diabetes. In our previous study, we showed that glucagon-like peptide-1 (GLP-1) receptor agonist has reno-protective effects through anti-inflammatory action. The mechanism of action of DPP-4 inhibitor is different from that of GLP-1 receptor agonists. It is not obvious whether DPP-4 inhibitor prevents the exacerbation of diabetic nephropathy through anti-inflammatory effects besides lowering blood glucose or not. The purpose of this study is to clarify the reno-protective effects of DPP-4 inhibitor through anti-inflammatory actions in the early diabetic nephropathy. Materials and methods: Five-week-old male Sprague–Dawley (SD) rats were divided into three groups; non-diabetes, diabetes and diabetes treated with DPP-4 inhibitor (PKF275-055; 3 mg/kg/day). PKF275-055 was administered orally for 8 weeks. Results: PKF275-055 increased the serum active GLP-1 concentration and the production of urinary cyclic AMP. PKF275-055 decreased urinary albumin excretion and ameliorated histological change of diabetic nephropathy. Macrophage infiltration was inhibited, and inflammatory molecules were down-regulated by PKF275-055 in the glomeruli. In addition, nuclear factor-κB (NF-κB) activity was suppressed in the kidney. Conclusions: These results indicate that DPP-4 inhibitor, PKF275-055, have reno-protective effects through anti-inflammatory action in the early stage of diabetic nephropathy. The endogenous biological active GLP-1 might be beneficial on diabetic nephropathy besides lowering blood glucose.

Dipeptidyl peptidase-4 inhibitor ameliorates early renal injury through its anti-inflammatory action in a rat model of type 1 diabetes

International Nuclear Information System (INIS)

Kodera, Ryo; Shikata, Kenichi; Takatsuka, Tetsuharu; Oda, Kaori; Miyamoto, Satoshi; Kajitani, Nobuo; Hirota, Daisho; Ono, Tetsuichiro; Usui, Hitomi Kataoka; Makino, Hirofumi

2014-01-01

Highlights: •DPP-4 inhibitor decreased urinary albumin excretion in a rat of type 1 diabetes. •DPP-4 inhibitor ameliorated histlogical changes of diabetic nephropathy. •DPP-4 inhibitor has reno-protective effects through anti-inflammatory action. •DPP-4 inhibitor is beneficial on diabetic nephropathy besides lowering blood glucose. -- Abstract: Introduction: Dipeptidyl peptidase-4 (DPP-4) inhibitors are incretin-based drugs in patients with type 2 diabetes. In our previous study, we showed that glucagon-like peptide-1 (GLP-1) receptor agonist has reno-protective effects through anti-inflammatory action. The mechanism of action of DPP-4 inhibitor is different from that of GLP-1 receptor agonists. It is not obvious whether DPP-4 inhibitor prevents the exacerbation of diabetic nephropathy through anti-inflammatory effects besides lowering blood glucose or not. The purpose of this study is to clarify the reno-protective effects of DPP-4 inhibitor through anti-inflammatory actions in the early diabetic nephropathy. Materials and methods: Five-week-old male Sprague–Dawley (SD) rats were divided into three groups; non-diabetes, diabetes and diabetes treated with DPP-4 inhibitor (PKF275-055; 3 mg/kg/day). PKF275-055 was administered orally for 8 weeks. Results: PKF275-055 increased the serum active GLP-1 concentration and the production of urinary cyclic AMP. PKF275-055 decreased urinary albumin excretion and ameliorated histological change of diabetic nephropathy. Macrophage infiltration was inhibited, and inflammatory molecules were down-regulated by PKF275-055 in the glomeruli. In addition, nuclear factor-κB (NF-κB) activity was suppressed in the kidney. Conclusions: These results indicate that DPP-4 inhibitor, PKF275-055, have reno-protective effects through anti-inflammatory action in the early stage of diabetic nephropathy. The endogenous biological active GLP-1 might be beneficial on diabetic nephropathy besides lowering blood glucose

INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions.

Science.gov (United States)

Cánepa, Eduardo T; Scassa, María E; Ceruti, Julieta M; Marazita, Mariela C; Carcagno, Abel L; Sirkin, Pablo F; Ogara, María F

2007-07-01

The cyclin D-Cdk4-6/INK4/Rb/E2F pathway plays a key role in controlling cell growth by integrating multiple mitogenic and antimitogenic stimuli. The members of INK4 family, comprising p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d), block the progression of the cell cycle by binding to either Cdk4 or Cdk6 and inhibiting the action of cyclin D. These INK4 proteins share a similar structure dominated by several ankyrin repeats. Although they appear to be structurally redundant and equally potent as inhibitors, the INK4 family members are differentially expressed during mouse development. The striking diversity in the pattern of expression of INK4 genes suggested that this family of cell cycle inhibitors might have cell lineage-specific or tissue-specific functions. The INK4 proteins are commonly lost or inactivated by mutations in diverse types of cancer, and they represent established or candidate tumor suppressors. Apart from their capacity to arrest cells in the G1-phase of the cell cycle they have been shown to participate in an increasing number of cellular processes. Given their emerging roles in fundamental physiological as well as pathological processes, it is interesting to explore the diverse roles for the individual INK4 family members in different functions other than cell cycle regulation. Extensive studies, over the past few years, uncover the involvement of INK4 proteins in senescence, apoptosis, DNA repair, and multistep oncogenesis. We will focus the discussion here on these unexpected issues.

Dipeptidyl peptidase-4 inhibitors in the management of type 2 diabetes: safety, tolerability, and efficacy

Directory of Open Access Journals (Sweden)

Mary Elizabeth Cox

2010-01-01

Full Text Available Mary Elizabeth Cox1, Jennifer Rowell1, Leonor Corsino1, Jennifer B Green1,21Department of Medicine, Division of Endocrinology, Metabolism, and Nutrition. Duke University Medical Center, Durham, NC, USA; 2Department of Medicine, Division of Endocrinology, Durham Veterans Affairs Medical Center, Durham, NC, USAAbstract: Although glycemic control is an important and effective way to prevent and minimize the worsening of diabetes-related complications, type 2 diabetes is a progressive disease which often proves difficult to manage. Most affected patients will eventually require therapy with multiple medications in order to reach appropriate glycemic targets. The dipeptidyl peptidase-4 (DPP-4 inhibitors constitute a relatively new class of oral medications for the treatment of type 2 diabetes, which has become widely incorporated into clinical practice. This review summarizes the available data on the efficacy, safety, and tolerability of these medications.Keywords: type 2 diabetes, pharmacotherapy, DPP-4 inhibitor, sitagliptin, vildagliptin, saxagliptin, alogliptin, linagliptin

Advance in Research on Mycobacterium tuberculosis FabG4 and Its Inhibitor

Directory of Open Access Journals (Sweden)

Debajyoti Dutta

2018-06-01

Full Text Available Increasing evidence from recent reports of drug-resistant mycobacterial strains poses a challenge worldwide. Drug-resistant strains often undergo mutations, adopt alternative pathways, and express drug efflux pumps to reduce or eliminate drug doses. Besides these intrinsic resistance mechanisms, bacteria can evade drug doses by forming biofilms. Biofilms are the concerted growth of adherent microorganisms, which can also be formed at the air-water interface. The growth is supported by the extracellular polymer matrix which is self-produced by the microorganisms. Reduced metabolic activity in a nutrient-deficient environment in the biofilm may cause the microorganisms to take alternative pathways that can make the microorganisms recalcitrant to the drug doses. Recent works have shown that Mycobacterium tuberculosis expresses several proteins during its growth in biofilm, those when deleted, did not show any effect on mycobacterial growth in normal nutrient-sufficient conditions. Studying these unconventional proteins in mycobacterial biofilms is therefore of utmost importance. In this article, I will discuss one such mycobacterial biofilm-related protein FabG4 that is recently shown to be important for mycobacterial survival in the presence of antibiotic stressors and limited nutrient condition. In an attempt to find more effective FabG4 inhibitors and its importance in biofilm forming M. tuberculosis, present knowledge about FabG4 and its known inhibitors are discussed. Based on the existing data, a putative role of FabG4 is also suggested.

Tyrosine kinase receptor inhibitor-targeted combined chemotherapy for metastatic bladder cancer

Directory of Open Access Journals (Sweden)

Chia-Lun Wu

2012-04-01

Full Text Available Overexpression of hypoxia-inducible factor-1 alpha is noted during the invasive and metastatic process of transitional cell carcinoma. It will upregulate vascular endothelial growth factor (VEGF and drive proliferation, invasiveness, metastasis, and antiapoptotic ability of cancer cells. We proposed that tyrosine kinase receptor inhibitor, sunitinib malate—(Sutent; Pfizer Inc., Taiwan, combined with chemotherapeutic drug may present synergistic cytotoxic enhancement to transitional cell carcinoma cells with subsequent inhibition of their cellular behaviors, including proliferation, invasiveness, and metastatic activity. The contents of VEGF-A in mouse bladder tumor cells (MBT-2 and culture medium were detected by quantification-polymerase chain reaction and Western blot individually. The inhibitory concentrations of various chemotherapeutic drugs, sunitinib, and their combination treatment in MBT-2 were determined by 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT assay. Microchamber transmembrane migration assay was applied in evaluation of the inhibitory effects of different dosages of sunitinib and combination treatment on tumor cells. The cell cycle and apoptosis were analyzed after combination therapy by flow cytometry. Variation in apoptotic pathway was elucidated by Western blot using specific antibodies with cleaved PARP and caspase-3. Metastatic animal model mimicked by tail vein injection of MBT-2 cells was used to evaluate the treatment efficiency in tumor weight and survival rate. The mRNA and protein level of VEGF-A in MBT-2 cells increased by 70% at 48 hours interval under hypoxia stress condition. In MTT assay, MBT-2 cells had shown the highest sensitivity to epirubicin. Sunitinib combined with epirubicin had shown a synergistic cytotoxic effect to MBT-2 cells. Sunitinib and its combination with epirubicin showed significant inhibition on MBT-2 cells migration in microchambers. G2/M phase arrest and

Ablation of the Ferroptosis Inhibitor Glutathione Peroxidase 4 in Neurons Results in Rapid Motor Neuron Degeneration and Paralysis.

Science.gov (United States)

Chen, Liuji; Hambright, William Sealy; Na, Ren; Ran, Qitao

2015-11-20

Glutathione peroxidase 4 (GPX4), an antioxidant defense enzyme active in repairing oxidative damage to lipids, is a key inhibitor of ferroptosis, a non-apoptotic form of cell death involving lipid reactive oxygen species. Here we show that GPX4 is essential for motor neuron health and survival in vivo. Conditional ablation of Gpx4 in neurons of adult mice resulted in rapid onset and progression of paralysis and death. Pathological inspection revealed that the paralyzed mice had a dramatic degeneration of motor neurons in the spinal cord but had no overt neuron degeneration in the cerebral cortex. Consistent with the role of GPX4 as a ferroptosis inhibitor, spinal motor neuron degeneration induced by Gpx4 ablation exhibited features of ferroptosis, including no caspase-3 activation, no TUNEL staining, activation of ERKs, and elevated spinal inflammation. Supplementation with vitamin E, another inhibitor of ferroptosis, delayed the onset of paralysis and death induced by Gpx4 ablation. Also, lipid peroxidation and mitochondrial dysfunction appeared to be involved in ferroptosis of motor neurons induced by Gpx4 ablation. Taken together, the dramatic motor neuron degeneration and paralysis induced by Gpx4 ablation suggest that ferroptosis inhibition by GPX4 is essential for motor neuron health and survival in vivo. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Characteristics of the Inhibition of Potato (Solanum tuberosum) Invertase by an Endogenous Proteinaceous Inhibitor in Potatoes

Science.gov (United States)

Bracho, Geracimo E.; Whitaker, John R.

1990-01-01

Effect of several parameters on inhibition of potato (Solanum tuberosum) invertase by its endogenous proteinaceous inhibitor was determined using homogeneous preparations of both proteins. The inhibitor and invertase formed an inactive complex with an observed association rate constant at pH 4.70 and 37°C of 8.82 × 102 per molar per second and a dissociation rate constant of 3.3 × 10−3 per minute. The inhibitor appeared to bind to invertase in more than one step. Initial interaction (measured by loss of invertase activity) was rapid, relatively weak, readily reversible (Ki of 2 × 10−6 molar) and noncompetitive with substrate at pH 4.70. Initial interaction was probably followed by isomerization to a tighter (Ki of 6.23 × 10−8 molar) complex, which dissociated slowly with a half-time of 3.5 hour. Interaction between enzyme and inhibitor appeared to be of ionic character and essentially pH independent between pH 3.5 and 7.4. PMID:16667286

Multicenter Evaluation of the Tolerability of Combined Treatment With PD-1 and CTLA-4 Immune Checkpoint Inhibitors and Palliative Radiation Therapy

Energy Technology Data Exchange (ETDEWEB)

Bang, Andrew [Department of Radiation Oncology, Brigham and Women' s Hospital/Dana-Farber Cancer Institute, Boston, Massachusetts (United States); Division of Radiation Oncology, University of Ottawa, Ottawa, Ontario (Canada); Wilhite, Tyler J. [Harvard Medical School, Boston, Massachusetts (United States); Pike, Luke R.G. [Harvard Radiation Oncology Program, Brigham and Women' s Hospital/Dana-Farber Cancer Institute, Boston, Massachusetts (United States); Cagney, Daniel N.; Aizer, Ayal A.; Taylor, Allison; Spektor, Alexander; Krishnan, Monica [Department of Radiation Oncology, Brigham and Women' s Hospital/Dana-Farber Cancer Institute, Boston, Massachusetts (United States); Ott, Patrick A. [Department of Medical Oncology and Center for Immuno-Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts (United States); Balboni, Tracy A. [Department of Radiation Oncology, Brigham and Women' s Hospital/Dana-Farber Cancer Institute, Boston, Massachusetts (United States); Hodi, F. Stephen [Department of Medical Oncology and Center for Immuno-Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts (United States); Schoenfeld, Jonathan D., E-mail: jdschoenfeld@partners.org [Department of Radiation Oncology, Brigham and Women' s Hospital/Dana-Farber Cancer Institute, Boston, Massachusetts (United States)

2017-06-01

Purpose: To analyze immune-related adverse events (ir-AEs) in patients treated with radiation and immune checkpoint blockade. Methods and Materials: We retrospectively reviewed records from patients with metastatic non-small cell lung cancer, melanoma, or renal cell cancer who received at least 1 cycle of a CTLA-4 or PD-1 inhibitor and radiation. Immune-related adverse events, defined using Common Terminology Criteria for Adverse Events version 4.0, were tabulated in relation to treatment variables, and associations with sequencing and timing were assessed. Results: We identified 133 patients, of whom 28 received a CTLA-4 inhibitor alone, 88 received a PD-1 inhibitor alone, and 17 received both classes of inhibitors either sequentially (n=13) or concurrently (n=4). Fifty-six patients received radiation within 14 days of an immune checkpoint inhibitor. Forty-six patients experienced at least 1 ir-AE (34.6%). Patients receiving both CTLA-4 and PD-1 inhibitors experienced more any-grade ir-AEs as compared with either individually (71% vs 29%, P=.0008). Any-grade ir-AEs occurred in 39% of patients in whom radiation was administered within 14 days of immunotherapy, compared with 23% of other patients (P=.06) and more often in patients who received higher equivalent dose in 2-Gy fractions (EQD2) EQD2 (P=.01). However, most toxicities were mild. There were no associations between site irradiated and specific ir-AEs. Conclusions: Our data suggest the combination of focal palliative radiation and CTLA-4 and/or PD-1 inhibitors is well tolerated, with manageable ir-AEs that did not seem to be associated with the particular site irradiated. Although conclusions are limited by the heterogeneity of patients and treatments, and future confirmatory studies are needed, this information can help guide clinical practice for patients receiving immune checkpoint therapy who require palliative radiation therapy.

Structure-Based Discovery of 4-(6-Methoxy-2-methyl-4-(quinolin-4-yl)-9 H -pyrimido[4,5- b ]indol-7-yl)-3,5-dimethylisoxazole (CD161) as a Potent and Orally Bioavailable BET Bromodomain Inhibitor

Energy Technology Data Exchange (ETDEWEB)

Zhao, Yujun; Bai, Longchuan; Liu, Liu; McEachern, Donna; Stuckey, Jeanne A.; Meagher, Jennifer L.; Yang, Chao-Yie; Ran, Xu; Zhou, Bing; Hu, Yang; Li, Xiaoqin; Wen, Bo; Zhao, Ting; Li, Siwei; Sun, Duxin; Wang, Shaomeng (Michigan)

2017-03-24

We have designed and synthesized 9H-pyrimido[4,5-b]indole-containing compounds to obtain potent and orally bioavailable BET inhibitors. By incorporation of an indole or a quinoline moiety to the 9H-pyrimido[4,5-b]indole core, we identified a series of small molecules showing high binding affinities to BET proteins and low nanomolar potencies in inhibition of cell growth in acute leukemia cell lines. One such compound, 4-(6-methoxy-2-methyl-4-(quinolin-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (31) has excellent microsomal stability and good oral pharmacokinetics in rats and mice. Orally administered, 31 achieves significant antitumor activity in the MV4;11 leukemia and MDA-MB-231 triple-negative breast cancer xenograft models in mice. Determination of the cocrystal structure of 31 with BRD4 BD2 provides a structural basis for its high binding affinity to BET proteins. Testing its binding affinities against other bromodomain-containing proteins shows that 31 is a highly selective inhibitor of BET proteins. Our data show that 31 is a potent, selective, and orally active BET inhibitor.

Protective effects of an aptamer inhibitor of neutrophil elastase in lung inflammatory injury

DEFF Research Database (Denmark)

Bless, N M; Smith, D; Charlton, J

1997-01-01

Neutrophils play an important part in the development of acute inflammatory injury. Human neutrophils contain high levels of the serine protease elastase, which is stored in azurophilic granules and is secreted in response to inflammatory stimuli. Elastase is capable of degrading many components...... of extracellular matrix [1-4] and has cytotoxic effects on endothelial cells [5-7] and airway epithelial cells. Three types of endogenous protease inhibitors control the activity of neutrophil elastase, including alpha-1 protease inhibitor (alpha-1PI), alpha-2 macroglobulin and secreted leukoproteinase inhibitor...... (SLPI) [8-10]. A disturbed balance between neutrophil elastase and these inhibitors has been found in various acute clinical conditions (such as adult respiratory syndrome and ischemia-reperfusion injury) and in chronic diseases. We investigated the effect of NX21909, a selected oligonucleotide (aptamer...

Sarcoid-like lung granulomas in a hemodialysis patient treated with a dipeptidyl peptidase-4 inhibitor.

Science.gov (United States)

Sada, Ken-Ei; Wada, Jun; Morinaga, Hiroshi; Tuchimochi, Shigeyuki; Uka, Mayu; Makino, Hirofumi

2014-04-01

It has been reported that the inhibition of dipeptidyl peptidase-4 (DPP-4)/CD26 on T-cells by DPP-4 enzymatic inhibitors suppresses lymphocyte proliferation and reduces the production of various cytokines, including tumor necrosis factor (TNF)-α. A 72-year-old female with diabetic nephropathy on hemodialysis developed multiple lung nodules following the administration of vildagliptin. A biopsy demonstrated the histology of granulomas without caseous necrosis. The discontinuation of vildagliptin resulted in the disappearance of the granulomas within 4 months. As granulomatosis often develops in patients under anti-TNF-α therapy, the accumulation of DPP-4 inhibitors or its metabolites is possibly linked to unrecognized complications, such as sarcoid-like lung granulomas.

The small molecule inhibitor YK-4-279 disrupts mitotic progression of neuroblastoma cells, overcomes drug resistance and synergizes with inhibitors of mitosis.

Science.gov (United States)

Kollareddy, Madhu; Sherrard, Alice; Park, Ji Hyun; Szemes, Marianna; Gallacher, Kelli; Melegh, Zsombor; Oltean, Sebastian; Michaelis, Martin; Cinatl, Jindrich; Kaidi, Abderrahmane; Malik, Karim

2017-09-10

Neuroblastoma is a biologically and clinically heterogeneous pediatric malignancy that includes a high-risk subset for which new therapeutic agents are urgently required. As well as MYCN amplification, activating point mutations of ALK and NRAS are associated with high-risk and relapsing neuroblastoma. As both ALK and RAS signal through the MEK/ERK pathway, we sought to evaluate two previously reported inhibitors of ETS-related transcription factors, which are transcriptional mediators of the Ras-MEK/ERK pathway in other cancers. Here we show that YK-4-279 suppressed growth and triggered apoptosis in nine neuroblastoma cell lines, while BRD32048, another ETV1 inhibitor, was ineffective. These results suggest that YK-4-279 acts independently of ETS-related transcription factors. Further analysis reveals that YK-4-279 induces mitotic arrest in prometaphase, resulting in subsequent cell death. Mechanistically, we show that YK-4-279 inhibits the formation of kinetochore microtubules, with treated cells showing a broad range of abnormalities including multipolar, fragmented and unseparated spindles, together leading to disrupted progression through mitosis. Notably, YK-4-279 does not affect microtubule acetylation, unlike the conventional mitotic poisons paclitaxel and vincristine. Consistent with this, we demonstrate that YK-4-279 overcomes vincristine-induced resistance in two neuroblastoma cell-line models. Furthermore, combinations of YK-4-279 with vincristine, paclitaxel or the Aurora kinase A inhibitor MLN8237/Alisertib show strong synergy, particularly at low doses. Thus, YK-4-279 could potentially be used as a single-agent or in combination therapies for the treatment of high-risk and relapsing neuroblastoma, as well as other cancers. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Synthesis, Pharmacological Profile and Docking Studies of New Sulfonamides Designed as Phosphodiesterase-4 Inhibitors.

Directory of Open Access Journals (Sweden)

Isabelle Karine da Costa Nunes

Full Text Available Prior investigations showed that increased levels of cyclic AMP down-regulate lung inflammatory changes, stimulating the interest in phosphodiesterase (PDE4 as therapeutic target. Here, we described the synthesis, pharmacological profile and docking properties of a novel sulfonamide series (5 and 6a-k designed as PDE4 inhibitors. Compounds were screened for their selectivity against the four isoforms of human PDE4 using an IMAP fluorescence polarized protocol. The effect on allergen- or LPS-induced lung inflammation and airway hyper-reactivity (AHR was studied in A/J mice, while the xylazine/ketamine-induced anesthesia test was employed as a behavioral correlate of emesis in rodents. As compared to rolipram, the most promising screened compound, 6a (LASSBio-448 presented a better inhibitory index concerning PDE4D/PDE4A or PDE4D/PDE4B. Accordingly, docking analyses of the putative interactions of LASSBio-448 revealed similar poses in the active site of PDE4A and PDE4C, but slight unlike orientations in PDE4B and PDE4D. LASSBio-448 (100 mg/kg, oral, 1 h before provocation, inhibited allergen-induced eosinophil accumulation in BAL fluid and lung tissue samples. Under an interventional approach, LASSBio-448 reversed ongoing lung eosinophilic infiltration, mucus exacerbation, peribronchiolar fibrosis and AHR by allergen provocation, in a mechanism clearly associated with blockade of pro-inflammatory mediators such as IL-4, IL-5, IL-13 and eotaxin-2. LASSBio-448 (2.5 and 10 mg/kg also prevented inflammation and AHR induced by LPS. Finally, the sulfonamide derivative was shown to be less pro-emetic than rolipram and cilomilast in the assay employed. These findings suggest that LASSBio-448 is a new PDE4 inhibitor with marked potential to prevent and reverse pivotal pathological features of diseases characterized by lung inflammation, such as asthma.

Effect of ketoconazole-mediated CYP3A4 inhibition on clinical pharmacokinetics of panobinostat (LBH589), an orally active histone deacetylase inhibitor

NARCIS (Netherlands)

A.P. Hamberg (Paul); M.M. Woo (Margaret M.); L.C. Chen (Lin-Chi); J. Verweij (Jaap); M.G. Porro; L. Zhao (Ling); W. Li (Weili); D.A.J. van der Biessen (Diane); H.S. Sharma (Hari); T. Hengelage (Thomas); M.J.A. de Jonge (Maja)

2011-01-01

textabstractPurpose: Panobinostat is partly metabolized by CYP3A4 in vitro. This study evaluated the effect of a potent CYP3A inhibitor, ketoconazole, on the pharmacokinetics and safety of panobinostat. Methods: Patients received a single panobinostat oral dose on day 1, followed by 4 days wash-out

Analysis of poly(ADP-Ribose polymerases in Arabidopsis telomere biology.

Directory of Open Access Journals (Sweden)

Kara A Boltz

Full Text Available Maintaining the length of the telomere tract at chromosome ends is a complex process vital to normal cell division. Telomere length is controlled through the action of telomerase as well as a cadre of telomere-associated proteins that facilitate replication of the chromosome end and protect it from eliciting a DNA damage response. In vertebrates, multiple poly(ADP-ribose polymerases (PARPs have been implicated in the regulation of telomere length, telomerase activity and chromosome end protection. Here we investigate the role of PARPs in plant telomere biology. We analyzed Arabidopsis thaliana mutants null for PARP1 and PARP2 as well as plants treated with the PARP competitive inhibitor 3-AB. Plants deficient in PARP were hypersensitive to genotoxic stress, and expression of PARP1 and PARP2 mRNA was elevated in response to MMS or zeocin treatment or by the loss of telomerase. Additionally, PARP1 mRNA was induced in parp2 mutants, and conversely, PARP2 mRNA was induced in parp1 mutants. PARP3 mRNA, by contrast, was elevated in both parp1 and parp2 mutants, but not in seedlings treated with 3-AB or zeocin. PARP mutants and 3-AB treated plants displayed robust telomerase activity, no significant changes in telomere length, and no end-to-end chromosome fusions. Although there remains a possibility that PARPs play a role in Arabidopsis telomere biology, these findings argue that the contribution is a minor one.

Analysis of Poly(ADP-Ribose) Polymerases in Arabidopsis Telomere Biology

Science.gov (United States)

Townley, Jennifer M.; Shippen, Dorothy E.

2014-01-01

Maintaining the length of the telomere tract at chromosome ends is a complex process vital to normal cell division. Telomere length is controlled through the action of telomerase as well as a cadre of telomere-associated proteins that facilitate replication of the chromosome end and protect it from eliciting a DNA damage response. In vertebrates, multiple poly(ADP-ribose) polymerases (PARPs) have been implicated in the regulation of telomere length, telomerase activity and chromosome end protection. Here we investigate the role of PARPs in plant telomere biology. We analyzed Arabidopsis thaliana mutants null for PARP1 and PARP2 as well as plants treated with the PARP competitive inhibitor 3-AB. Plants deficient in PARP were hypersensitive to genotoxic stress, and expression of PARP1 and PARP2 mRNA was elevated in response to MMS or zeocin treatment or by the loss of telomerase. Additionally, PARP1 mRNA was induced in parp2 mutants, and conversely, PARP2 mRNA was induced in parp1 mutants. PARP3 mRNA, by contrast, was elevated in both parp1 and parp2 mutants, but not in seedlings treated with 3-AB or zeocin. PARP mutants and 3-AB treated plants displayed robust telomerase activity, no significant changes in telomere length, and no end-to-end chromosome fusions. Although there remains a possibility that PARPs play a role in Arabidopsis telomere biology, these findings argue that the contribution is a minor one. PMID:24551184

Physiologically based pharmacokinetic and pharmacodynamic modeling of an antagonist (SM-406/AT-406) of multiple inhibitor of apoptosis proteins (IAPs) in a mouse xenograft model of human breast cancer.

Science.gov (United States)

Zhang, Tao; Li, Yanyan; Zou, Peng; Yu, Jing-yu; McEachern, Donna; Wang, Shaomeng; Sun, Duxin

2013-09-01

The inhibitors of apoptosis proteins (IAPs) are a class of key apoptosis regulators overexpressed or dysregulated in cancer. SM-406/AT-406 is a potent and selective small molecule mimetic of Smac that antagonizes the inhibitor of apoptosis proteins (IAPs). A physiologically based pharmacokinetic and pharmacodynamic (PBPK-PD) model was developed to predict the tissue concentration-time profiles of SM-406, the related onco-protein levels in tumor, and the tumor growth inhibition in a mouse model bearing human breast cancer xenograft. In the whole body physiologically based pharmacokinetic (PBPK) model for pharmacokinetics characterization, a well stirred (perfusion rate-limited) model was used to describe SM-406 pharmacokinetics in the lung, heart, kidney, intestine, liver and spleen, and a diffusion rate-limited (permeability limited) model was used for tumor. Pharmacodynamic (PD) models were developed to correlate the SM-406 concentration in tumor to the cIAP1 degradation, pro-caspase 8 decrease, CL-PARP accumulation and tumor growth inhibition. The PBPK-PD model well described the experimental pharmacokinetic data, the pharmacodynamic biomarker responses and tumor growth. This model may be helpful to predict tumor and plasma SM-406 concentrations in the clinic. Copyright © 2013 John Wiley & Sons, Ltd.

Spotlight on olaparib in the treatment of BRCA-mutated ovarian cancer: design, development and place in therapy

Directory of Open Access Journals (Sweden)

Lorusso D

2018-05-01

Full Text Available Domenica Lorusso, Elisa Tripodi, Giuseppa Maltese, Stefano Lepori, Ilaria Sabatucci, Giorgio Bogani, Francesco Raspagliesi Gynecologic Oncology Unit, Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Italy Abstract: Epithelial ovarian cancer is the sixth most common cancer among women worldwide and the first cause of death among gynecological malignancies. Most of the patients present recurrent disease and unfortunately cannot be cured. The unsatisfactory results obtained with salvage chemotherapy have elicited investigators to search for novel biological agents capable of achieving a better control of the disease. In the setting of homologous recombination deficiency, the DNA errors that occur cannot be accurately repaired, and the treatment with poly(ADP-ribose polymerase (PARP inhibition results in definitive cell death in a process called synthetic lethality. As a result of two positive clinical trials, Olaparib was approved in 2014 by U.S. Food and Drug Administration and European Medicines Agency as the first-in-class PARP inhibitor. Olaparib is effective and well tolerated in homologous recombination deficient patients. Several studies with Olaparib have been conducted in the recurrent setting either as maintenance in platinum-responsive patients or as a single agent. Ongoing trials are focused on the use of olaparib as maintenance in the first-line ovarian cancer setting alone or in combination with antiangiogenic agents. Future perspectives will probably investigate the association of olaparib with novel agents as check-point inhibitors and PI3K-AKT inhibitors. The PARP inhibitor era is just at the beginning. Keywords: olaparib, ovarian cancer, PARP inhibitors, homologous recombination deficiency, BRCA mutation

Effects of Dipeptidyl Peptidase-4 Inhibitors on Hyperglycemia and Blood Cyclosporine Levels in Renal Transplant Patients with Diabetes: A Pilot Study

Directory of Open Access Journals (Sweden)

Jaehyun Bae

2016-03-01

Full Text Available BackgroundThe use of dipeptidyl peptidase-4 (DPP-4 inhibitors is increasing among renal transplant patients with diabetes. However, the glucose-lowering efficacies of various DPP-4 inhibitors and their effects on blood cyclosporine levels have not been fully investigated. We compared the glucose-lowering efficacies of DPP 4 inhibitors and evaluate their effects on the blood levels of cyclosporine in renal transplant recipients with diabetes.MethodsSixty-five renal allograft recipients who received treatment with DPP-4 inhibitors (vildagliptin, sitagliptin, or linagliptin following kidney transplant were enrolled. The glucose-lowering efficacies of the DPP-4 inhibitors were compared according to the changes in the hemoglobin A1c (HbA1c levels after 3 months of treatment. Changes in the trough levels of the cyclosporine were also assessed 2 months after treatment with each DPP-4 inhibitor.ResultsHbA1c significantly decreased in the linagliptin group in comparison with other DPP-4 inhibitors (vildagliptin –0.38%±1.03%, sitagliptin –0.53%±0.95%, and linagliptin –1.40±1.34; P=0.016. Cyclosporine trough levels were significantly increased in the sitagliptin group compared with vildagliptin group (30.62±81.70 ng/mL vs. –24.22±53.54 ng/mL, P=0.036. Cyclosporine trough levels were minimally changed in patients with linagliptin.ConclusionLinagliptin demonstrates superior glucose-lowering efficacy and minimal effect on cyclosporine trough levels in comparison with other DPP-4 inhibitors in kidney transplant patients with diabetes.

Plasminogen Activator Inhibitor-1 (PAI-1) gene 4G/5G alleles frequency distribution in the Lebanese population.

Science.gov (United States)

Shammaa, Dina M R; Sabbagh, Amira S; Taher, Ali T; Zaatari, Ghazi S; Mahfouz, Rami A R

2008-09-01

Plasminogen activator inhibitor-1 (PAI-1) is an inhibitor of fibrinolysis. Increased plasma PAI-1 levels play an essential role in the pathogenesis of cardiovascular risk and other diseases associated with thrombosis. The 4G/5G polymorphism of the PAI-1 promoter region has been extensively studied in different populations. We studied 160 healthy unrelated Lebanese individuals using a reverse hybridization PCR assay to detect the 5G/5G, 4G/5G and, 4G/4G genotypes of the PAI-1 gene and the frequencies of the 4G and 5G alleles. We found that 4G/5G genotype was the most prevalent (45.6%) followed by 5G/5G (36.9%) and 4G/4G (17.5%). The frequencies of the 4G and 5G alleles were calculated to be 0.403 and 0.597, respectively. Compared to other ethnic communities, the Lebanese population was found to harbour a relatively high prevalence of the rare 4G allele. This, in turn, may predispose this population to develop cardiovascular diseases and other thrombotic clinical conditions. This study aids to enhance our understanding of the genetic features of the Lebanese population.

Poly(ADP-Ribose) Polymerase-1: A Novel Therapeutic Target in Necrotizing Enterocolitis

Science.gov (United States)

Giannone, Peter J.; Alcamo, Alicia A.; Schanbacher, Brandon L.; Nankervis, Craig A.; Besner, Gail E.; Bauer, John A.

2011-01-01

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of infancy, afflicting 11% of infants born 22–28 weeks gestational age. Both inflammation and oxidation may be involved in NEC pathogenesis through reactive nitrogen species production, protein oxidation and DNA damage. Poly(ADP-ribose) polymerase-1 (PARP-1) is a critical enzyme activated to facilitate DNA repair using nicotinamide adenine dinucleotide (NAD+) as a substrate. However, in the presence of severe oxidative stress and DNA damage, PARP-1 over-activation may ensue, depleting cells of NAD+ and ATP, killing them by metabolic catastrophe. Here we tested the hypothesis that NO dysregulation in intestinal epithelial cells during NEC leads to marked PARP-1 expression and that administration of a PARP-1 inhibitor (nicotinamide) attenuates intestinal injury in a newborn rat model of NEC. In this model, 56% of control pups developed NEC (any stage), versus 14% of pups receiving nicotinamide. Forty-four percent of control pups developed high-grade NEC (grades 3–4), whereas only 7% of pups receiving nicotinamide developed high-grade NEC. Nicotinamide treatment protects pups against intestinal injury incurred in the newborn rat NEC model. We speculate that PARP-1 over-activation in NEC may drive mucosal cell death in this disease and that PARP-1 may be a novel therapeutic target in NEC. PMID:21399558

Discovery of potent 1H-imidazo[4,5-b]pyridine-based c-Met kinase inhibitors via mechanism-directed structural optimization.

Science.gov (United States)

An, Xiao-De; Liu, Hongyan; Xu, Zhong-Liang; Jin, Yi; Peng, Xia; Yao, Ying-Ming; Geng, Meiyu; Long, Ya-Qiu

2015-02-01

Starting from our previously identified novel c-Met kinase inhibitors bearing 1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-one scaffold, a global structural exploration was conducted to furnish an optimal binding motif for further development, directed by the enzyme inhibitory mechanism. First round SAR study picked two imidazonaphthyridinone frameworks with 1,8- and 3,5-disubstitution pattern as class I and class II c-Met kinase inhibitors, respectively. Further structural optimization on type II inhibitors by truncation of the imidazonaphthyridinone core and incorporation of an N-phenyl cyclopropane-1,1-dicarboxamide pharmacophore led to the discovery of novel imidazopyridine-based c-Met kinase inhibitors, displaying nanomolar enzyme inhibitory activity and improved Met kinase selectivity. More significantly, the new chemotype c-Met kinase inhibitors effectively inhibited Met phosphorylation and its downstream signaling as well as the proliferation of Met-dependent EBC-1 human lung cancer cells at submicromolar concentrations. Copyright © 2014 Elsevier Ltd. All rights reserved.