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Sample records for parp-1 inhibitor involvement

  1. Combinations of PARP Inhibitors with Temozolomide Drive PARP1 Trapping and Apoptosis in Ewing's Sarcoma.

    Science.gov (United States)

    Gill, Sonja J; Travers, Jon; Pshenichnaya, Irina; Kogera, Fiona A; Barthorpe, Syd; Mironenko, Tatiana; Richardson, Laura; Benes, Cyril H; Stratton, Michael R; McDermott, Ultan; Jackson, Stephen P; Garnett, Mathew J

    2015-01-01

    Ewing's sarcoma is a malignant pediatric bone tumor with a poor prognosis for patients with metastatic or recurrent disease. Ewing's sarcoma cells are acutely hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibition and this is being evaluated in clinical trials, although the mechanism of hypersensitivity has not been directly addressed. PARP inhibitors have efficacy in tumors with BRCA1/2 mutations, which confer deficiency in DNA double-strand break (DSB) repair by homologous recombination (HR). This drives dependence on PARP1/2 due to their function in DNA single-strand break (SSB) repair. PARP inhibitors are also cytotoxic through inhibiting PARP1/2 auto-PARylation, blocking PARP1/2 release from substrate DNA. Here, we show that PARP inhibitor sensitivity in Ewing's sarcoma cells is not through an apparent defect in DNA repair by HR, but through hypersensitivity to trapped PARP1-DNA complexes. This drives accumulation of DNA damage during replication, ultimately leading to apoptosis. We also show that the activity of PARP inhibitors is potentiated by temozolomide in Ewing's sarcoma cells and is associated with enhanced trapping of PARP1-DNA complexes. Furthermore, through mining of large-scale drug sensitivity datasets, we identify a subset of glioma, neuroblastoma and melanoma cell lines as hypersensitive to the combination of temozolomide and PARP inhibition, potentially identifying new avenues for therapeutic intervention. These data provide insights into the anti-cancer activity of PARP inhibitors with implications for the design of treatment for Ewing's sarcoma patients with PARP inhibitors.

  2. Research progress of PARP-1 inhibitors in antitumor drugs and radionuclide markers

    International Nuclear Information System (INIS)

    Zhao Lingzhou; Zhang Huabei

    2011-01-01

    Poly(ADP-ribose)polymerase (PARP) is a new target in the cancer treatment nowadays. PARP not only can repair DNA damage, regulate and control transcription, maintain the stability of intracellular environment and genome, regulate the process of cell survival and death, but also is the main transcription factor in the development of inflammation and the process of cancer. To inhibit PARP activity can reduce the DNA repair function in tumor cells, and increase the sensibility to DNA damage agents, so as to improve the efficacy of radiation therapy and chemotherapy for tumor. A number of studies have suggested that, whether used alone or combination with other chemotherapy drugs, PARP inhibitors show the potential in the anti-tumor therapeutic areas. In this paper, PARP-1 inhibitors were reviewed in antitumor research progress. According to the stage of development , PARP-1 inhibitors are classified. Several representative PARP-1 inhibitors, in clinical trials, with potential clinical value were introduced. Positron emission tomography (PET), uses the main short half-life elementary in human body as tracer, and at the molecular level, achieve the no wound, quantitative and dynamic observation about the different changes of metabolites or drugs in the body. PET is the most advanced contemporary video diagnostic technology, and this paper simply introduce the research progress of PARP-1 inhibitors labeled with radioactive nuclides. (authors)

  3. PARP1 inhibitors: contemporary attempts at their use in anticancer therapy and future perspective

    Directory of Open Access Journals (Sweden)

    Ewelina Wiśnik

    2016-04-01

    Full Text Available Current cancer therapies are based mainly on the use of compounds that cause DNA damage. Unfortunately, even the combination therapies do not give rewarding effects, due to the high efficiency of DNA damage repair mechanisms in tumor cells. Therefore, the present studies should be focused on proteins that are involved in DNA repair systems. Poly(ADP-ribose polymerase-1 is an example of a protein commonly known as an enzyme that plays a role in the detection of DNA damage and repair. Activation of PARP1 in response to DNA damage leads to poly-ADP-ribosylation of proteins contributing to DNA repair systems, therefore facilitating the maintenance of genome stability. On the other hand, inhibition of PARP1 enzyme results in the accumulation of DNA damage, which in turn contributes to cell death. Studies on inhibitors of PARP1 are still ongoing, and some of them are currently in the third phase of clinical trials. To date, only one representative of the PARP1 inhibitors, called olaparib, has been approved for anti-cancer therapy in the EU and the USA. Moreover, a growing body of evidence indicates a role of this protein in various intracellular processes such as bioenergetics, proliferation, regulation of gene expression, cell death as well as immunoregulation. A number of different intracellular processes regulated by PARP1 give rise to potential wider use of PARP1 inhibitors in treatment of other diseases, including immune or autoimmune disorders.

  4. 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.

  5. 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

  6. Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors.

    Science.gov (United States)

    Thorsell, Ann-Gerd; Ekblad, Torun; Karlberg, Tobias; Löw, Mirjam; Pinto, Ana Filipa; Trésaugues, Lionel; Moche, Martin; Cohen, Michael S; Schüler, Herwig

    2017-02-23

    Selective inhibitors could help unveil the mechanisms by which inhibition of poly(ADP-ribose) polymerases (PARPs) elicits clinical benefits in cancer therapy. We profiled 10 clinical PARP inhibitors and commonly used research tools for their inhibition of multiple PARP enzymes. We also determined crystal structures of these compounds bound to PARP1 or PARP2. Veliparib and niraparib are selective inhibitors of PARP1 and PARP2; olaparib, rucaparib, and talazoparib are more potent inhibitors of PARP1 but are less selective. PJ34 and UPF1069 are broad PARP inhibitors; PJ34 inserts a flexible moiety into hydrophobic subpockets in various ADP-ribosyltransferases. XAV939 is a promiscuous tankyrase inhibitor and a potent inhibitor of PARP1 in vitro and in cells, whereas IWR1 and AZ-6102 are tankyrase selective. Our biochemical and structural analysis of PARP inhibitor potencies establishes a molecular basis for either selectivity or promiscuity and provides a benchmark for experimental design in assessment of PARP inhibitor effects.

  7. Inhibition of matrix metalloproteinase-2 by PARP inhibitors

    International Nuclear Information System (INIS)

    Nicolescu, Adrian C.; Holt, Andrew; Kandasamy, Arulmozhi D.; Pacher, Pal; Schulz, Richard

    2009-01-01

    Matrix metalloproteinase-2 (MMP-2), a ubiquitously expressed zinc-dependent endopeptidase, and poly(ADP-ribosyl) polymerase (PARP), a nuclear enzyme regulating DNA repair, are activated by nitroxidative stress associated with various pathologies. As MMP-2 plays a detrimental role in heart injuries resulting from enhanced nitroxidative stress, where PARP and MMP inhibitors are beneficial, we hypothesized that PARP inhibitors may affect MMP-2 activity. Using substrate degradation assays to determine MMP-2 activity we found that four PARP inhibitors (3-AB, PJ-34, 5-AIQ, and EB-47) inhibited 64 kDa MMP-2 in a concentration-dependent manner. The IC 50 values of PJ-34 and 5-AIQ were in the high micromolar range and comparable to those of known MMP-2 inhibitors doxycycline, minocycline or o-phenanthroline, whereas those for 3-AB and EB-47 were in the millimolar range. Co-incubation of PARP inhibitors with doxycycline showed an additive inhibition of MMP-2 that was significant for 3-AB alone. These data demonstrate that the protective effects of some PARP inhibitors may include inhibition of MMP-2 activity.

  8. Inhibition of matrix metalloproteinase-2 by PARP inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Nicolescu, Adrian C.; Holt, Andrew; Kandasamy, Arulmozhi D. [Departments of Pharmacology and Pediatrics, Cardiovascular Research Centre, University of Alberta, Edmonton, Alta., Canada T6G 2S2 (Canada); Pacher, Pal [National Institutes of Health, NIAAA, Laboratory of Physiologic Studies, Bethesda, MD (United States); Schulz, Richard, E-mail: richard.schulz@ualberta.ca [Departments of Pharmacology and Pediatrics, Cardiovascular Research Centre, University of Alberta, Edmonton, Alta., Canada T6G 2S2 (Canada)

    2009-10-02

    Matrix metalloproteinase-2 (MMP-2), a ubiquitously expressed zinc-dependent endopeptidase, and poly(ADP-ribosyl) polymerase (PARP), a nuclear enzyme regulating DNA repair, are activated by nitroxidative stress associated with various pathologies. As MMP-2 plays a detrimental role in heart injuries resulting from enhanced nitroxidative stress, where PARP and MMP inhibitors are beneficial, we hypothesized that PARP inhibitors may affect MMP-2 activity. Using substrate degradation assays to determine MMP-2 activity we found that four PARP inhibitors (3-AB, PJ-34, 5-AIQ, and EB-47) inhibited 64 kDa MMP-2 in a concentration-dependent manner. The IC{sub 50} values of PJ-34 and 5-AIQ were in the high micromolar range and comparable to those of known MMP-2 inhibitors doxycycline, minocycline or o-phenanthroline, whereas those for 3-AB and EB-47 were in the millimolar range. Co-incubation of PARP inhibitors with doxycycline showed an additive inhibition of MMP-2 that was significant for 3-AB alone. These data demonstrate that the protective effects of some PARP inhibitors may include inhibition of MMP-2 activity.

  9. BET Bromodomain Inhibition Synergizes with PARP Inhibitor in Epithelial Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Sergey Karakashev

    2017-12-01

    Full Text Available PARP inhibition is known to be an effective clinical strategy in BRCA mutant cancers, but PARP inhibition has not been applied to BRCA-proficient tumors. Here, we show the synergy of BET bromodomain inhibition with PARP inhibition in BRCA-proficient ovarian cancers due to mitotic catastrophe. Treatment of BRCA-proficient ovarian cancer cells with the BET inhibitor JQ1 downregulated the G2-M cell-cycle checkpoint regulator WEE1 and the DNA-damage response factor TOPBP1. Combining PARP inhibitor Olaparib with the BET inhibitor, we observed a synergistic increase in DNA damage and checkpoint defects, which allowed cells to enter mitosis despite the accumulation of DNA damage, ultimately causing mitotic catastrophe. Moreover, JQ1 and Olaparib showed synergistic suppression of growth of BRCA-proficient cancer in vivo in a xenograft ovarian cancer mouse model. Our findings indicate that a combination of BET inhibitor and PARP inhibitor represents a potential therapeutic strategy for BRCA-proficient cancers.

  10. PARPi-FL - a Fluorescent PARP1 Inhibitor for Glioblastoma Imaging

    Directory of Open Access Journals (Sweden)

    Christopher P. Irwin

    2014-05-01

    Full Text Available New intravital optical imaging technologies have revolutionized our understanding of mammalian biology and continue to evolve rapidly. However, there are only a limited number of imaging probes available to date. In this study, we investigated in mouse models of glioblastoma whether a fluorescent small molecule inhibitor of the DNA repair enzyme PARP1, PARPi-FL, can be used as an imaging agent to detect glioblastomas in vivo. We demonstrated that PARPi-FL has appropriate biophysical properties, low toxicity at concentrations used for imaging, high stability in vivo, and accumulates selectively in glioblastomas due to high PARP1 expression. Importantly, subcutaneous and orthotopic glioblastoma xenografts were imaged with high contrast clearly defining tumor tissue from normal surrounding tissue. This research represents a step toward exploring and developing PARPi-FL as an optical intraoperative imaging agent for PARP1 in the clinic.

  11. 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

    Pancreatic cancer has a dismal prognosis and is currently the fourth leading cause of cancer-related death in developed countries. The inhibition of poly(ADP-ribose) polymerase-1 (Parp-1), the major protein responsible for poly(ADP-ribosy)lation in response to DNA damage, has emerged as a promising treatment for several tumour types. Here we aimed to elucidate the involvement of Parp-1 in pancreatic tumour progression. We assessed Parp-1 protein expression in normal, preneoplastic and pancreatic tumour samples from humans and from K-Ras- and c-myc-driven mouse models of pancreatic cancer. Parp-1 was highly expressed in acinar cells in normal and cancer tissues. In contrast, ductal cells expressed very low or undetectable levels of this protein, both in a normal and in a tumour context. The Parp-1 expression pattern was similar in human and mouse samples, thereby validating the use of animal models for further studies. To determine the in vivo effects of Parp-1 depletion on pancreatic cancer progression, Ela-myc-driven pancreatic tumour development was analysed in a Parp-1 knock-out background. Loss of Parp-1 resulted in increased tumour necrosis and decreased proliferation, apoptosis and angiogenesis. Interestingly, Ela-myc:Parp-1(-/-) mice displayed fewer ductal tumours than their Ela-myc:Parp-1(+/+) counterparts, suggesting that Parp-1 participates in promoting acinar-to-ductal metaplasia, a key event in pancreatic cancer initiation. Moreover, impaired macrophage recruitment can be responsible for the ADM blockade found in the Ela-myc:Parp-1(-/-) mice. Finally, molecular analysis revealed that Parp-1 modulates ADM downstream of the Stat3-MMP7 axis and is also involved in transcriptional up-regulation of the MDM2, VEGFR1 and MMP28 cancer-related genes. In conclusion, the expression pattern of Parp-1 in normal and cancer tissue and the in vivo functional effects of Parp-1 depletion point to a novel role for this protein in pancreatic carcinogenesis and shed light

  12. 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.

  13. PARP inhibitor rucaparib induces changes in NAD levels in cells and liver tissues as assessed by MRS.

    Science.gov (United States)

    Almeida, Gilberto S; Bawn, Carlo M; Galler, Martin; Wilson, Ian; Thomas, Huw D; Kyle, Suzanne; Curtin, Nicola J; Newell, David R; Maxwell, Ross J

    2017-09-01

    Poly(adenosine diphosphate ribose) polymerases (PARPs) are multifunctional proteins which play a role in many cellular processes. Namely, PARP1 and PARP2 have been shown to be involved in DNA repair, and therefore are valid targets in cancer treatment with PARP inhibitors, such as rucaparib, currently in clinical trials. Proton magnetic resonance spectroscopy ( 1 H-MRS) was used to study the impact of rucaparib in vitro and ex vivo in liver tissue from mice, via quantitative analysis of nicotinamide adenosine diphosphate (NAD + ) spectra, to assess the potential of MRS as a biomarker of the PARP inhibitor response. SW620 (colorectal) and A2780 (ovarian) cancer cell lines, and PARP1 wild-type (WT) and PARP1 knock-out (KO) mice, were treated with rucaparib, temozolomide (methylating agent) or a combination of both drugs. 1 H-MRS spectra were obtained from perchloric acid extracts of tumour cells and mouse liver. Both cell lines showed an increase in NAD + levels following PARP inhibitor treatment in comparison with temozolomide treatment. Liver extracts from PARP1 WT mice showed a significant increase in NAD + levels after rucaparib treatment compared with untreated mouse liver, and a significant decrease in NAD + levels in the temozolomide-treated group. The combination of rucaparib and temozolomide did not prevent the NAD + depletion caused by temozolomide treatment. The 1 H-MRS results show that NAD + levels can be used as a biomarker of PARP inhibitor and methylating agent treatments, and suggest that in vivo measurement of NAD + would be valuable. Copyright © 2017 John Wiley & Sons, Ltd.

  14. 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...

  15. Imidazoquinolinone, imidazopyridine, and isoquinolindione derivatives as novel and potent inhibitors of the poly(ADP-ribose) polymerase (PARP): a comparison with standard PARP inhibitors.

    Science.gov (United States)

    Eltze, Tobias; Boer, Rainer; Wagner, Thomas; Weinbrenner, Steffen; McDonald, Michelle C; Thiemermann, Christoph; Bürkle, Alexander; Klein, Thomas

    2008-12-01

    We have identified three novel structures for inhibitors of the poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA and implicated in DNA repair, apoptosis, organ dysfunction or necrosis. 2-[4-(5-Methyl-1H-imidazol-4-yl)-piperidin-1-yl]-4,5-dihydro-imidazo[4,5,1-i,j]quinolin-6-one (BYK49187), 2-(4-pyridin-2-yl-phenyl)-4,5-dihydro-imidazo[4,5,1-i,j]quinolin-6-one (BYK236864), 6-chloro-8-hydroxy-2,3-dimethyl-imidazo-[1,2-alpha]-pyridine (BYK20370), and 4-(1-methyl-1H-pyrrol-2-ylmethylene)-4H-isoquinolin-1,3-dione (BYK204165) inhibited cell-free recombinant human PARP-1 with pIC(50) values of 8.36, 7.81, 6.40, and 7.35 (pK(i) 7.97, 7.43, 5.90, and 7.05), and murine PARP-2 with pIC(50) values of 7.50, 7.55, 5.71, and 5.38, respectively. BYK49187, BYK236864, and BYK20370 displayed no selectivity for PARP-1/2, whereas BYK204165 displayed 100-fold selectivity for PARP-1. The IC(50) values for inhibition of poly(ADP-ribose) synthesis in human lung epithelial A549 and cervical carcinoma C4I cells as well in rat cardiac myoblast H9c2 cells after PARP activation by H(2)O(2) were highly significantly correlated with those at cell-free PARP-1 (r(2) = 0.89-0.96, P < 0.001) but less with those at PARP-2 (r(2) = 0.78-0.84, P < 0.01). The infarct size caused by coronary artery occlusion and reperfusion in the anesthetized rat was reduced by 22% (P < 0.05) by treatment with BYK49187 (3 mg/kg i.v. bolus and 3 mg/kg/h i.v. during 2-h reperfusion), whereas the weaker PARP inhibitors, BYK236864 and BYK20370, were not cardioprotective. In conclusion, the imidazoquinolinone BYK49187 is a potent inhibitor of human PARP-1 activity in cell-free and cellular assays in vitro and reduces myocardial infarct size in vivo. The isoquinolindione BYK204165 was found to be 100-fold more selective for PARP-1. Thus, both compounds might be novel and valuable tools for investigating PARP-1-mediated effects.

  16. Novel targeted therapeutics: inhibitors of MDM2, ALK and PARP

    Directory of Open Access Journals (Sweden)

    Hsueh Chung-Tsen

    2011-04-01

    Full Text Available Abstract We reviewed preclinical data and clinical development of MDM2 (murine double minute 2, ALK (anaplastic lymphoma kinase and PARP (poly [ADP-ribose] polymerase inhibitors. MDM2 binds to p53, and promotes degradation of p53 through ubiquitin-proteasome degradation. JNJ-26854165 and RO5045337 are 2 small-molecule inhibitors of MDM2 in clinical development. ALK is a transmembrane protein and a member of the insulin receptor tyrosine kinases. EML4-ALK fusion gene is identified in approximately 3-13% of non-small cell lung cancer (NSCLC. Early-phase clinical studies with Crizotinib, an ALK inhibitor, in NSCLC harboring EML4-ALK have demonstrated promising activity with high response rate and prolonged progression-free survival. PARPs are a family of nuclear enzymes that regulates the repair of DNA single-strand breaks through the base excision repair pathway. Randomized phase II study has shown adding PARP-1 inhibitor BSI-201 to cytotoxic chemotherapy improves clinical outcome in patients with triple-negative breast cancer. Olaparib, another oral small-molecule PARP inhibitor, demonstrated encouraging single-agent activity in patients with advanced breast or ovarian cancer. There are 5 other PARP inhibitors currently under active clinical investigation.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    Highlights: • PARP-1 is not a key regulator of photochemotherapy. • The PARP inhibitor PJ-34 sensitizes cells to UVA-induced phototoxicity by a PARP independent mechanism. • Photosensitization by PJ-34 is associated with increased ROS production and DNA damage. • Cells sensitized by PJ-34 undergo caspase-mediated apoptosis. - Abstract: 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 (2.5 J/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

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

    Energy Technology Data Exchange (ETDEWEB)

    Lakatos, Petra; Hegedűs, Csaba [Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Debrecen (Hungary); Salazar Ayestarán, Nerea; Juarranz, Ángeles [Department of Biology, Faculty of Sciences, Universidad Autónoma of Madrid, 28049-Madrid (Spain); Kövér, Katalin E. [Department of Inorganic and Analytical Chemistry, Faculty of Sciences, University of Debrecen, Debrecen (Hungary); Szabó, Éva [Department of Dermatology, Faculty of Medicine, University of Debrecen, Debrecen (Hungary); Virág, László, E-mail: lvirag@med.unideb.hu [Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Debrecen (Hungary); MTA-DE Cell Biology and Signaling Research Group, Debrecen (Hungary)

    2016-08-15

    Highlights: • PARP-1 is not a key regulator of photochemotherapy. • The PARP inhibitor PJ-34 sensitizes cells to UVA-induced phototoxicity by a PARP independent mechanism. • Photosensitization by PJ-34 is associated with increased ROS production and DNA damage. • Cells sensitized by PJ-34 undergo caspase-mediated apoptosis. - Abstract: 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 (2.5 J/cm{sup 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

  19. PARP inhibitors protect against sex- and AAG-dependent alkylation-induced neural degeneration.

    Science.gov (United States)

    Allocca, Mariacarmela; Corrigan, Joshua J; Fake, Kimberly R; Calvo, Jennifer A; Samson, Leona D

    2017-09-15

    Alkylating agents are commonly used to treat cancer. Although base excision repair (BER) is a major pathway for repairing DNA alkylation damage, under certain conditions, the initiation of BER produces toxic repair intermediates that damage healthy tissues. The initiation of BER by the alkyladenine DNA glycosylase (AAG, a.k.a. MPG) can mediate alkylation-induced cytotoxicity in specific cells in the retina and cerebellum of male mice. Cytotoxicity in both wild-type and Aag -transgenic ( AagTg ) mice is abrogated in the absence of Poly(ADP-ribose) polymerase-1 (PARP1). Here, we tested whether PARP inhibitors can also prevent alkylation-induced retinal and cerebellar degeneration in male and female WT and AagTg mice. Importantly, we found that WT mice display sex-dependent alkylation-induced retinal damage (but not cerebellar damage), with WT males being more sensitive than females. Accordingly, estradiol treatment protects males against alkylation-induced retinal degeneration. In AagTg male and female mice, the alkylation-induced tissue damage in both the retina and cerebellum is exacerbated and the sex difference in the retina is abolished. PARP inhibitors, much like Parp1 gene deletion, protect against alkylation-induced AAG-dependent neuronal degeneration in WT and AagTg mice, regardless of the gender, but their efficacy in preventing alkylation-induced neuronal degeneration depends on PARP inhibitor characteristics and doses. The recent surge in the use of PARP inhibitors in combination with cancer chemotherapeutic alkylating agents might represent a powerful tool for obtaining increased therapeutic efficacy while avoiding the collateral effects of alkylating agents in healthy tissues.

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

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    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.

  1. 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,

  2. 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

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

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    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.

  4. 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

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

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    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.

  6. Rucaparib: a novel PARP inhibitor for BRCA advanced ovarian cancer

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    Colombo I

    2018-03-01

    Full Text Available Ilaria Colombo, Stephanie Lheureux, Amit Manulal Oza Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada Abstract: Rucaparib is a potent small-molecule inhibitor of poly (ADP-ribose polymerase (PARP proteins (PARP-1, PARP-2 and PARP-3 that play an important role in repairing DNA damage and maintaining genomic stability. Tumors with mutations in BRCA1/2 or other homologous recombination deficiency (HRD genes are particularly sensitive to PARP inhibitors because of “synthetic lethality”, whereby a therapeutic agent can take advantage of an intrinsic weakness in DNA repair. Rucaparib has been investigated in several preclinical and clinical studies showing promising activity in BRCA-mutant and BRCA–wild-type epithelial ovarian cancers (EOCs. Dose-escalation Phase I studies have established the recommended Phase II dose to be 600 mg twice a day for oral rucaparib. Phase II and III studies have defined its role as treatment for BRCA-mutant recurrent high-grade EOC and as maintenance treatment for platinum-sensitive relapsed EOC following response to platinum-based chemotherapy. Genomic loss of heterozygosity has also been investigated as a potential signature of HRD and as a potential predictive biomarker of response. Treatment-induced adverse events (AEs have been observed in almost all patients treated with rucaparib, but mainly lower grade; with the most common being nausea, vomiting, asthenia/fatigue, anemia and transient transaminitis. The majority of AEs occurred early in treatment, were transient and have been easily managed with supportive treatment, dose interruption or discontinuation. This review will analyze the results of clinical trials investigating efficacy and safety of rucaparib in patients with ovarian cancer. Keywords: rucaparib, ovarian cancer, BRCA mutations, homologous recombination deficiency, maintenance treatment, PARP inhibitor

  7. Effect of MRE11 loss on PARP-inhibitor sensitivity in endometrial cancer in vitro.

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    Romana Koppensteiner

    Full Text Available To evaluate the frequency of MRE11/RAD50/NBS1 (MRN-complex loss of protein expression in endometrial cancers (EC and to determine whether loss of MRE11 renders the cancer cells sensitive to Poly(ADP-ribose polymerase (PARP-inhibitory treatment.MRN expression was examined in 521 samples of endometrial carcinomas and in 10 cancer cell lines. A putative mutation hotspot in the form of an intronic poly(T allele in MRE11 was sequenced in selected cases (n = 26. Sensitivity to the PARP-inhibitor, BMN673 was tested in colony formation assays before and after MRE11 silencing using siRNA. Homologous recombination (HR DNA repair was evaluated by RAD51-foci formation assay upon irradiation and drug treatment.Loss of MRE11 protein was found in 30.7% of EC tumours and significantly associated with loss of RAD50, NBS1 and mismatch repair protein expression. One endometrial cell line showed a markedly reduced MRE11 expression due to a homozygous poly(T mutation of MRE11, thereby exhibiting an increased sensitivity to BMN673. MRE11 depletion sensitizes MRE11 expressing EC cell lines to the treatment with BMN673. The increased sensitivity to PARP-inhibition correlates with reduced RAD51 foci formation upon ionizing radiation in MRE11-depleted cells.Loss of the MRE11 protein predicts sensitivity to PARP-inhibitor sensitivity in vitro, defining it as an additional synthetic lethal gene with PARP. The high incidence of MRE11 loss in ECs can be potentially exploited for PARP-inhibitor therapy. Furthermore, MRE11 protein expression using immunohistochemistry could be investigated as a predictive biomarker for PARP-inhibitor treatment.

  8. Sigma-2 ligands and PARP inhibitors synergistically trigger cell death in breast cancer cells

    International Nuclear Information System (INIS)

    McDonald, Elizabeth S.; Mankoff, Julia; Makvandi, Mehran; Chu, Wenhua; Chu, Yunxiang; Mach, Robert H.; Zeng, Chenbo

    2017-01-01

    The sigma-2 receptor is overexpressed in proliferating cells compared to quiescent cells and has been used as a target for imaging solid tumors by positron emission tomography. Recent work has suggested that the sigma-2 receptor may also be an effective therapeutic target for cancer therapy. Poly (ADP-ribose) polymerase (PARP) is a family of enzymes involved in DNA damage response. In this study, we looked for potential synergy of cytotoxicity between PARP inhibitors and sigma-2 receptor ligands in breast cancer cell lines. We showed that the PARP inhibitor, YUN3-6, sensitized mouse breast cancer cell line, EMT6, to sigma-2 receptor ligand (SV119, WC-26, and RHM-138) induced cell death determined by cell viability assay and colony forming assay. The PARP inhibitor, olaparib, sensitized tumor cells to a different sigma-2 receptor ligand SW43-induced apoptosis and cell death in human triple negative cell line, MDA-MB-231. Olaparib inhibited PARP activity and cell proliferation, and arrested cells in G2/M phase of the cell cycle in MDA-MB-231 cells. Subsequently cells became sensitized to SW43 induced cell death. In conclusion, the combination of sigma-2 receptor ligands and PARP inhibitors appears to hold promise for synergistically triggering cell death in certain types of breast cancer cells and merits further investigation. - Highlights: • PARPi, YUN3-6 and olaparib, and σ2 ligands, SV119 and SW43, were evaluated. • Mouse and human breast cancer cells, EMT6 and MDA-MB-231 respectively, were used. • YUN3-6 and SV119 synergistically triggered cell death in EMT6 cells. • Olaparib and SW43 additively triggered cell death in MDA-MB-231 cells. • Olaparib arrested cells in G2/M in MDA-MB-231 cells.

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

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    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.

  10. 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

    The post-translational modifications of DNA repair proteins and histone remodeling factors by poly(ADP-ribose)ylation and phosphorylation are essential for the maintenance of DNA integrity and chromatin structure, and in particular in response to DNA damaging produced by ionizing radiation (IR). Amongst the proteins implicated in these two processes are the poly(ADP-ribose) polymerase -1 (PARP-1) and PARP-2, and the cyclin-dependent kinase Cdk5: PARP-1 and 2 are involved in DNA single strand break (SSB) repair (SSBR) and Cdk5 depletion has been linked with increased cell sensitivity to PARP inhibition. We have shown by using HeLa cells stably depleted for either CdK5 or PARP-2, that the recruitment profile of PARP-1 and XRCC-1, two proteins involved in the short-patch (SP) SSBR sub-pathway, to DNA damage sites is sub-maximal and that of PCNA, a protein involved in the long-patch (LP) repair pathway, is increased in the absence of Cdk5 and decreased in the absence of PARP-2 suggesting that both Cdk5 and PARP-2 are involved in both SSBR sub-pathways. PARP-2 and Cdk5 also impact on the poly(ADP-ribose) levels in cells as in the absence of Cdk5 a hyper-activation of PARP-1 was found and in the absence of PARP-2 a reduction in poly(ADP-ribose) glyco-hydrolase (PARG) activity was seen. However, in spite of these changes no impact on the repair of SSBs induced by IR was seen in either the Cdk5 or PARP-2 depleted cells (Cdk5 KD or PARP-2 KD cells) but, interestingly, increased radiation sensitivity in terms of cell killing was noted in the Cdk5 depleted cells. We also found that Cdk5, PARP-2 and PARG were all implicated in the regulation of the recruitment and the dissociation of the chromatin-remodeling factor ALC1 from DNA damage sites suggesting a role for these three proteins in changes in chromatin structure after DNA photo-damage. These results, taken together with the observation that PARP-1 recruitment is sub-optimal in both Cdk5 KD and PARP-2 KD cells, show that

  11. 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

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    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.

  12. 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.

  13. 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)

  14. 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

  15. 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.

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

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    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.

  17. 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.

  18. ATM-Deficient Colorectal Cancer Cells Are Sensitive to the PARP Inhibitor Olaparib.

    Science.gov (United States)

    Wang, Chen; Jette, Nicholas; Moussienko, Daniel; Bebb, D Gwyn; Lees-Miller, Susan P

    2017-04-01

    The ataxia telangiectasia mutated (ATM) protein kinase plays a central role in the cellular response to DNA damage. Loss or inactivation of both copies of the ATM gene (ATM) leads to ataxia telangiectasia, a devastating childhood condition characterized by neurodegeneration, immune deficiencies, and cancer predisposition. ATM is also absent in approximately 40% of mantle cell lymphomas (MCLs), and we previously showed that MCL cell lines with loss of ATM are sensitive to poly-ADP ribose polymerase (PARP) inhibitors. Next-generation sequencing of patient tumors has revealed that ATM is altered in many human cancers including colorectal, lung, prostate, and breast. Here, we show that the colorectal cancer cell line SK-CO-1 lacks detectable ATM protein expression and is sensitive to the PARP inhibitor olaparib. Similarly, HCT116 colorectal cancer cells with shRNA depletion of ATM are sensitive to olaparib, and depletion of p53 enhances this sensitivity. Moreover, HCT116 cells are sensitive to olaparib in combination with the ATM inhibitor KU55933, and sensitivity is enhanced by deletion of p53. Together our studies suggest that PARP inhibitors may have potential for treating colorectal cancer with ATM dysfunction and/or colorectal cancer with mutation of p53 when combined with an ATM kinase inhibitor. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  19. 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

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

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    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

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

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    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.

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

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    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.

  3. Structural Implications for Selective Targeting of PARPs.

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    Steffen, Jamin D; Brody, Jonathan R; Armen, Roger S; Pascal, John M

    2013-12-20

    Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that use NAD(+) as a substrate to synthesize polymers of ADP-ribose (PAR) as post-translational modifications of proteins. PARPs have important cellular roles that include preserving genomic integrity, telomere maintenance, transcriptional regulation, and cell fate determination. The diverse biological roles of PARPs have made them attractive therapeutic targets, which have fueled the pursuit of small molecule PARP inhibitors. The design of PARP inhibitors has matured over the past several years resulting in several lead candidates in clinical trials. PARP inhibitors are mainly used in clinical trials to treat cancer, particularly as sensitizing agents in combination with traditional chemotherapy to reduce side effects. An exciting aspect of PARP inhibitors is that they are also used to selectivity kill tumors with deficiencies in DNA repair proteins (e.g., BRCA1/2) through an approach termed "synthetic lethality." In the midst of the tremendous efforts that have brought PARP inhibitors to the forefront of modern chemotherapy, most clinically used PARP inhibitors bind to conserved regions that permits cross-selectivity with other PARPs containing homologous catalytic domains. Thus, the differences between therapeutic effects and adverse effects stemming from pan-PARP inhibition compared to selective inhibition are not well understood. In this review, we discuss current literature that has found ways to gain selectivity for one PARP over another. We furthermore provide insights into targeting other domains that make up PARPs, and how new classes of drugs that target these domains could provide a high degree of selectivity by affecting specific cellular functions. A clear understanding of the inhibition profiles of PARP inhibitors will not only enhance our understanding of the biology of individual PARPs, but may provide improved therapeutic options for patients.

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

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    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.

  5. PARP Inhibitors in Ovarian Cancer.

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    Mittica, Gloria; Ghisoni, Eleonora; Giannone, Gaia; Genta, Sofia; Aglietta, Massimo; Sapino, Anna; Valabrega, Giorgio

    2018-03-05

    Treatment of Epithelial Ovarian Cancer (EOC), historically based on surgery and platinum doublet chemotherapy, is associated with high risk of relapse and poor prognosis for recurrent disease. In this landscape, the innovative treatment with PARP inhibitors (PARPis) demonstrated an outstanding activity in EOC, and is currently changing clinical practice in BRCA mutant patients. To highlight the mechanism of action, pharmacokinetics, clinical activity, indications and current strategies of development of Olaparib, Niraparib, Rucaparib, Talazoparib and Veliparib, the 5 most relevant PARPis. We performed a review on Pubmed using 'ovarian cancer' and the name of each PARPi (PARP inhibitor) discussed in the review as Medical Subject Headings (MeSH) keywords. The same search was performed on "clinicaltrial.gov" to identify ongoing clinical trials and on "google.com/patents" and "uspto.gov" for recent patents exploring PARPIs in ovarian cancer. Olaparib, Niraparib and Rucaparib are already approved for treatment of recurrent EOC and their indications are partially overlapping. Talazoparib and Veliparib are promising PARPis, but currently under investigation in early phase trials. Several studies are evaluating PARPis in monotherapy or in associations, in a wide range of settings (i.e. first line, neoadjuvant, platinum-sensitive and resistant disease). PARPis are valuable options in patients with recurrent ovarian cancer with promising activity in different stages of this disease. Further studies are required to better define optimal clinical settings, predictors of response beyond BRCA mutations and strategies to overcome secondary resistance of PARPis therapy in EOC. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  6. 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)

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

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    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.

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

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    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.

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

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    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.

  10. 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)

  11. 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

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

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    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.

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

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    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.

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

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    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.

  15. 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

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

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    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.

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

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    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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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.

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

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    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.

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

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    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.

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

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    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.

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

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    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.

  2. 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.

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

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    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.

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

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    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.

  5. 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}.

  6. 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.

  7. 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

  8. Radiosensitization by PARP inhibition to proton beam irradiation in cancer cells

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    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.

  9. 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

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

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    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

  11. 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

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

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    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.

  13. 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...

  14. 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.

  15. 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)

  16. Cross talk between poly(ADP-ribose) polymerase 1 methylation and oxidative stress involved in the toxic effect of anatase titanium dioxide nanoparticles

    Science.gov (United States)

    Bai, Wenlin; Chen, Yujiao; Gao, Ai

    2015-01-01

    Given the tremendous growth in the application of titanium dioxide nanoparticles (TNPs), concerns about the potential health hazards of TNPs to humans have been raised. Poly(ADP-ribose) polymerase 1 (PARP-1), a highly conserved DNA-binding protein, is involved in many molecular and cellular processes. Limited data demonstrated that certain nanomaterials induced the aberrant hypermethylation of PARP-1. However, the mechanism involved in TNP-induced PARP-1 abnormal methylation has not been studied. A549 cells were incubated with anatase TNPs (22.1 nm) for 24 hours pretreatment with or without methyltransferase inhibitor 5-aza-2′-deoxycytidine and the reactive oxygen species (ROS) scavenger α-lipoic acid to assess the possible role of methylation and ROS in the toxic effect of TNPs. After TNPs characterization, a battery of assays was performed to evaluate the toxic effect of TNPs, PARP-1 methylation status, and oxidative damage. Results showed that TNPs decreased the cell viability in a dose-dependent manner, in accordance with the increase of lactate dehydrogenase activity, which indicated membrane damage of cells. Similar to the high level of PARP-1 methylation, the generation of ROS was significantly increased after exposure to TNPs for 24 hours. Furthermore, α-lipoic acid decreased TNP-induced ROS generation and then attenuated TNP-triggered PARP-1 hypermethylation. Meanwhile, 5-aza-2′-deoxycytidine simultaneously decreased the ROS generation induced by TNPs, resulting in the decline of PARP-1 methylation. In summary, TNPs triggered the aberrant hypermethylation of the PARP-1 promoter and there was a cross talk between oxidative stress and PARP-1 methylation in the toxic effect of TNPs. PMID:26366077

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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...

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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....

  18. 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...

  19. 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.

  20. 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

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

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    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

  2. 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.

  3. 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

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

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    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

  5. 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.

  6. 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

  7. 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...

  8. 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.

  9. 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.

  10. 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.

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

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    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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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...

  18. 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).

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. ARTD1/PARP1 Negatively Regulates Glycolysis by Inhibiting Hexokinase 1 Independent of NAD+ Depletion

    Directory of Open Access Journals (Sweden)

    Elise Fouquerel

    2014-09-01

    Full Text Available ARTD1 (PARP1 is a key enzyme involved in DNA repair through the synthesis of poly(ADP-ribose (PAR in response to strand breaks, and it plays an important role in cell death following excessive DNA damage. ARTD1-induced cell death is associated with NAD+ depletion and ATP loss; however, the molecular mechanism of ARTD1-mediated energy collapse remains elusive. Using real-time metabolic measurements, we compared the effects of ARTD1 activation and direct NAD+ depletion. We found that ARTD1-mediated PAR synthesis, but not direct NAD+ depletion, resulted in a block to glycolysis and ATP loss. We then established a proteomics-based PAR interactome after DNA damage and identified hexokinase 1 (HK1 as a PAR binding protein. HK1 activity is suppressed following nuclear ARTD1 activation and binding by PAR. These findings help explain how prolonged activation of ARTD1 triggers energy collapse and cell death, revealing insight into the importance of nucleus-to-mitochondria communication via ARTD1 activation.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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 ...

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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)

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. The adaptor SASH1 acts through NOTCH1 and its inhibitor DLK1 in a 3D model of lumenogenesis involving CEACAM1.

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    Stubblefield, Kandis; Chean, Jennifer; Nguyen, Tung; Chen, Charng-Jui; Shively, John E

    2017-10-15

    CEACAM1 transfection into breast cancer cells restores lumen formation in a 3D culture model. Among the top up-regulated genes that were associated with restoration of lumen formation, the adaptor protein SASH1 was identified. Furthermore, SASH1 was shown to be critical for lumen formation by RNAi inhibition. Upon analyzing the gene array from CEACAM1/MCF7 cells treated with SASH1 RNAi, DLK1, an inhibitor of NOTCH1 signaling, was found to be down-regulated to the same extent as SASH1. Subsequent treatment of CEACAM1/MCF7 cells with RNAi to DLK1 also inhibited lumen formation, supporting its association with SASH1. In agreement with the role of DLK1 as a NOTCH1 inhibitor, NOTCH1, as well as its regulated genes HES1 and HEY1, were down-regulated in CEACAM1/MCF7 cells by the action of DLK1 RNAi, and up-regulated by SASH1 RNAi. When CEACAM1/MCF7 cells were treated with a γ-secretase inhibitor known to inhibit NOTCH signaling, lumen formation was inhibited. We conclude that restoration of lumen formation by CEACAM1 regulates the NOTCH1 signaling pathway via the adaptor protein SASH1 and the NOTCH1 inhibitor DLK1. These data suggest that the putative involvement of NOTCH1 as a tumor-promoting gene in breast cancer may depend on its lack of regulation in cancer, whereas its involvement in normal lumen formation requires activation of its expression, and subsequently, inhibition of its signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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    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.

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

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    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.

  2. 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.

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    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

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

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

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    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.

  5. Poly(ADP-ribosepolymerase-1 modulates microglial responses to amyloid β

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    Kauppinen Tiina M

    2011-11-01

    Full Text Available Abstract Background Amyloid β (Aβ accumulates in Alzheimer's disease (AD brain. Microglial activation also occurs in AD, and this inflammatory response may contribute to disease progression. Microglial activation can be induced by Aβ, but the mechanisms by which this occurs have not been defined. The nuclear enzyme poly(ADP-ribose polymerase-1 (PARP-1 regulates microglial activation in response to several stimuli through its interactions with the transcription factor, NF-κB. The purpose of this study was to evaluate whether PARP-1 activation is involved in Aβ-induced microglial activation, and whether PARP-1 inhibition can modify microglial responses to Aβ. Methods hAPPJ20 mice, which accumulate Aβ with ageing, were crossed with PARP-1-/- mice to assess the effects of PARP-1 depletion on microglial activation, hippocampal synaptic integrity, and cognitive function. Aβ peptide was also injected into brain of wt and PARP-1-/- mice to directly determine the effects of PARP-1 on Aβ-induced microglial activation. The effect of PARP-1 on Aβ-induced microglial cytokine production and neurotoxicity was evaluated in primary microglia cultures and in microglia-neuron co-cultures, utilizing PARP-1-/- cells and a PARP-1 inhibitor. NF-κB activation was evaluated in microglia infected with a lentivirus reporter gene. Results The hAPPJ20 mice developed microglial activation, reduced hippocampal CA1 calbindin expression, and impaired novel object recognition by age 6 months. All of these features were attenuated in hAPPJ20/PARP-1-/- mice. Similarly, Aβ1-42 injected into mouse brain produced a robust microglial response in wild-type mice, and this was blocked in mice lacking PARP-1 expression or activity. Studies using microglial cultures showed that PARP-1 activity was required for Aβ-induced NF-κB activation, morphological transformation, NO release, TNFα release, and neurotoxicity. Conversely, PARP-1 inhibition increased release of the

  6. 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

  7. Targeting poly(ADP-ribose)polymerase1 in neurological diseases: A promising trove for new pharmacological interventions to enter clinical translation.

    Science.gov (United States)

    Sriram, Chandra Shekhar; Jangra, Ashok; Kasala, Eshvendar Reddy; Bodduluru, Lakshmi Narendra; Bezbaruah, Babul Kumar

    2014-10-01

    The highly conserved abundant nuclear protein poly(ADP-ribose)polymerase1 (PARP1) functions at the center of cellular stress response and is mainly implied in DNA damage repair mechanism. Apart from its involvement in DNA damage repair, it does sway multiple vital cellular processes such as cell death pathways, cell aging, insulator function, chromatin modification, transcription and mitotic apparatus function. Since brain is the principal organ vulnerable to oxidative stress and inflammatory responses, upon stress encounters robust DNA damage can occur and intense PARP1 activation may result that will lead to various CNS diseases. In the context of soaring interest towards PARP1 as a therapeutic target for newer pharmacological interventions, here in the present review, we are attempting to give a silhouette of the role of PARP1 in the neurological diseases and the potential of its inhibitors to enter clinical translation, along with its structural and functional aspects. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. 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.

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

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    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.

  10. 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.

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

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    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.

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

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    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.

  13. Evaluation of the neuronal apoptotic pathways involved in cytoskeletal disruption-induced apoptosis.

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    Jordà, Elvira G; Verdaguer, Ester; Jimenez, Andrés; Arriba, S Garcia de; Allgaier, Clemens; Pallàs, Mercè; Camins, Antoni

    2005-08-01

    The cytoskeleton is critical to neuronal functioning and survival. Cytoskeletal alterations are involved in several neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. We studied the possible pathways involved in colchicine-induced apoptosis in cerebellar granule neurons (CGNs). Although colchicine evoked an increase in caspase-3, caspase-6 and caspase-9 activation, selective caspase inhibitors did not attenuate apoptosis. Inhibitors of other cysteine proteases such as PD150606 (a calpain-specific inhibitor), Z-Phe-Ala fluoromethyl ketone (a cathepsins-inhibitors) and N(alpha)-p-tosyl-l-lysine chloromethyl ketone (serine-proteases inhibitor) also had no effect on cell death/apoptosis induced by colchicine. However, BAPTA-AM 10 microM (intracellular calcium chelator) prevented apoptosis mediated by cytoskeletal alteration. These data indicate that calcium modulates colchicine-induced apoptosis in CGNs. PARP-1 inhibitors did not prevent apoptosis mediated by colchicine. Finally, colchicine-induced apoptosis in CGNs was attenuated by kenpaullone, a cdk5 inhibitor. Kenpaullone and indirubin also prevented cdk5/p25 activation mediated by colchicine. These findings indicate that cytoskeletal alteration can compromise cdk5 activation, regulating p25 formation and suggest that cdk5 inhibitors attenuate apoptosis mediated by cytoskeletal alteration. The present data indicate the potential therapeutic value of drugs that prevent the formation of p25 for the treatment of neurodegenerative disorders.

  14. 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

  15. 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.

  16. Possible involvement of 12-lipoxygenase activation in glucose-deprivation/reload-treated neurons.

    Science.gov (United States)

    Nagasawa, Kazuki; Kakuda, Taichi; Higashi, Youichirou; Fujimoto, Sadaki

    2007-12-18

    The aim of this study was to clarify whether 12-lipoxygenase (12-LOX) activation was involved in reactive oxygen species (ROS) generation, extensive poly(ADP-ribose) polymerase (PARP) activation and neuronal death induced by glucose-deprivation, followed by glucose-reload (GD/R). The decrease of neuronal viability and accumulation of poly(ADP-ribose) induced by GD/R were prevented 3-aminobenzamide, a representative PARP inhibitor, demonstrating this treatment protocol caused the same oxidative stress with the previously reported one. The PARP activation, ROS generation and decrease of neuron viability induced by GD/R treatment were almost completely abolished by an extracellular zinc chelator, CaEDTA. p47(phox), a cytosolic component of NADPH oxidase was translocated the membrane fraction by GD/R, indicating its activation, but it did not generate detectable ROS. Surprisingly, pharmacological inhibition of NADPH oxidase with apocynin and AEBSF further decreased the decreased neuron viability induced by GD/R. On the other hand, AA861, a 12-LOX inhibitor, prevented ROS generation and decrease of neuron viability caused by GD/R. Interestingly, an antioxidant, N-acetyl-l-cysteine rescued the neurons from GD/R-induced oxidative stress, implying effectiveness of antioxidant administration. These findings suggested that activation of 12-LOX, but not NADPH oxidase, following to zinc release might play an important role in ROS generation and decrease of viability in GD/R-treated neurons.

  17. 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.

  18. 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.

  19. 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

  20. Small molecule inhibitors uncover synthetic genetic interactions of human flap endonuclease 1 (FEN1 with DNA damage response genes.

    Directory of Open Access Journals (Sweden)

    Thomas A Ward

    Full Text Available Flap endonuclease 1 (FEN1 is a structure selective endonuclease required for proficient DNA replication and the repair of DNA damage. Cellularly active inhibitors of this enzyme have previously been shown to induce a DNA damage response and, ultimately, cell death. High-throughput screens of human cancer cell-lines identify colorectal and gastric cell-lines with microsatellite instability (MSI as enriched for cellular sensitivity to N-hydroxyurea series inhibitors of FEN1, but not the PARP inhibitor olaparib or other inhibitors of the DNA damage response. This sensitivity is due to a synthetic lethal interaction between FEN1 and MRE11A, which is often mutated in MSI cancers through instabilities at a poly(T microsatellite repeat. Disruption of ATM is similarly synthetic lethal with FEN1 inhibition, suggesting that disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. These are likely a result of the accumulation of aberrant replication forks, that accumulate as a consequence of a failure in Okazaki fragment maturation, as inhibition of FEN1 is toxic in cells disrupted for the Fanconi anemia pathway and post-replication repair. Furthermore, RAD51 foci accumulate as a consequence of FEN1 inhibition and the toxicity of FEN1 inhibitors increases in cells disrupted for the homologous recombination pathway, suggesting a role for homologous recombination in the resolution of damage induced by FEN1 inhibition. Finally, FEN1 appears to be required for the repair of damage induced by olaparib and cisplatin within the Fanconi anemia pathway, and may play a role in the repair of damage associated with its own disruption.

  1. Does plasminogen activator inhibitor-1 drive lymphangiogenesis?

    DEFF Research Database (Denmark)

    Bruyère, Françoise; Melen-Lamalle, Laurence; Blacher, Silvia

    2010-01-01

    The purpose of this study is to explore the function of plasminogen activator inhibitor-1 (PAI-1) during pathological lymphangiogenesis. PAI-1, the main physiological inhibitor of plasminogen activators is involved in pathological angiogenesis at least by controlling extracellular proteolysis and...

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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)

  7. BRCA1/2 mutation analysis in 41 ovarian cell lines reveals only one functionally deleterious BRCA1 mutation.

    LENUS (Irish Health Repository)

    Stordal, Britta

    2013-06-01

    Mutations in BRCA1\\/2 increase the risk of developing breast and ovarian cancer. Germline BRCA1\\/2 mutations occur in 8.6-13.7% of unselected epithelial ovarian cancers, somatic mutations are also frequent. BRCA1\\/2 mutated or dysfunctional cells may be sensitive to PARP inhibition by synthetic lethality. The aim of this study is to comprehensively characterise the BRCA1\\/2 status of a large panel of ovarian cancer cell lines available to the research community to assist in biomarker studies of novel drugs and in particular of PARP inhibitors. The BRCA1\\/2 genes were sequenced in 41 ovarian cell lines, mRNA expression of BRCA1\\/2 and gene methylation status of BRCA1 was also examined. The cytotoxicity of PARP inhibitors olaparib and veliparib was examined in 20 cell lines. The cell line SNU-251 has a deleterious BRCA1 mutation at 5564G > A, and is the only deleterious BRCA1\\/2 mutant in the panel. Two cell lines (UPN-251 and PEO1) had deleterious mutations as well as additional reversion mutations that restored the protein functionality. Heterozygous mutations in BRCA1\\/2 were relatively common, found in 14.6% of cell lines. BRCA1 was methylated in two cell lines (OVCAR8, A1847) and there was a corresponding decrease in gene expression. The BRCA1 methylated cell lines were more sensitive to PARP inhibition than wild-type cells. The SNU-251 deleterious mutant was more sensitive to PARP inhibition, but only in a long-term exposure to correct for its slow growth rate. Cell lines derived from metastatic disease are significantly more resistant to veliparib (2.0 fold p = 0.03) compared to those derived from primary tumours. Resistance to olaparib and veliparib was correlated Pearsons-R 0.5393, p = 0.0311. The incidence of BRCA1\\/2 deleterious mutations 1\\/41 cell lines derived from 33 different patients (3.0%) is much lower than the population incidence. The reversion mutations and high frequency of heterozygous mutations suggest that there is a selective

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. Identification of BRCA1-like triple-negative breast cancers by quantitative multiplex-ligation-dependent probe amplification (MLPA) analysis of BRCA1-associated chromosomal regions: a validation study

    International Nuclear Information System (INIS)

    Gross, Eva; Tinteren, Harm van; Li, Zhou; Raab, Sandra; Meul, Christina; Avril, Stefanie; Laddach, Nadja; Aubele, Michaela; Propping, Corinna; Gkazepis, Apostolos; Schmitt, Manfred; Meindl, Alfons; Nederlof, Petra M.; Kiechle, Marion; Lips, Esther H.

    2016-01-01

    Triple-negative breast cancer (TNBC) with a BRCA1-like molecular signature has been demonstrated to remarkably respond to platinum-based chemotherapy and might be suited for a future treatment with poly(ADP-ribose)polymerase (PARP) inhibitors. In order to rapidly assess this signature we have previously developed a multiplex-ligation-dependent probe amplification (MLPA)-based assay. Here we present an independent validation of this assay to confirm its important clinical impact. One-hundred-forty-four TNBC tumor specimens were analysed by the MLPA-based “BRCA1-like” test. Classification into BRCA1-like vs. non-BRCA1-like samples was performed by our formerly established nearest shrunken centroids classifier. Data were subsequently compared with the BRCA1-mutation/methylation status of the samples. T-lymphocyte infiltration and expression of the main target of PARP inhibitors, PARP1, were assessed on a subset of samples by immunohistochemistry. Data acquisition and interpretation was performed in a blinded manner. In the studied TNBC cohort, 63 out of 144 (44 %) tumors were classified into the BRCA1-like category. Among these, the MLPA test correctly predicted 15 out of 18 (83 %) samples with a pathogenic BRCA1-mutation and 20 of 22 (91 %) samples exhibiting BRCA1-promoter methylation. Five false-negative samples were observed. We identified high lymphocyte infiltration as one possible basis for misclassification. However, two falsely classified BRCA1-mutated tumors were also characterized by rather non-BRCA1-associated histopathological features such as borderline ER expression. The BRCA1-like vs. non-BRCA1-like signature was specifically enriched in high-grade (G3) cancers (90 % vs. 58 %, p = 0.0004) and was also frequent in tumors with strong (3+) nuclear PARP1 expression (37 % vs. 16 %; p = 0.087). This validation study confirmed the good performance of the initial MLPA assay which might thus serve as a valuable tool to select patients for platinum

  14. Identification of poly(ADP-ribose) polymerase-1 as the OXPHOS-generated ATP sensor of nuclei of animal cells

    International Nuclear Information System (INIS)

    Kun, Ernest; Kirsten, Eva; Hakam, Alaeddin; Bauer, Pal I.; Mendeleyev, Jerome

    2008-01-01

    Our results show that in the intact normal animal cell mitochondrial ATP is directly connected to nuclear PARP-1 by way of a specific adenylate kinase enzymatic path. This mechanism is demonstrated in two models: (a) by its inhibition with a specific inhibitor of adenylate kinase, and (b) by disruption of ATP synthesis through uncoupling of OXPHOS. In each instance the de-inhibited PARP-1 is quantitatively determined by enzyme kinetics. The nuclear binding site of PARP-1 is Topo I, and is identified as a critical 'switchpoint' indicating the nuclear element that connects OXPHOS with mRNA synthesis in real time. The mitochondrial-nuclear PARP-1 pathway is not operative in cancer cells

  15. 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.

  16. 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.

  17. 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.

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

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    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.

  19. The Role of Poly(ADP-ribose Polymerase-1 in Rheumatoid Arthritis

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    Samuel García

    2015-01-01

    Full Text Available Poly(ADP-ribose polymerase-1 (PARP-1 is a nuclear enzyme with a crucial role in the maintenance of genomic stability. In addition to the role of PARP-1 in DNA repair, multiple studies have also demonstrated its involvement in several inflammatory diseases, such as septic shock, asthma, atherosclerosis, and stroke, as well as in cancer. In these diseases, the pharmacological inhibition of PARP-1 has shown a beneficial effect, suggesting that PARP-1 regulates their inflammatory processes. In recent years, we have studied the role of PARP-1 in rheumatoid arthritis, as have other researchers, and the results have shown that PARP-1 has an important function in the development of this disease. This review summarizes current knowledge on the effects of PARP-1 in rheumatoid arthritis.

  20. 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

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

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    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.

  2. Loss of CHD1 causes DNA repair defects and enhances prostate cancer therapeutic responsiveness

    DEFF Research Database (Denmark)

    Kari, Vijayalakshmi; Mansour, Wael Yassin; Raul, Sanjay Kumar

    2016-01-01

    The CHD1 gene, encoding the chromo-domain helicase DNA-binding protein-1, is one of the most frequently deleted genes in prostate cancer. Here, we examined the role of CHD1 in DNA double-strand break (DSB) repair in prostate cancer cells. We show that CHD1 is required for the recruitment of Ct......-homologous end joining. Together, we provide evidence for a previously unknown role of CHD1 in DNA DSB repair via HR and show that CHD1 depletion sensitizes cells to PARP inhibitors, which has potential therapeutic relevance. Our findings suggest that CHD1 deletion, like BRCA1/2 mutation in ovarian cancer, may...... serve as a marker for prostate cancer patient stratification and the utilization of targeted therapies such as PARP inhibitors, which specifically target tumors with HR defects....

  3. Involvement of poly(ADP-ribose polymerase-1 in development of spinal cord injury in Chinese individuals: a Chinese clinical study

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    Meng Q

    2017-12-01

    Full Text Available Qing-Tao Meng,* Guang Yang,* Ren-Bo Li, Jing-Xin Nie, Wei Zhou, Hong-De Yu, Bo Chen, Li Jiang, Jing-Bo Shang Department of Spine Surgery, The Third People’s Hospital of Dalian, Dalian, People’s Republic of China *These authors contributed equally to this work Objective: We aimed to evaluate whether the polymorphism of poly(ADP-ribose polymerase-1 (PARP-1 is involved as potential risk factor in the development of spinal cord injury (SCI among Chinese individuals.Patients and methods: Patients with a confirmed diagnosis of SCI (other than traumatic injury and healthy individuals with no clinical symptoms of SCI were enrolled at Spinal Cord Injury Care Center, The Third People’s Hospital of Dalian, China. Genetic polymorphisms were studied in plasma samples by polymerase chain reaction-restriction fragment length polymorphism assay.Results: A total of 130 Chinese patients with SCI and 130 healthy Chinese individuals were included. We found that patients with the GG genotype (odds ratio [OR]: 4.09, 95% confidence interval [CI] 2.42–6.90, P<0.001 and carriers of the G allele (OR 3.96, 95% CI 2.33–6.74, P<0.0001 were at high risk of developing SCI. A del/ins polymorphism of the NF-κB1 gene (OR 3.32, 95% CI 1.96–5.61, P<0.001 was also found to be associated with SCI.Conclusion: Our study suggests that PARP-1 polymorphisms are involved in the development of SCI in Chinese individuals. Thus, PARP-1 polymorphisms can be considered as one of the potential risk factors for developing SCI. Keywords: spinal cord injury, poly(ADP-ribose polymerase-1, polymorphism 

  4. Parthanatos, a messenger of death

    Science.gov (United States)

    David, Karen Kate; Andrabi, Shaida Ahmad; Dawson, Ted Murray; Dawson, Valina Lynn

    2015-01-01

    Poly-ADP-ribose polymerase-1 (PARP-1)'s multiple roles in the cell span from maintaining life to inducing death. The processes PARP-1 is involved in include, but are not limited to DNA repair, DNA transcription, mitosis, and cell death. Of PARP-1's different cellular functions, its active role in cell death is of particular interest to designing therapies for diseases. Genetic deletion of PARP-1 revealed that PARP-1 over activation underlies cell death in experimental models of stroke, diabetes, inflammation and neurodegeneration. Since interfering with PARP-1 mediated cell death will be clinically beneficial, great effort has been invested into designing PARP-1 inhibitors and understanding mechanisms downstream of PARP-1 over activation. PARP-1 overactivation may kill by depleting cellular energy through nicotinamide adenine dinucleotide (NAD+) consumption, and by releasing the cell death effector apoptosis-inducing factor (AIF). Unexpectedly, recent evidence shows that poly-ADP ribose (PAR) polymer itself, and not the consumption of NAD+ is the source of cytotoxicity. Thus, PAR polymer acts as a cell death effector downstream of PARP-1-mediated cell death signaling. We coined the term parthanatos after Thanatos, the personification of death in Greek mythology, to refer to PAR-mediated cell death. In this review, we will summarize the proposed mechanisms by which PARP-1 overactivation kills. We will present evidence for parthanatos, and the questions raised by these recent findings. It is evident that further understanding of parthanatos opens up new avenues for therapy in ameliorating diseases related to PARP-1 over activation. PMID:19273119

  5. Biochemical and Biophysical Methods for Analysis of Poly(ADP-Ribose) Polymerase 1 and Its Interactions with Chromatin

    Energy Technology Data Exchange (ETDEWEB)

    Chassé, Maggie H.; Muthurajan, Uma M.; Clark, Nicholas J.; Kramer, Michael A.; Chakravarthy, Srinivas; Irving, Thomas; Luger, Karolin [Children; (IIT); (Colorado); (Amgen)

    2018-01-18

    Poly (ADP-Ribose) Polymerase I (PARP-1) is a first responder to DNA damage and participates in the regulation of gene expression. The interaction of PARP-1 with chromatin and DNA is complex and involves at least two different modes of interaction. In its enzymatically inactive state, PARP-1 binds native chromatin with similar affinity as it binds free DNA ends. Automodification of PARP-1 affects interaction with chromatin and DNA to different extents. Here we describe a series of biochemical and biophysical techniques to quantify and dissect the different binding modes of PARP-1 with its various substrates. The techniques listed here allow for high throughput and quantitative measurements of the interaction of different PARP-1 constructs (inactive and automodified) with chromatin and DNA damage models.

  6. In vivo repair of DNA damage induced by X-rays in the early stages of mouse fertilization, and the influence of maternal PARP1 ablation

    Energy Technology Data Exchange (ETDEWEB)

    Pacchierotti, F., E-mail: francesca.pacchierotti@enea.it [Unit of Radiation Biology and Human Health, ENEA CR Casaccia, Via Anguillarese 301, 00123 Rome (Italy); Ranaldi, R. [Unit of Radiation Biology and Human Health, ENEA CR Casaccia, Via Anguillarese 301, 00123 Rome (Italy); Derijck, A.A.; Heijden, G.W. van der; Boer, P. de [Radboud University Nijmegen Medical Centre, Department of Obstetrics and Gynaecology, P.O. Box 9101, 6500 HB Nijmegen (Netherlands)

    2011-09-01

    Highlights: {yields} We measure {gamma}H2AX and chromosome aberrations in mouse zygotes irradiated in vivo. {yields} We compare effects between zygotes obtained from wild type or Parp1 knockout females. {yields} The rate of chromosome aberrations is as high as that previously induced in vitro. {yields} The rate of radiation-induced {gamma}H2AX foci is lower than that measured in other cells. {yields} Without Parp1 there are more {gamma}H2AX foci but chromosome aberration rate is unaffected. - Abstract: The early pronucleus stage of the mouse zygote has been characterised in vitro as radiosensitive, due to a high rate of induction of chromosome-type chromosome abnormalities (CA). We have investigated the repair of irradiation induced double strand DNA breaks in vivo by {gamma}H2AX foci and first cleavage metaphase analysis. Breaks were induced in sperm and in the early zygote stages comprising sperm chromatin remodelling and early pronucleus expansion. Moreover, the role of PARP1 in the formation and repair of spontaneous and radiation-induced double strand breaks in the zygote was evaluated by comparing observations in C57BL/6J and PARP1 genetically ablated females. The results confirmed in vivo that the rate of chromosome aberration induction by X-rays was approximately 3-fold higher in the zygote than in mouse lymphocytes. This finding was related to a diminished efficiency of double strand break signalling, as shown by a lower rate of {gamma}H2AX radiation-induced foci compared to that measured in most other somatic cell types. The spontaneous frequency of CA in PARP1 depleted zygotes was slightly but significantly higher than in wild type zygotes. Also, these zygotes showed some impairment of the radiation-induced DNA Damage Response when exposed closer to the start of S-phase, revealed by a higher number of {gamma}H2AX foci and a longer cell cycle delay. The rate of chromosome aberrations, however, was not elevated over that of wild type zygotes, possibly

  7. Combining RNA interference and kinase inhibitors against cell signalling components involved in cancer

    International Nuclear Information System (INIS)

    O'Grady, Michael; Raha, Debasish; Hanson, Bonnie J; Bunting, Michaeline; Hanson, George T

    2005-01-01

    The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including oncogenic transformation. The tyrosine kinases of the epidermal growth factor receptor (EGFR) constitute the beginning of one signal transduction cascade leading to AP-1 activation and are known to control cell proliferation and differentiation. Drug discovery efforts targeting this receptor and other pathway components have centred on monoclonal antibodies and small molecule inhibitors. Resistance to such inhibitors has already been observed, guiding the prediction of their use in combination therapies with other targeted agents such as RNA interference (RNAi). This study examines the use of RNAi and kinase inhibitors for qualification of components involved in the EGFR/AP-1 pathway of ME180 cells, and their inhibitory effects when evaluated individually or in tandem against multiple components of this important disease-related pathway. AP-1 activation was assessed using an ME180 cell line stably transfected with a beta-lactamase reporter gene under the control of AP-1 response element following epidermal growth factor (EGF) stimulation. Immunocytochemistry allowed for further quantification of small molecule inhibition on a cellular protein level. RNAi and RT-qPCR experiments were performed to assess the amount of knockdown on an mRNA level, and immunocytochemistry was used to reveal cellular protein levels for the targeted pathway components. Increased potency of kinase inhibitors was shown by combining RNAi directed towards EGFR and small molecule inhibitors acting at proximal or distal points in the pathway. After cellular stimulation with EGF and analysis at the level of AP-1 activation using a β-lactamase reporter gene, a 10–12 fold shift or 2.5–3 fold shift toward greater potency in the IC 50 was observed for EGFR and MEK-1 inhibitors, respectively, in the presence of RNAi targeting EGFR. EGFR pathway components were qualified as

  8. 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.

  9. Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 or PARP

    Energy Technology Data Exchange (ETDEWEB)

    Stenerl& #246; w, Bo; Karlsson, Karin H.; Radulescu, Irina; Rydberg, Bjorn; Stenerlow, Bo

    2008-04-29

    Ionizing radiation induces a variety of different DNA lesions: in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have previously shown that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites (HLS) within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of HLS on DSB induction and repair, four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for bi-phasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements the fraction of fast rejoining decreased to less than 50% of the total. However, neither the half-times of the fast (t{sub 1/2} = 7-8 min) or slow (t{sub 1/2} = 2.5 h) DSB rejoining were changed significantly. At t=0 the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSB/cell/Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all tested cells, including M059K cells treated with wortmannin or DNA-PKcs defect M059J cells. Furthermore, cells lacking XRCC1 or Poly(ADP-ribose) polymerase-1 (PARP-1) rejoined both total DSBs and heat-released DSBs similar to normal cells. In summary, the presence of heat-labile sites have a substantial impact on DSB induction yields and DSB rejoining rates measured by pulsed-field gel electrophoresis, and HLS repair is independent of DNA-PKcs, XRCC1 and PARP.

  10. BRCA1 Expression Is Epigenetically Repressed in Sporadic Ovarian Cancer Cells by Overexpression of C-Terminal Binding Protein 2

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    Taymaa May

    2013-06-01

    Full Text Available INTRODUCTION: Ovarian cancer is the leading cause of mortality from gynecological malignancy despite advancements in novel therapeutics. We have recently demonstrated that the transcriptional co-repressor C-terminal binding protein 2 (CtBP2 is overexpressed in epithelial ovarian carcinoma. MATERIALS AND METHODS: Reverse-transcribed cDNA from CtBP2 wild-type and knockdown ovarian cancer cell lines was hybridized to Affymetrix Gene 1.0 ST microarrays, and differentially expressed genes were studied. Immunohistochemical analysis of CtBP2 and BRCA1 staining of ovarian tissues was performed. Chromatin immunoprecipitation (ChIP and luciferase assays were carried out. The effect of the drugs 4-methylthio-2-oxobutyric acid (MTOB and poly(ADP-ribose polymerase (PARP inhibitor Olaparib on CtBP2 wild-type and knockdown cell lines was examined using methylthiazol tetrazolium assays and an xCELLigence System. RESULTS: Eighty-five genes involved in DNA repair, mitotic checkpoint, nucleosome assembly, and the BRCA1 network were differentially regulated by CtBP2 expression. ChIP and luciferase reporter assays using a BRCA1 promoter-regulated luciferase construct indicated that the CtBP2 complex binds the BRCA1 promoter and represses BRCA1 transcription. Immunohistochemistry illustrated a significant inverse CtBP2 and BRCA1 expression in a panel of malignant ovarian tumor tissues. The CtBP2 inhibitor MTOB suppressed ovarian cancer cell survival in a CtBP2-dependent manner. Ovarian cancer cells with CtBP2 knockdown did not display increased sensitivity to the PARP inhibitor Olaparib. CONCLUSION: CtBP2 is an ovarian cancer oncogene that may play a significant role in epigenetically silencing BRCA1 function in sporadic epithelial ovarian cancer. CtBP2-specific inhibitors, such as MTOB, may be effective adjunct therapies in the management of patients with CtBP2-positive ovarian carcinoma.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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....

  16. 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.

  17. 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.

  18. Medicinal chemistry insights in the discovery of novel LSD1 inhibitors.

    Science.gov (United States)

    Wang, Xueshun; Huang, Boshi; Suzuki, Takayoshi; Liu, Xinyong; Zhan, Peng

    2015-01-01

    LSD1 is an epigenetic modulator associated with transcriptional regulation of genes involved in a broad spectrum of key cellular processes, and its activity is often altered under pathological conditions. LSD1 inhibitors are considered to be candidates for therapy of cancer, viral diseases and neurodegeneration. Many LSD1 inhibitors with various scaffolds have been disclosed, and a few potent molecules are in different stages of clinical development. In this review, we summarize recent biological findings on the roles of LSD1 and the current understanding of the clinical significance of LSD1, and focus on the medicinal chemistry strategies used in the design and development of LSD1 inhibitors as drug-like epigenetic modulators since 2012, including a brief consideration of structure-activity relationships.

  19. 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

  20. Differential effect of EGFR inhibitors on tamoxifen-resistant breast cancer cells.

    Science.gov (United States)

    Kim, Sangmin; Lee, Jeongmin; Oh, Soo Jin; Nam, Seok Jin; Lee, Jeong Eon

    2015-09-01

    Although tamoxifen is the most common and effective therapy for treatment of estrogen receptor-α (ER-α) breast cancer patients, resistance of endocrine therapy occurs, either de novo or acquired during therapy. Here, we investigated the clinical value of epidermal growth factor receptor (EGFR) in tamoxifen-resistant (TamR) patients and the differential effect of EGFR inhibitors, neratinib and gefitinib, on TamR breast cancer cell model. The morphology of TamR MCF7 cells showed mesenchymal phenotypes and did not induce cell death by tamoxifen treatment compared with tamoxifen‑sensitive (TamS) MCF7 cells. In addition, mesenchymal marker proteins, including N-cadherin (N-cad), fibronectin (FN), and Slug, significantly increased in TamR cells. In contrast, ER-α and E-cadherin (E-cad) were greatly decreased. We also found that the levels of EGFR and HER2 expression were increased in TamR cells. Furthermore, we observed that EGFR expression was directly involved with poor prognosis of tamoxifen-treated breast cancer patients using the GSE1378 date set. Thus, we treated TamR and TamS cells with EGFR inhibitors, neratinib and gefitinib, respectively. Interestingly, neratinib induced apoptotic cell death of TamR but not gefitinib. Cleaved PARP-1 expression was also increased by neratinib treatment in TamR cells. Therefore, we suggest that neratinib may be a potential therapeutic drug for treating TamR breast cancer.

  1. Design, synthesis, X-ray studies, and biological evaluation of novel macrocyclic HIV-1 protease inhibitors involving the P1'-P2' ligands

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Arun K.; Sean Fyvie, W.; Brindisi, Margherita; Steffey, Melinda; Agniswamy, Johnson; Wang, Yuan-Fang; Aoki, Manabu; Amano, Masayuki; Weber, Irene T.; Mitsuya, Hiroaki

    2017-11-01

    Design, synthesis, and evaluation of a new class of HIV-1 protease inhibitors containing diverse flexible macrocyclic P1'-P2' tethers are reported. Inhibitor 5a with a pyrrolidinone-derived macrocycle exhibited favorable enzyme inhibitory and antiviral activity (Ki = 13.2 nM, IC50 = 22 nM). Further incorporation of heteroatoms in the macrocyclic skeleton provided macrocyclic inhibitors 5m and 5o. These compounds showed excellent HIV-1 protease inhibitory (Ki = 62 pM and 14 pM, respectively) and antiviral activity (IC50 = 5.3 nM and 2.0 nM, respectively). Inhibitor 5o also remained highly potent against a DRV-resistant HIV-1 variant.

  2. Differentiation-Associated Downregulation of Poly(ADP-Ribose Polymerase-1 Expression in Myoblasts Serves to Increase Their Resistance to Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Gábor Oláh

    Full Text Available Poly(ADP-ribose polymerase 1 (PARP-1, the major isoform of the poly (ADP-ribose polymerase family, is a constitutive nuclear and mitochondrial protein with well-recognized roles in various essential cellular functions such as DNA repair, signal transduction, apoptosis, as well as in a variety of pathophysiological conditions including sepsis, diabetes and cancer. Activation of PARP-1 in response to oxidative stress catalyzes the covalent attachment of the poly (ADP-ribose (PAR groups on itself and other acceptor proteins, utilizing NAD+ as a substrate. Overactivation of PARP-1 depletes intracellular NAD+ influencing mitochondrial electron transport, cellular ATP generation and, if persistent, can result in necrotic cell death. Due to their high metabolic activity, skeletal muscle cells are particularly exposed to constant oxidative stress insults. In this study, we investigated the role of PARP-1 in a well-defined model of murine skeletal muscle differentiation (C2C12 and compare the responses to oxidative stress of undifferentiated myoblasts and differentiated myotubes. We observed a marked reduction of PARP-1 expression as myoblasts differentiated into myotubes. This alteration correlated with an increased resistance to oxidative stress of the myotubes, as measured by MTT and LDH assays. Mitochondrial function, assessed by measuring mitochondrial membrane potential, was preserved under oxidative stress in myotubes compared to myoblasts. Moreover, basal respiration, ATP synthesis, and the maximal respiratory capacity of mitochondria were higher in myotubes than in myoblasts. Inhibition of the catalytic activity of PARP-1 by PJ34 (a phenanthridinone PARP inhibitor exerted greater protective effects in undifferentiated myoblasts than in differentiated myotubes. The above observations in C2C12 cells were also confirmed in a rat-derived skeletal muscle cell line (L6. Forced overexpression of PARP1 in C2C12 myotubes sensitized the cells to oxidant

  3. 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.

  4. 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.

  5. 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.

  6. Notch signaling protects retina from nuclear factor-kB- and poly-ADP-ribosepolymerase-mediated apoptosis under high-glucose stimulation

    Institute of Scientific and Technical Information of China (English)

    Xiuhong Qin1; Zhenzhen Zhang2; Haitao Xu1; and Yazhen Wu1

    2011-01-01

    Proliferative diabetic retinopathy,the primary cause of vision loss in adults,is one of serious microvascular complications caused by diabetes.Both poly-ADP-ribosepolymerase (PARP) and nuclear factor (NF)-kB signaling are involved in the injury process.Injury activates PARP,which in turn potentiates NF-kB activation and causes cell apoptosis.Like the NF-kB pathway,Notch1 signaling plays a key role in the regulation of cell proliferation,differentiation,and apoptosis.However,the connections between these signaling pathways are not well understood.In this study,we used both streptozotocin (STZ)-induced diabetic mice and human retinal vascular endothelial cells (HRVECs) cultured in high glucose to detect these relationships.We found that apoptosis was increased in both STZinduced diabetic mice and high-glucose-treated HRVECs,which was due to increased activation of PARP,cleaved caspase3,and reduced expression of Notch1 and p-Akt.The results of Notch1 overexpression and knockdown indicated that Notch1 signaling participated in the interaction of PARP and p50,and inhibited PARP- and p50-mediated apoptosis directly.These phenomena could be blocked by pretreatment with the PI3K inhibitor wortmannin via reducing p-Akt levels.Thus,our study demonstrated that Notch1 signaling protects cells from PARP- and NF-kB-induced apoptosis under high glucose through the activation of Akt.

  7. Involvement of caspase-2 and caspase-9 in endoplasmic reticulum stress-induced apoptosis: A role for the IAPs

    International Nuclear Information System (INIS)

    Cheung, Herman H.; Lynn Kelly, N.; Liston, Peter; Korneluk, Robert G.

    2006-01-01

    Dysregulation of apoptosis is involved in a wide spectrum of disease ranging from proliferative to degenerative disorders. An emerging area of study in apoptosis is the critical contribution of the endoplasmic reticulum (ER) in both mitochondrial and ER specific apoptosis pathways. Here we show that brefeldin A and tunicamycin-mediated ER stress lead to caspase-dependent apoptosis involving caspase-2. Confocal microscopy and subcellular fractionation indicate that caspase-2 is localized to the ER, and following ER stress, the processing of caspase-2 and -9 is an early event preceding the activation of caspase-3 and -7 and the cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP). Inhibition and silencing of either caspase-2 or caspase-9 suppress ER stress-induced apoptosis, as demonstrated by annexin V binding. Similarly, transduction with an adenovirus encoding either Inhibitors of Apoptosis (IAP) protein HIAP1/c-IAP2 or HIAP2/c-IAP1 also suppresses ER stress-induced apoptosis. However, among HIAP1, HIAP2 and XIAP, only HIAP2 binds and inhibits caspase-2. Our results thus indicate a novel mechanism by which HIAP2 can regulate ER-initiated apoptosis by modulating the activity of caspase-2

  8. Discovery of natural mouse serum derived HIV-1 entry inhibitor(s).

    Science.gov (United States)

    Wei, M; Chen, Y; Xi, J; Ru, S; Ji, M; Zhang, D; Fang, Q; Tang, B

    Among rationally designed human immunodeficiency virus 1 (HIV-1) inhibitors, diverse natural factors have showed as potent anti-HIV activity in human blood. We have discovered that the boiled supernatant of healthy mouse serum could suppress HIV-1 entry, and exhibited reduced inhibitory activity after trypsin digestion. Further analysis demonstrated that only the fraction containing 10-25 K proteins could inhibit HIV-1 mediated cell-cell fusion. These results suggest that the 10-25 K protein(s) is novel natural HIV-1 entry inhibitor(s). Our findings provide important information about novel natural HIV entry inhibitors in mouse serum.

  9. Positive transcriptional regulation of the human micro opioid receptor gene by poly(ADP-ribose) polymerase-1 and increase of its DNA binding affinity based on polymorphism of G-172 -> T.

    Science.gov (United States)

    Ono, Takeshi; Kaneda, Toshio; Muto, Akihiro; Yoshida, Tadashi

    2009-07-24

    Micro opioid receptor (MOR) agonists such as morphine are applied widely in clinical practice as pain therapy. The effects of morphine through MOR, such as analgesia and development of tolerance and dependence, are influenced by individual specificity. Recently, we analyzed single nucleotide polymorphisms on the human MOR gene to investigate the factors that contribute to individual specificity. In process of single nucleotide polymorphisms analysis, we found that specific nuclear proteins bound to G(-172) --> T region in exon 1 in MOR gene, and its affinity to DNA was increased by base substitution from G(-172) to T(-172). The isolated protein was identified by mass spectrometry and was confirmed by Western blotting to be poly(ADP-ribose) polymerase-1 (PARP-1). The overexpressed PARP-1 bound to G(-172) --> T and enhanced the transcription of reporter vectors containing G(-172) and T(-172). Furthermore, PARP-1 inhibitor (benzamide) decreased PARP-1 binding to G(-172) --> T without affecting mRNA or protein expression level of PARP-1 and down-regulated the subsequent MOR gene expression in SH-SY5Y cells. Moreover, we found that tumor necrosis factor-alpha enhanced MOR gene expression as well as increased PARP-1 binding to the G(-172) --> T region and G(-172) --> T-dependent transcription in SH-SY5Y cells. These effects were also inhibited by benzamide. In this study, our data suggest that PARP-1 positively regulates MOR gene transcription via G(-172) --> T, which might influence individual specificity in therapeutic opioid effects.

  10. 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.

  11. The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays.

    Directory of Open Access Journals (Sweden)

    Marlien Pieters

    Full Text Available Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g, platelet-containing (352 g and platelet-rich plasma (200 g were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation. Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly

  12. Escape from Human Immunodeficiency Virus Type 1 (HIV-1 Entry Inhibitors

    Directory of Open Access Journals (Sweden)

    Carol D. Weiss

    2012-12-01

    Full Text Available The human immunodeficiency virus (HIV enters cells through a series of molecular interactions between the HIV envelope protein and cellular receptors, thus providing many opportunities to block infection. Entry inhibitors are currently being used in the clinic, and many more are under development. Unfortunately, as is the case for other classes of antiretroviral drugs that target later steps in the viral life cycle, HIV can become resistant to entry inhibitors. In contrast to inhibitors that block viral enzymes in intracellular compartments, entry inhibitors interfere with the function of the highly variable envelope glycoprotein as it continuously adapts to changing immune pressure and available target cells in the extracellular environment. Consequently, pathways and mechanisms of resistance for entry inhibitors are varied and often involve mutations across the envelope gene. This review provides a broad overview of entry inhibitor resistance mechanisms that inform our understanding of HIV entry and the design of new inhibitors and vaccines.

  13. 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.

  14. Discovery and study of novel protein tyrosine phosphatase 1B inhibitors

    Science.gov (United States)

    Zhang, Qian; Chen, Xi; Feng, Changgen

    2017-10-01

    Protein tyrosine phosphatase 1B (PTP1B) is considered to be a target for therapy of type II diabetes and obesity. So it is of great significance to take advantage of a computer aided drug design protocol involving the structured-based virtual screening with docking simulations for fast searching small molecule PTP1B inhibitors. Based on optimized complex structure of PTP1B bound with specific inhibitor of IX1, structured-based virtual screening against a library of natural products containing 35308 molecules, which was constructed based on Traditional Chinese Medicine database@ Taiwan (TCM database@ Taiwan), was conducted to determine the occurrence of PTP1B inhibitors using the Lubbock module and CDOCKER module from Discovery Studio 3.1 software package. The results were further filtered by predictive ADME simulation and predictive toxic simulation. As a result, 2 good drug-like molecules, namely para-benzoquinone compound 1 and Clavepictine analogue 2 were identified ultimately with the dock score of original inhibitor (IX1) and the receptor as a threshold. Binding model analyses revealed that these two candidate compounds have good interactions with PTP1B. The PTP1B inhibitory activity of compound 2 hasn't been reported before. The optimized compound 2 has higher scores and deserves further study.

  15. 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.

  16. Critical role of poly(ADP-ribose) polymerase-1 in modulating the mode of cell death caused by continuous oxidative stress.

    Science.gov (United States)

    Son, Young-Ok; Kook, Sung-Ho; Jang, Yong-Suk; Shi, Xianglin; Lee, Jeong-Chae

    2009-11-01

    Continuously generated hydrogen peroxide (H(2)O(2)) inhibits typical apoptosis and instead initiates a caspase-independent, apoptosis-inducing factor (AIF)-mediated pyknotic cell death. This may be related to H(2)O(2)-mediated DNA damage and subsequent ATP depletion, although the exact mechanisms by which the mode of cell death is decided after H(2)O(2) exposure are still unclear. Accumulated evidence and our previous data led us to hypothesize that continuously generated H(2)O(2), not an H(2)O(2) bolus, induces severe DNA damage, signaling poly(ADP-ribose) polymerase-1 (PARP-1) activation, ATP depletion, and eventually caspase-independent cell death. Results from the present study support that H(2)O(2) generated continuously by glucose oxidase causes excessive DNA damage and PARP-1 activation. Blockage of PARP-1 by a siRNA transfection or by pharmacological inhibitor resulted in the significant inhibition of ATP depletion, loss of mitochondrial membrane potential, nuclear translocation of AIF and endonuclease G, and eventually conversion to caspase-dependent apoptosis. Overall, the current study demonstrates the different roles of PARP-1 inhibition in modulation of cell death according to the method of H(2)O(2) exposure, that is, continuous generation versus a direct addition. (c) 2009 Wiley-Liss, Inc.

  17. Engagement of Components of DNA-Break Repair Complex and NFκB in Hsp70A1A Transcription Upregulation by Heat Shock.

    Science.gov (United States)

    Hazra, Joyita; Mukherjee, Pooja; Ali, Asif; Poddar, Soumita; Pal, Mahadeb

    2017-01-01

    An involvement of components of DNA-break repair (DBR) complex including DNA-dependent protein kinase (DNA-PK) and poly-ADP-ribose polymerase 1 (PARP-1) in transcription regulation in response to distinct cellular signalling has been revealed by different laboratories. Here, we explored the involvement of DNA-PK and PARP-1 in the heat shock induced transcription of Hsp70A1A. We find that inhibition of both the catalytic subunit of DNA-PK (DNA-PKc), and Ku70, a regulatory subunit of DNA-PK holo-enzyme compromises transcription of Hsp70A1A under heat shock treatment. In immunoprecipitation based experiments we find that Ku70 or DNA-PK holoenzyme associates with NFκB. This NFκB associated complex also carries PARP-1. Downregulation of both NFκB and PARP-1 compromises Hsp70A1A transcription induced by heat shock treatment. Alteration of three bases by site directed mutagenesis within the consensus κB sequence motif identified on the promoter affected inducibility of Hsp70A1A transcription by heat shock treatment. These results suggest that NFκB engaged with the κB motif on the promoter cooperates in Hsp70A1A activation under heat shock in human cells as part of a DBR complex including DNA-PK and PARP-1.

  18. 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.

  19. Chemotherapeutics-resistance "arms" race: An update on mechanisms involved in resistance limiting EGFR inhibitors in lung cancer.

    Science.gov (United States)

    Singh, Pankaj Kumar; Silakari, Om

    2017-10-01

    Clinical reports suggest that EGFR-mutated lung cancer usually respond significantly towards small molecule tyrosine kinase inhibitors. Same studies also report the eventual development of acquired resistance within a median time interval of 9 to 14months. One of the major mechanisms involved in this acquired resistance was found to be a secondary point mutation at gate-keeper residue, EGFR T790M. However, there are other recent studies which disclose the role of few other novel key players such as, ZEB1, TOPK etc., in the development of tolerance towards the EGFR TKI's, along with other commonly known mechanisms, such as amplification of signalling pathways such as, c-MET, Erbb2, AXL, additional acquired secondary mutations (PIK3CA, BRAF), or phenotypic transformation (small cell or epithelial to mesenchymal transitions). Interestingly, a recent study showed development of resistance via another point mutation, C797S, in case of tumors which were previously resistant and were administered agents capable of overcoming T790M gatekeeper mutation based resistance. Thus, raising serious concern over the direction of drug development involving tyrosine kinases such as EGFR. Current approaches focussing on development of third generation inhibitors, dual inhibitors or inhibitors of HSP90 have shown significant activity but do not answer the long term question of resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Co-targeting Deoxyribonucleic Acid–Dependent Protein Kinase and Poly(Adenosine Diphosphate-Ribose) Polymerase-1 Promotes Accelerated Senescence of Irradiated Cancer Cells

    International Nuclear Information System (INIS)

    Azad, Arun; Bukczynska, Patricia; Jackson, Susan; Haput, Ygal; Cullinane, Carleen; McArthur, Grant A.; Solomon, Benjamin

    2014-01-01

    Purpose: To examine the effects of combined blockade of DNA-dependent protein kinase (DNA-PK) and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) on accelerated senescence in irradiated H460 and A549 non-small cell lung cancer cells. Methods and Materials: The effects of KU5788 and AG014699 (inhibitors of DNA-PK and PARP-1, respectively) on clonogenic survival, DNA double-strand breaks (DSBs), apoptosis, mitotic catastrophe, and accelerated senescence in irradiated cells were examined in vitro. For in vivo experiments, H460 xenografts established in athymic nude mice were treated with BEZ235 (a DNA-PK, ATM, and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) and AG014699 to determine effects on proliferation, DNA DSBs, and accelerated senescence after radiation. Results: Compared with either inhibitor alone, combination treatment with KU57788 and AG014699 reduced postradiation clonogenic survival and significantly increased persistence of Gamma-H2AX (γH2AX) foci in irradiated H460 and A549 cells. Notably, these effects coincided with the induction of accelerated senescence in irradiated cells as reflected by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression, and senescence-associated cytokine secretion. In irradiated H460 xenografts, concurrent therapy with BEZ235 and AG014699 resulted in sustained Gamma-H2AX (γH2AX) staining and prominent β-galactosidase activity. Conclusion: Combined DNA-PK and PARP-1 blockade increased tumor cell radiosensitivity and enhanced the prosenescent properties of ionizing radiation in vitro and in vivo. These data provide a rationale for further preclinical and clinical testing of this therapeutic combination

  1. Co-targeting Deoxyribonucleic Acid–Dependent Protein Kinase and Poly(Adenosine Diphosphate-Ribose) Polymerase-1 Promotes Accelerated Senescence of Irradiated Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Azad, Arun, E-mail: arun.azad@bccancer.bc.ca [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Department of Pathology, St. Vincent' s Hospital, University of Melbourne, Parkville, Victoria (Australia); Bukczynska, Patricia; Jackson, Susan [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Haput, Ygal; Cullinane, Carleen [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria (Australia); McArthur, Grant A.; Solomon, Benjamin [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Division of Cancer Medicine, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Department of Medicine, St. Vincent' s Hospital, University of Melbourne, Parkville, Victoria (Australia); Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria (Australia)

    2014-02-01

    Purpose: To examine the effects of combined blockade of DNA-dependent protein kinase (DNA-PK) and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) on accelerated senescence in irradiated H460 and A549 non-small cell lung cancer cells. Methods and Materials: The effects of KU5788 and AG014699 (inhibitors of DNA-PK and PARP-1, respectively) on clonogenic survival, DNA double-strand breaks (DSBs), apoptosis, mitotic catastrophe, and accelerated senescence in irradiated cells were examined in vitro. For in vivo experiments, H460 xenografts established in athymic nude mice were treated with BEZ235 (a DNA-PK, ATM, and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) and AG014699 to determine effects on proliferation, DNA DSBs, and accelerated senescence after radiation. Results: Compared with either inhibitor alone, combination treatment with KU57788 and AG014699 reduced postradiation clonogenic survival and significantly increased persistence of Gamma-H2AX (γH2AX) foci in irradiated H460 and A549 cells. Notably, these effects coincided with the induction of accelerated senescence in irradiated cells as reflected by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression, and senescence-associated cytokine secretion. In irradiated H460 xenografts, concurrent therapy with BEZ235 and AG014699 resulted in sustained Gamma-H2AX (γH2AX) staining and prominent β-galactosidase activity. Conclusion: Combined DNA-PK and PARP-1 blockade increased tumor cell radiosensitivity and enhanced the prosenescent properties of ionizing radiation in vitro and in vivo. These data provide a rationale for further preclinical and clinical testing of this therapeutic combination.

  2. Synthetic Routes to N-9 Alkylated 8-Oxoguanines; Weak Inhibitors of the Human DNA Glycosylase OGG1

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    Tushar R. Mahajan

    2015-09-01

    Full Text Available The human 8-oxoguanine DNA glycosylase OGG1 is involved in base excision repair (BER, one of several DNA repair mechanisms that may counteract the effects of chemo- and radiation therapy for the treatment of cancer. We envisage that potent inhibitors of OGG1 may be found among the 9-alkyl-8-oxoguanines. Thus we explored synthetic routes to 8-oxoguanines and examined these as OGG1 inhibitors. The best reaction sequence started from 6-chloroguanine and involved N-9 alkylation, C-8 bromination, and finally simultaneous hydrolysis of both halides. Bromination before N-alkylation should only be considered when the N-substituent is not compatible with bromination conditions. The 8-oxoguanines were found to be weak inhibitors of OGG1. 6-Chloro-8-oxopurines, byproducts in the hydrolysis of 2,6-halopurines, turned out to be slightly better inhibitors than the corresponding 8-oxoguanines.

  3. Autophagy regulated by prolyl isomerase Pin1 and phospho-Ser-GSK3αβ involved in protection of oral squamous cell carcinoma against cadmium toxicity

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    So, Keum-Young [Department of Anesthesiology and Pain Medicine College of Dentistry, Chosun University, 309 Pilmundaero, Dong-gu, Gwangju 501-759 (Korea, Republic of); Ahn, Sang-Gun [Department of Pathology, College of Dentistry, Chosun University, 309 Pilmundaero, Dong-gu, Gwangju 501-759 (Korea, Republic of); Oh, Seon-Hee, E-mail: seonh@chosun.ac.kr [Department of Premedicine, School of Medicine, College of Dentistry, Chosun University, 309 Pilmundaero, Dong-gu, Gwangju 501-759 (Korea, Republic of)

    2015-10-23

    Prolyl isomerase Pin1 plays an important role in cell proliferation and is overexpressed in many human tumors. However, its role in autophagy induction remains undefined. Here we show that Pin1 regulates cell survival via autophagy in cadmium (Cd)-exposed oral squamous cell carcinoma (OSCC). OSCC exposure to Cd induced autophagy, as demonstrated by the formation of green fluorescent punctae in transfected cells expressing GFP-conjugated microtubule-associated protein light chain 3 (LC3) and by LC3 flux in the presence of autophagy inhibitors. Suppression of Atg5 enhanced Cd-induced apoptosis, indicating that autophagy is involved in cell protection. In dose–response experiments, cleavage of procaspase-3, PARP-1, and LC3-II was induced by Cd with an IC{sub 50} of 45 μM. Expression of Pin1 was decreased at or above the Cd IC{sub 50} value and was inversely correlated with the level of phospho(p)-Ser-GSK3αβ. Genetic or pharmacologic inhibition of Pin1 suppressed Cd-induced autophagy, but increased p-Akt-mediated p-Ser-GSK3αβ; this was reversed by overexpression of Pin1. However, suppression of GSK3αβ inhibited Cd-induced autophagy and induced apoptosis, which could be reversed by overexpression of GSK3β. The PI3K inhibitor Ly294002 blocked p-Akt-mediated increases in p-Ser-GSK3αβ and autophagy and induced apoptosis. Therefore, p-Ser-GSK3αβ can directly regulate Cd-induced autophagy, although its function is suppressed by Pin1. Collectively, the present results indicate that targeting Pin1 and GSK3αβ at the same time could be an effective therapeutic tool for Cd-induced carcinogenesis. - Highlights: • Pin1 regulated autophagy to protect cells from cadmium toxicity. • Pin1 suppression inhibited cadmium-induced autophagy and induced apoptosis. • Pin1 inhibited the function of p-Ser-GSK3αβ in autophagy regulation. • p-Ser-GSK3αβ regulated autophagy independently of Pin1.

  4. Autophagy regulated by prolyl isomerase Pin1 and phospho-Ser-GSK3αβ involved in protection of oral squamous cell carcinoma against cadmium toxicity

    International Nuclear Information System (INIS)

    So, Keum-Young; Ahn, Sang-Gun; Oh, Seon-Hee

    2015-01-01

    Prolyl isomerase Pin1 plays an important role in cell proliferation and is overexpressed in many human tumors. However, its role in autophagy induction remains undefined. Here we show that Pin1 regulates cell survival via autophagy in cadmium (Cd)-exposed oral squamous cell carcinoma (OSCC). OSCC exposure to Cd induced autophagy, as demonstrated by the formation of green fluorescent punctae in transfected cells expressing GFP-conjugated microtubule-associated protein light chain 3 (LC3) and by LC3 flux in the presence of autophagy inhibitors. Suppression of Atg5 enhanced Cd-induced apoptosis, indicating that autophagy is involved in cell protection. In dose–response experiments, cleavage of procaspase-3, PARP-1, and LC3-II was induced by Cd with an IC_5_0 of 45 μM. Expression of Pin1 was decreased at or above the Cd IC_5_0 value and was inversely correlated with the level of phospho(p)-Ser-GSK3αβ. Genetic or pharmacologic inhibition of Pin1 suppressed Cd-induced autophagy, but increased p-Akt-mediated p-Ser-GSK3αβ; this was reversed by overexpression of Pin1. However, suppression of GSK3αβ inhibited Cd-induced autophagy and induced apoptosis, which could be reversed by overexpression of GSK3β. The PI3K inhibitor Ly294002 blocked p-Akt-mediated increases in p-Ser-GSK3αβ and autophagy and induced apoptosis. Therefore, p-Ser-GSK3αβ can directly regulate Cd-induced autophagy, although its function is suppressed by Pin1. Collectively, the present results indicate that targeting Pin1 and GSK3αβ at the same time could be an effective therapeutic tool for Cd-induced carcinogenesis. - Highlights: • Pin1 regulated autophagy to protect cells from cadmium toxicity. • Pin1 suppression inhibited cadmium-induced autophagy and induced apoptosis. • Pin1 inhibited the function of p-Ser-GSK3αβ in autophagy regulation. • p-Ser-GSK3αβ regulated autophagy independently of Pin1.

  5. 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

  6. Analysis of poly(ADP-Ribose polymerases in Arabidopsis telomere biology.

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    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.

  7. 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

  8. The combination of olaparib and camptothecin for effective radiosensitization

    International Nuclear Information System (INIS)

    Miura, Katsutoshi; Sakata, Koh-ichi; Someya, Masanori; Matsumoto, Yoshihisa; Matsumoto, Hideki; Takahashi, Akihisa; Hareyama, Masato

    2012-01-01

    Poly (ADP-ribose) polymerase-1 (PARP-1) is a key enzyme involved in the repair of radiation-induced single-strand DNA breaks. PARP inhibitors such as olaparib (KU-0059436, AZD-2281) enhance tumor sensitivity to radiation and to topoisomerase I inhibitors like camptothecin (CPT). Olaparib is an orally bioavailable inhibitor of PARP-1 and PARP-2 that has been tested in multiple clinical trials. The purpose of this study was to investigate the characteristics of the sensitizing effect of olaparib for radiation and CPT in order to support clinical application of this agent. DLD-1 cells (a human colorectal cancer cell line) and H1299 cells (a non-small cell lung cancer cell line) with differences of p53 gene status were used. The survival of these cells was determined by clonogenic assay after treatment with drugs and X-ray irradiation. The γH2AX focus formation assay was performed to examine the influence of olaparib on induction and repair of double-stranded DNA breaks after exposure to radiation or CPT. A radiosensitizing effect of olaparib was seen even at 0.01 μM. Its radiosensitizing effect after exposure for 2 h was similar to that after 24 h. H1299 cells with depletion or mutation of p53 were more radioresistant than H1299 cells with wild-type p53. However, similar enhancement of radiosensitization by olaparib was observed with all of the tested cell lines regardless of the p53 status. Olaparib also sensitized cells to CPT. This sensitizing effect was seen at low concentrations of olaparib such as 0.01 μM, and its sensitizing effect was the same at both 0.01 μM and 1 μM. The combination of olaparib and CPT had a stronger radiosensitizing effect. The results of the γH2AX focus assay corresponded with the clonogenic assay findings. Olaparib enhanced sensitivity to radiation and CPT at low concentrations and after relatively short exposure times such as 2 h. The radiosensitizing effect of olaprib was not dependent on the p53 status of tumor cells. These

  9. Combination of gefitinib and DNA methylation inhibitor decitabine exerts synergistic anti-cancer activity in colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Yun-feng Lou

    Full Text Available Despite recent advances in the treatment of human colon cancer, the chemotherapy efficacy against colon cancer is still unsatisfactory. In the present study, effects of concomitant inhibition of the epidermal growth factor receptor (EGFR and DNA methyltransferase were examined in human colon cancer cells. We demonstrated that decitabine (a DNA methyltransferase inhibitor synergized with gefitinib (an EGFR inhibitor to reduce cell viability and colony formation in SW1116 and LOVO cells. However, the combination of the two compounds displayed minimal toxicity to NCM460 cells, a normal human colon mucosal epithelial cell line. The combination was also more effective at inhibiting the AKT/mTOR/S6 kinase pathway. In addition, the combination of decitabine with gefitinib markedly inhibited colon cancer cell migration. Furthermore, gefitinib synergistically enhanced decitabine-induced cytotoxicity was primarily due to apoptosis as shown by Annexin V labeling that was attenuated by z-VAD-fmk, a pan caspase inhibitor. Concomitantly, cell apoptosis resulting from the co-treatment of gefitinib and decitabine was accompanied by induction of BAX, cleaved caspase 3 and cleaved PARP, along with reduction of Bcl-2 compared to treatment with either drug alone. Interestingly, combined treatment with these two drugs increased the expression of XIAP-associated factor 1 (XAF1 which play an important role in cell apoptosis. Moreover, small interfering RNA (siRNA depletion of XAF1 significantly attenuated colon cancer cells apoptosis induced by the combination of the two drugs. Our findings suggested that gefitinib in combination with decitabine exerted enhanced cell apoptosis in colon cancer cells were involved in mitochondrial-mediated pathway and induction of XAF1 expression. In conclusion, based on the observations from our study, we suggested that the combined administration of these two drugs might be considered as a novel therapeutic regimen for treating colon

  10. Role of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in psychological stress and depression.

    Science.gov (United States)

    Tsai, Shih-Jen

    2017-12-22

    Major depressive disorder is a common illness worldwide, but the pathogenesis of the disorder remains incompletely understood. The tissue-type plasminogen activator-plasminogen proteolytic cascade is highly expressed in the brain regions involved in mood regulation and neuroplasticity. Accumulating evidence from animal and human studies suggests that tissue-type plasminogen activator and its chief inhibitor, plasminogen activator inhibitor-1, are related to stress reaction and depression. Furthermore, the neurotrophic hypothesis of depression postulates that compromised neurotrophin brain-derived neurotrophic factor (BDNF) function is directly involved in the pathophysiology of depression. In the brain, the proteolytic cleavage of proBDNF, a BDNF precursor, to mature BDNF through plasmin represents one mechanism that can change the direction of BDNF action. We also discuss the implications of tissue-type plasminogen activator and plasminogen activator inhibitor-1 alterations as biomarkers for major depressive disorder. Using drugs that increase tissue-type plasminogen activator or decrease plasminogen activator inhibitor-1 levels may open new avenues to develop conceptually novel therapeutic strategies for depression treatment.

  11. 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

  12. Interactions of plasminogen activator inhibitor-1 with vitronectin involve an extensive binding surface and induce mutual conformational rearrangements

    DEFF Research Database (Denmark)

    Blouse, Grant E; Dupont, Daniel Miotto; Schar, Christine R

    2009-01-01

    In order to explore early events during the association of plasminogen activator inhibitor-1 (PAI-1) with its cofactor vitronectin, we have applied a robust strategy that combines protein engineering, fluorescence spectroscopy, and rapid reaction kinetics. Fluorescence stopped-flow experiments de...

  13. The association of TP53 mutations with the resistance of colorectal carcinoma to the insulin-like growth factor-1 receptor inhibitor picropodophyllin

    International Nuclear Information System (INIS)

    Wang, Quan; Wei, Feng; Lv, Guoyue; Li, Chunsheng; Liu, Tongjun; Hadjipanayis, Costas G; Zhang, Guikai; Hao, Chunhai; Bellail, Anita C

    2013-01-01

    There is growing evidence indicating the insulin-like growth factor 1 receptor (IGF-1R) plays a critical role in the progression of human colorectal carcinomas. IGF-1R is an attractive drug target for the treatment of colon cancer. Picropodophyllin (PPP), of the cyclolignan family, has recently been identified as an IGF-1R inhibitor. The aim of this study is to determine the therapeutic response and mechanism after colorectal carcinoma treatment with PPP. Seven colorectal carcinoma cell lines were treated with PPP. Following treatment, cells were analyzed for growth by a cell viability assay, sub-G1 apoptosis by flow cytometry, caspase cleavage and activation of AKT and extracellular signal-regulated kinase (ERK) by western blot analysis. To examine the in vivo therapeutic efficacy of PPP, mice implanted with human colorectal carcinoma xenografts underwent PPP treatment. PPP treatment blocked the phosphorylation of IGF-1R, AKT and ERK and inhibited the growth of TP53 wild-type but not mutated colorectal carcinoma cell lines. The treatment of PPP also induced apoptosis in TP53 wild-type cells as evident by the presence of sub-G1 cells and the cleavage of caspase-9, caspase-3, DNA fragmentation factor-45 (DFF45), poly (ADP-ribose) polymerase (PARP), and X-linked inhibitor of apoptosis protein (XIAP). The loss of BAD phosphorylation in the PPP-treated TP53 wild type cells further suggested that the treatment induced apoptosis through the BAD-mediated mitochondrial pathway. In contrast, PPP treatment failed to induce the phosphorylation of AKT and ERK and caspase cleavage in TP53 mutated colorectal carcinoma cell lines. Finally, PPP treatment suppressed the growth of xenografts derived from TP53 wild type but not mutated colorectal carcinoma cells. We report the association of TP53 mutations with the resistance of treatment of colorectal carcinoma cells in culture and in a xenograft mouse model with the IGF-1R inhibitor PPP. TP53 mutations often occur in colorectal

  14. Potent nonnucleoside reverse transcriptase inhibitors target HIV-1 Gag-Pol.

    Directory of Open Access Journals (Sweden)

    Anna Figueiredo

    2006-11-01

    Full Text Available Nonnucleoside reverse transcriptase inhibitors (NNRTIs target HIV-1 reverse transcriptase (RT by binding to a pocket in RT that is close to, but distinct, from the DNA polymerase active site and prevent the synthesis of viral cDNA. NNRTIs, in particular, those that are potent inhibitors of RT polymerase activity, can also act as chemical enhancers of the enzyme's inter-subunit interactions. However, the consequences of this chemical enhancement effect on HIV-1 replication are not understood. Here, we show that the potent NNRTIs efavirenz, TMC120, and TMC125, but not nevirapine or delavirdine, inhibit the late stages of HIV-1 replication. These potent NNRTIs enhanced the intracellular processing of Gag and Gag-Pol polyproteins, and this was associated with a decrease in viral particle production from HIV-1-transfected cells. The increased polyprotein processing is consistent with premature activation of the HIV-1 protease by NNRTI-enhanced Gag-Pol multimerization through the embedded RT sequence. These findings support the view that Gag-Pol multimerization is an important step in viral assembly and demonstrate that regulation of Gag-Pol/Gag-Pol interactions is a novel target for small molecule inhibitors of HIV-1 production. Furthermore, these drugs can serve as useful probes to further understand processes involved in HIV-1 particle assembly and maturation.

  15. HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors

    DEFF Research Database (Denmark)

    Vanangamudi, Murugesan; Poongavanam, Vasanthanathan; Namasivayam, Vigneshwaran

    2017-01-01

    BACKGROUND: Design of inhibitors for HIV-1 reverse transcriptase inhibition (HIV-1 RT) is one of the successful chemotherapies for the treatment of HIV infection. Among the inhibitors available for HIV-1 RT, non-nucleoside reverse transcriptase inhibitors (NNRTIs) have shown to be very promising......: The conformation dependent-alignment based (CoMFA and CoMSIA) methods have been proven very successful ligand based strategy in the drug design. Here, CoMFA and CoMSIA studies reported for structurally distinct NNRTIs including thiazolobenzimidazole, dipyridodiazepinone, 1,1,3-trioxo [1,2,4]-thiadiazine...

  16. Identification of BRCA1-deficient ovarian cancers

    DEFF Research Database (Denmark)

    Skytte, Anne-Bine; Waldstrøm, Marianne; Rasmussen, Anders Aamann

    2011-01-01

    of offering genetic counseling and due to beneficial effects of PARP inhibitor treatment in this group. Since DNA sequencing is expensive and time-consuming efforts have been devoted to develop more indirect methods for BRCA screening that can improve the selection of patients for sequence-based BRCA testing....... Design. BRCA1-immunohistochemistry (IHC), fluorescence in-situ hybridization (FISH) and methylation analyses were performed on formalin-fixed, paraffin-embedded ovarian cancer tissue. Sample: 54 ovarian cancers; 15 BRCA1 cancers, 4 BRCA2 cancers, 10 cancers from patients with a family history...

  17. 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.

  18. 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.

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

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    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.

  20. 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.

  1. 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.

  2. Hydrofluoric Acid-Based Derivatization Strategy To Profile PARP-1 ADP-Ribosylation by LC-MS/MS.

    Science.gov (United States)

    Gagné, Jean-Philippe; Langelier, Marie-France; Pascal, John M; Poirier, Guy G

    2018-06-11

    Despite significant advances in the development of mass spectrometry-based methods for the identification of protein ADP-ribosylation, current protocols suffer from several drawbacks that preclude their widespread applicability. Given the intrinsic heterogeneous nature of poly(ADP-ribose), a number of strategies have been developed to generate simple derivatives for effective interrogation of protein databases and site-specific localization of the modified residues. Currently, the generation of spectral signatures indicative of ADP-ribosylation rely on chemical or enzymatic conversion of the modification to a single mass increment. Still, limitations arise from the lability of the poly(ADP-ribose) remnant during tandem mass spectrometry, the varying susceptibilities of different ADP-ribose-protein bonds to chemical hydrolysis, or the context dependence of enzyme-catalyzed reactions. Here, we present a chemical-based derivatization method applicable to the confident identification of site-specific ADP-ribosylation by conventional mass spectrometry on any targeted amino acid residue. Using PARP-1 as a model protein, we report that treatment of ADP-ribosylated peptides with hydrofluoric acid generates a specific +132 Da mass signature that corresponds to the decomposition of mono- and poly(ADP-ribosylated) peptides into ribose adducts as a consequence of the cleavage of the phosphorus-oxygen bonds.

  3. Binding of the Inhibitor Protein IF1 to Bovine F1-ATPase

    Science.gov (United States)

    Bason, John V.; Runswick, Michael J.; Fearnley, Ian M.; Walker, John E.

    2011-01-01

    In the structure of bovine F1-ATPase inhibited with residues 1–60 of the bovine inhibitor protein IF1, the α-helical inhibitor interacts with five of the nine subunits of F1-ATPase. In order to understand the contributions of individual amino acid residues to this complex binding mode, N-terminal deletions and point mutations have been introduced, and the binding properties of each mutant inhibitor protein have been examined. The N-terminal region of IF1 destabilizes the interaction of the inhibitor with F1-ATPase and may assist in removing the inhibitor from its binding site when F1Fo-ATPase is making ATP. Binding energy is provided by hydrophobic interactions between residues in the long α-helix of IF1 and the C-terminal domains of the βDP-subunit and βTP-subunit and a salt bridge between residue E30 in the inhibitor and residue R408 in the C-terminal domain of the βDP-subunit. Several conserved charged amino acids in the long α-helix of IF1 are also required for establishing inhibitory activity, but in the final inhibited state, they are not in contact with F1-ATPase and occupy aqueous cavities in F1-ATPase. They probably participate in the pathway from the initial interaction of the inhibitor and the enzyme to the final inhibited complex observed in the structure, in which two molecules of ATP are hydrolysed and the rotor of the enzyme turns through two 120° steps. These findings contribute to the fundamental understanding of how the inhibitor functions and to the design of new inhibitors for the systematic analysis of the catalytic cycle of the enzyme. PMID:21192948

  4. TOPBP1 regulates RAD51 phosphorylation and chromatin loading and determines PARP inhibitor sensitivity

    DEFF Research Database (Denmark)

    Moudry, Pavel; Watanabe, Kenji; Wolanin, Kamila M.

    2016-01-01

    to chromatin and formation of RAD51 foci, but without affecting the upstream HR steps of DNA end resection and RPA loading. Furthermore, TOPBP1 BRCT domains 7/8 are essential for RAD51 foci formation. Mechanistically, TOPBP1 physically binds PLK1 and promotes PLK1 kinase-mediated phosphorylation of RAD51...

  5. 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

  6. Tofacitinib and analogs as inhibitors of the histone kinase PRK1 (PKN1).

    Science.gov (United States)

    Ostrovskyi, Dmytro; Rumpf, Tobias; Eib, Julia; Lumbroso, Alexandre; Slynko, Inna; Klaeger, Susan; Heinzlmeir, Stephanie; Forster, Michael; Gehringer, Matthias; Pfaffenrot, Ellen; Bauer, Silke Mona; Schmidtkunz, Karin; Wenzler, Sandra; Metzger, Eric; Kuster, Bernhard; Laufer, Stefan; Schüle, Roland; Sippl, Wolfgang; Breit, Bernhard; Jung, Manfred

    2016-09-01

    The histone kinase PRK1 has been identified as a potential target to combat prostate cancer but selective PRK1 inhibitors are lacking. The US FDA -approved JAK1-3 inhibitor tofacitinib also potently inhibits PRK1 in vitro. We show that tofacitinib also inhibits PRK1 in a cellular setting. Using tofacitinib as a starting point for structure-activity relationship studies, we identified a more potent and another more selective PRK1 inhibitor compared with tofacitinib. Furthermore, we found two potential PRK1/JAK3-selectivity hotspots. The identified inhibitors and the selectivity hotspots lay the basis for the development of selective PRK1 inhibitors. The identification of PRK1, but also of other cellular tofacitinib targets, has implications on its clinical use and on future development of tofacitinib-like JAK inhibitors. [Formula: see text].

  7. Flavonoids Are Inhibitors of Human Organic Anion Transporter 1 (OAT1)–Mediated Transport

    Science.gov (United States)

    An, Guohua; Wang, Xiaodong

    2014-01-01

    Organic anion transporter 1 (OAT1) has been reported to be involved in the nephrotoxicity of many anionic xenobiotics. As current clinically used OAT1 inhibitors are often associated with safety issues, identifying potent OAT1 inhibitors with little toxicity is of great value in reducing OAT1-mediated drug nephrotoxicity. Flavonoids are a class of polyphenolic compounds with exceptional safety records. Our objective was to evaluate the effects of 18 naturally occurring flavonoids, and some of their glycosides, on the uptake of para-aminohippuric acid (PAH) in both OAT1-expressing and OAT1-negative LLC-PK1 cells. Most flavonoid aglycones produced substantial decreases in PAH uptake in OAT1-expressing cells. Among the flavonoids screened, fisetin, luteolin, morin, and quercetin exhibited the strongest effect and produced complete inhibition of OAT1-mediated PAH uptake at a concentration of 50 μM. Further concentration-dependent studies revealed that both morin and luteolin are potent OAT1 inhibitors, with IC50 values of flavonoid aglycones, all flavonoid glycosides had negligible or small effects on OAT1. In addition, the role of OAT1 in the uptake of fisetin, luteolin, morin, and quercetin was investigated and fisetin was found to be a substrate of OAT1. Taken together, our results indicate that flavonoids are a novel class of OAT1 modulators. Considering the high consumption of flavonoids in the diet and in herbal products, OAT1-mediated flavonoid-drug interactions may be clinically relevant. Further investigation is warranted to evaluate the nephroprotective role of flavonoids in relation to drug-induced nephrotoxicity mediated by the OAT1 pathway. PMID:25002746

  8. Poly (ADP-ribose polymerase 1 is required for protein localization to Cajal body.

    Directory of Open Access Journals (Sweden)

    Elena Kotova

    2009-02-01

    Full Text Available Recently, the nuclear protein known as Poly (ADP-ribose Polymerase1 (PARP1 was shown to play a key role in regulating transcription of a number of genes and controlling the nuclear sub-organelle nucleolus. PARP1 enzyme is known to catalyze the transfer of ADP-ribose to a variety of nuclear proteins. At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear. Therefore, we investigated the roles of PARP1 auto ADP-ribosylation in dynamic nuclear processes during development. Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB by protein non-covalent interaction with pADPr in vivo. We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose, PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

  9. The combination of olaparib and camptothecin for effective radiosensitization

    Directory of Open Access Journals (Sweden)

    Miura Katsutoshi

    2012-04-01

    Full Text Available Abstract Background Poly (ADP-ribose polymerase-1 (PARP-1 is a key enzyme involved in the repair of radiation-induced single-strand DNA breaks. PARP inhibitors such as olaparib (KU-0059436, AZD-2281 enhance tumor sensitivity to radiation and to topoisomerase I inhibitors like camptothecin (CPT. Olaparib is an orally bioavailable inhibitor of PARP-1 and PARP-2 that has been tested in multiple clinical trials. The purpose of this study was to investigate the characteristics of the sensitizing effect of olaparib for radiation and CPT in order to support clinical application of this agent. Methods DLD-1 cells (a human colorectal cancer cell line and H1299 cells (a non-small cell lung cancer cell line with differences of p53 gene status were used. The survival of these cells was determined by clonogenic assay after treatment with drugs and X-ray irradiation. The γH2AX focus formation assay was performed to examine the influence of olaparib on induction and repair of double-stranded DNA breaks after exposure to radiation or CPT. Results A radiosensitizing effect of olaparib was seen even at 0.01 μM. Its radiosensitizing effect after exposure for 2 h was similar to that after 24 h. H1299 cells with depletion or mutation of p53 were more radioresistant than H1299 cells with wild-type p53. However, similar enhancement of radiosensitization by olaparib was observed with all of the tested cell lines regardless of the p53 status. Olaparib also sensitized cells to CPT. This sensitizing effect was seen at low concentrations of olaparib such as 0.01 μM, and its sensitizing effect was the same at both 0.01 μM and 1 μM. The combination of olaparib and CPT had a stronger radiosensitizing effect. The results of the γH2AX focus assay corresponded with the clonogenic assay findings. Conclusion Olaparib enhanced sensitivity to radiation and CPT at low concentrations and after relatively short exposure times such as 2 h. The radiosensitizing effect of olaprib

  10. Poly(ADP-ribose) polymerase-1 inhibits ATM kinase activity in DNA damage response

    International Nuclear Information System (INIS)

    Watanabe, Fumiaki; Fukazawa, Hidesuke; Masutani, Mitsuko; Suzuki, Hiroshi; Teraoka, Hirobumi; Mizutani, Shuki; Uehara, Yoshimasa

    2004-01-01

    DNA double-strand breaks (DSB) mobilize DNA-repair machinery and cell cycle checkpoint by activating the ataxia-telangiectasia (A-T) mutated (ATM). Here we show that ATM kinase activity is inhibited by poly(ADP-ribose) polymerase-1 (PARP-1) in vitro. It was shown by biochemical fractionation procedure that PARP-1 as well as ATM increases at chromatin level after induction of DSB with neocarzinostatin (NCS). Phosphorylation of histone H2AX on serine 139 and p53 on serine 15 in Parp-1 knockout (Parp-1 -/- ) mouse embryonic fibroblasts (MEF) was significantly induced by NCS treatment compared with MEF derived from wild-type (Parp-1 +/+ ) mouse. NCS-induced phosphorylation of histone H2AX on serine 139 in Parp-1 -/- embryonic stem cell (ES) clones was also higher than that in Parp-1 +/+ ES clone. Furthermore, in vitro, PARP-1 inhibited phosphorylation of p53 on serine 15 and 32 P-incorporation into p53 by ATM in a DNA-dependent manner. These results suggest that PARP-1 negatively regulates ATM kinase activity in response to DSB

  11. 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)

  12. The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays

    NARCIS (Netherlands)

    M. Pieters (Marlien); S.A. Barnard (Sunelle A.); D.T. Loots (Du Toit); D.C. Rijken (Dingeman)

    2017-01-01

    textabstractDue to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen

  13. Tissue inhibitor of matrix metalloproteinase-1 suppresses apoptosis of mouse bone marrow stromal cell line MBA-1.

    Science.gov (United States)

    Guo, L-J; Luo, X-H; Xie, H; Zhou, H-D; Yuan, L-Q; Wang, M; Liao, E-Y

    2006-05-01

    We investigated the action of tissue inhibitor of metalloproteinase-1 (TIMP-1) on apoptosis and differentiation of mouse bone marrow stromal cell line MBA-1. TIMP-1 did not affect alkaline phosphatase (ALP) activity, suggesting that it is not involved in osteoblastic differentiation in MBA-1 cells. However, TIMP-1 inhibited MBA-1 apoptosis induced by serum deprivation in a dose-dependent manner. Our study also showed increased Bcl-2 protein expression and decreased Bax protein expression with TIMP-1 treatment. TIMP-1 decreased cytochrome c release and caspase-3 activation in MBA-1 cells. TIMP-1 activated phosphatidylinositol 3-kinase (PI3-kinase) and c-Jun N-terminal kinase (JNK), and the PI3-kinase inhibitor LY294002 or the JNK inhibitor SP600125 abolished its antiapoptotic activity. To investigate whether antiapoptotic action of TIMP-1 was mediated through its inhibition on MMP activities, we constructed mutant TIMP-1 by side-directed mutagenesis, which abolished the inhibitory activity of MMPs by deletion of Cys1 to Ala4. Wild-type TIMP-1 and mutant TIMP-1 expression plasmids were transfected in MBA-1 cells, and results showed that mutant TIMP-1 still protected the induced MBA-1 cell against apoptosis. These data suggest that TIMP-1 antiapoptotic actions are mediated via the PI3-kinase and JNK signaling pathways and independent of TIMP-1 inhibition of MMP activities.

  14. Role of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in psychological stress and depression

    OpenAIRE

    Tsai, Shih-Jen

    2017-01-01

    Major depressive disorder is a common illness worldwide, but the pathogenesis of the disorder remains incompletely understood. The tissue-type plasminogen activator-plasminogen proteolytic cascade is highly expressed in the brain regions involved in mood regulation and neuroplasticity. Accumulating evidence from animal and human studies suggests that tissue-type plasminogen activator and its chief inhibitor, plasminogen activator inhibitor-1, are related to stress reaction and depression. Fur...

  15. Poly(ADP-ribose) polymerase-1 and its cleavage products differentially modulate cellular protection through NF-kB-dependent signaling

    Science.gov (United States)

    Castri, Paola; Lee, Yang-ja; Ponzio, Todd; Maric, Dragan; Spatz, Maria; Bembry, Joliet; Hallenbeck, John

    2014-01-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) and its cleavage products regulate cell viability and NF-kB activity when expressed in neurons. PARP-1 cleavage generates a 24kDa (PARP-124) and an 89kDa fragment (PARP-189). Compared to WT (PARP-1WT), the expression of an uncleavable PARP-1 (PARP-1UNCL) or of PARP-124 conferred protection from oxygen/glucose deprivation (OGD) or OGD/restoration of oxygen and glucose (ROG) damage in vitro, whereas expression of PARP-189 was cytotoxic. Viability experiments were performed in SH-SY5Y, a human neuroblastoma cell line, as well as in rat primary cortical neurons. Following OGD, the higher viability in the presence of PARP-1UNCL or PARP-124 was not accompanied with decreased formation of poly(ADP-riboses) or higher NAD levels. PARP-1 is a known cofactor for NF-kB, hence we investigated whether PARP-1 cleavage influences the inflammatory response. All PARP-1 constructs mimicked PARP-1WT in regards to induction of NF-kB translocation into the nucleus and its increased activation during ischemic challenge. However, expression of PARP-189 construct induced significantly higher NF-kB activity than PARP-1WT; and the same was true for NF-kB-dependent iNOS promoter binding activity. At a protein level, PARP-1UNCL and PARP-124 decreased iNOS (and lower levels of iNOS transcript) and COX-2, and increased Bcl-xL. The increased levels of NF-kB and iNOS transcriptional activities, seen with cytotoxic PARP-189, were accompanied by higher protein expression of COX-2 and iNOS (and higher levels of iNOS transcript) and lower protein expression of Bcl-xL. Taken together, these findings suggest that PARP-1 cleavage products may regulate cellular viability and inflammatory responses in opposing ways during in vitro models of “ischemia”. PMID:24333653

  16. XRCC1 coordinates disparate responses and multiprotein repair complexes depending on the nature and context of the DNA damage

    DEFF Research Database (Denmark)

    Hanssen-Bauer, Audun; Solvang-Garten, Karin; Sundheim, Ottar

    2011-01-01

    . We demonstrate that the laser dose used for introducing DNA damage determines the repertoire of DNA repair proteins recruited. Furthermore, we demonstrate that recruitment of POLß and PNK to regions irradiated with low laser dose requires XRCC1 and that inhibition of PARylation by PARP......-inhibitors only slightly reduces the recruitment of XRCC1, PNK, or POLß to sites of DNA damage. Recruitment of PCNA and FEN-1 requires higher doses of irradiation and is enhanced by XRCC1, as well as by accumulation of PARP-1 at the site of DNA damage. These data improve our understanding of recruitment of BER......XRCC1 is a scaffold protein capable of interacting with several DNA repair proteins. Here we provide evidence for the presence of XRCC1 in different complexes of sizes from 200 to 1500 kDa, and we show that immunoprecipitates using XRCC1 as bait are capable of complete repair of AP sites via both...

  17. 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.

  18. PD-1/PD-L1 Inhibitors for Immuno-oncology: From Antibodies to Small Molecules.

    Science.gov (United States)

    Geng, Qiaohong; Jiao, Peifu; Jin, Peng; Su, Gaoxing; Dong, Jinlong; Yan, Bing

    2018-02-12

    The recent regulatory approvals of immune checkpoint protein inhibitors, such as ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, and avelumab ushered a new era in cancer therapy. These inhibitors do not attack tumor cells directly but instead mobilize the immune system to re-recognize and eradicate tumors, which endows them with unique advantages including durable clinical responses and substantial clinical benefits. PD-1/PD-L1 inhibitors, a pillar of immune checkpoint protein inhibitors, have demonstrated unprecedented clinical efficacy in more than 20 cancer types. Besides monoclonal antibodies, diverse PD- 1/PD-L1 inhibiting candidates, such as peptides, small molecules have formed a powerful collection of weapons to fight cancer. The goal of this review is to summarize and discuss the current PD-1/PD-L1 inhibitors including candidates under clinical development, their molecular interactions with PD-1 or PD-L1, the disclosed structureactivity relationships of peptides and small molecules as inhibitors. Current PD-1/PD-L1 inhibitors under clinical development are exclusively dominated by antibodies. The molecular interactions of therapeutic antibodies with PD-1 or PD-L1 have been gradually elucidated for the design of novel inhibitors. Various peptides and traditional small molecules have been investigated in preclinical model to discover novel PD-1/PD-L1 inhibitors. Peptides and small molecules may play an important role in immuno-oncology because they may bind to multiple immune checkpoint proteins via rational design, opening opportunity for a new generation of novel PD-1/PD-L1 inhibitors. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. 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.

  20. 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.

  1. Thermodynamic and kinetic characterization of hydroxyethylamine β-secretase-1 inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Mondal, Kalyani; Regnstrom, Karin; Morishige, Winse; Barbour, Robin; Probst, Gary; Xu, Ying-Zi; Artis, Dean R.; Yao, Nanhua; Beroza, Paul; Bova, Michael P., E-mail: mpbova2001@yahoo.com

    2013-11-15

    Highlights: •Kinetic and thermodynamic characterization of 10 hydroxyethylamine BACE-1 inhibitors. •Equilibrium binding of inhibitors was enthalpy driven for BACE-1. •Negative entropy of binding was observed towards BACE-1, but not Cathepsin-D. •Structural analysis demonstrates ligand binding induces a major conformational change. •Structural analysis and SPR analysis corroborate induced fit and negative entropy of binding. -- Abstract: Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting millions of people. β-Secretase-1 (BACE-1), an enzyme involved in the processing of the amyloid precursor protein (APP) to form Aβ, is a well validated target for AD. Herein, the authors characterize 10 randomly selected hydroxyethylamine (HEA) BACE-1 inhibitors in terms of their association and dissociation rate constants and thermodynamics of binding using surface plasmon resonance (SPR). Rate constants of association (k{sub a}) measured at 25 °C ranged from a low of 2.42 × 10{sup 4} M{sup −1} s{sup −1} to the highest value of 8.3 × 10{sup 5} M{sup −1} s{sup −1}. Rate constants of dissociation (k{sub d}) ranged from 1.09 × 10{sup −4} s{sup −1} (corresponding to a residence time of close to three hours), to the fastest of 0.028 s{sup −1}. Three compounds were selected for further thermodynamic analysis where it was shown that equilibrium binding was enthalpy driven while unfavorable entropy of binding was observed. Structural analysis revealed that upon ligand binding, the BACE-1flap folds down over the bound ligand causing an induced fit. The maximal difference between alpha carbon positions in the open and closed conformations of the flap was over 5 Å. Thus the negative entropy of binding determined using SPR analysis was consistent with an induced fit observed by structural analysis.

  2. Parthanatos, a messenger of death

    OpenAIRE

    David, Karen Kate; Andrabi, Shaida Ahmad; Dawson, Ted Murray; Dawson, Valina Lynn

    2009-01-01

    Poly-ADP-ribose polymerase-1 (PARP-1)'s multiple roles in the cell span from maintaining life to inducing death. The processes PARP-1 is involved in include, but are not limited to DNA repair, DNA transcription, mitosis, and cell death. Of PARP-1's different cellular functions, its active role in cell death is of particular interest to designing therapies for diseases. Genetic deletion of PARP-1 revealed that PARP-1 over activation underlies cell death in experimental models of stroke, diabet...

  3. CSF-1R Inhibitor Development: Current Clinical Status.

    Science.gov (United States)

    Peyraud, Florent; Cousin, Sophie; Italiano, Antoine

    2017-09-05

    Colony-stimulating factor 1 receptor (CSF-1R) and its ligands, CSF-1 and interleukin 34 (IL-34), regulate the function and survival of tumor-associated macrophages, which are involved in tumorigenesis and in the suppression of antitumor immunity. Moreover, the CSF-1R/CSF-1 axis has been implicated in the pathogenesis of pigmented villonodular synovitis (PVNS), a benign tumor of the synovium. As advanced or metastatic malignant solid tumors and relapsed/refractory PVNS remain unresolved therapeutic problems, new approaches are needed to improve the outcome of patients with these conditions. In solid tumors, targeting CSF-1R via either small molecules or antibodies has shown interesting results in vitro but limited antitumor activity in vivo. Concerning PVNS, clinical trials assessing CSF-1R inhibitors have revealed promising initial outcomes. Blocking CSF-1/CSF-1R signaling represents a promising immunotherapy approach and several new potential combination therapies for future clinical testing.

  4. Mechanism for the differentiation of EoL-1 cells into eosinophils by histone deacetylase inhibitors.

    Science.gov (United States)

    Kaneko, Motoko; Ishihara, Kenji; Takahashi, Aki; Hong, Jangja; Hirasawa, Noriyasu; Zee, Okpyo; Ohuchi, Kazuo

    2007-01-01

    EoL-1 cells have a FIP1L1-PDGFRA fusion gene which causes the transformation of eosinophilic precursor cells into leukemia cells. Recently, we suggested that the induction of differentiation of EoL-1 cells into eosinophils by the HDAC inhibitors apicidin and n-butyrate is due to the continuous inhibition of HDACs. However, neither apicidin nor n-butyrate inhibited the expression of FIP1L1-PDGFRA mRNA, although both these inhibitors suppressed cell proliferation. Therefore, in this study, we analyzed whether the levels of FIP1L1-PDGFRalpha protein and phosphorylated-Stat5 involved in the signaling for the proliferation of EoL-1 cells are attenuated by HDAC inhibitors. EoL-1 cells were incubated in the presence of apicidin, TSA or n-butyrate. FIP1L1-PDGFRalpha and phosphorylated-Stat5 were detected by Western blotting. Treatment of EoL-1 cells with apicidin at 100 nM or n-butyrate at 500 microM decreased the levels of FIP1L1-PDGFRalpha protein and phosphorylated-Stat5, while that with trichostatin A at 30 nM did not. The decrease in the level of FIP1L1-PDGFRalpha protein caused by apicidin and n-butyrate might be one of the mechanisms by which EoL-1 cells are induced to differentiate into eosinophils by these HDAC inhibitors.

  5. Identification of a D-amino acid decapeptide HIV-1 entry inhibitor

    International Nuclear Information System (INIS)

    Boggiano, Cesar; Jiang Shibo; Lu Hong; Zhao Qian; Liu Shuwen; Binley, James; Blondelle, Sylvie E.

    2006-01-01

    Entry of human immunodeficiency virus type 1 (HIV-1) virion into host cells involves three major steps, each being a potential target for the development of entry inhibitors: gp120 binding to CD4, gp120-CD4 complex interacting with a coreceptor, and gp41 refolding to form a six-helix bundle. Using a D-amino acid decapeptide combinatorial library, we identified peptide DC13 as having potent HIV-1 fusion inhibitory activity, and effectively inhibiting infection by several laboratory-adapted and primary HIV-1 strains. While DC13 did not block binding of gp120 to CD4, nor disrupt the gp41 six-helix bundle formation, it effectively blocked the binding of an anti-CXCR4 monoclonal antibody and chemokine SDF-1α to CXCR4-expressing cells. However, because R5-using primary viruses were also neutralized, the antiviral activity of DC13 implies additional mode(s) of action. These results suggest that DC13 is a useful HIV-1 coreceptor antagonist for CXCR4 and, due to its biostability and simplicity, may be of value for developing a new class of HIV-1 entry inhibitors

  6. Targeting Mcl-1 for Radiosensitization of Pancreatic Cancers

    Directory of Open Access Journals (Sweden)

    Dongping Wei

    2015-02-01

    Full Text Available In order to identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer, we previously conducted an RNAi library screen of 8,800 genes. We identified Mcl-1 (myeloid cell leukemia-1, an anti-apoptotic member of the Bcl-2 family, as a target for sensitizing pancreatic cancer cells to chemoradiation. In the present study we investigated Mcl-1 inhibition by either genetic or pharmacological approaches as a radiosensitizing strategy in pancreatic cancer cells. Mcl-1 depletion by siRNA produced significant radiosensitization in BxPC-3 and Panc-1 cells in association with Caspase-3 activation and PARP cleavage, but only minimal radiosensitization in MiaPaCa-2 cells. We next tested the ability of the recently identified, selective, small molecule inhibitor of Mcl-1, UMI77, to radiosensitize in pancreatic cancer cells. UMI77 caused dissociation of Mcl-1 from the pro-apoptotic protein Bak and produced significant radiosensitization in BxPC-3 and Panc-1 cells, but minimal radiosensitization in MiaPaCa-2 cells. Radiosensitization by UMI77 was associated with Caspase-3 activation and PARP cleavage. Importantly, UMI77 did not radiosensitize normal small intestinal cells. In contrast, ABT-737, an established inhibitor of Bcl-2, Bcl-XL, and Bcl-w, failed to radiosensitize pancreatic cancer cells suggesting the unique importance of Mcl-1 relative to other Bcl-2 family members to radiation survival in pancreatic cancer cells. Taken together, these results validate Mcl-1 as a target for radiosensitization of pancreatic cancer cells and demonstrate the ability of small molecules which bind the canonical BH3 groove of Mcl-1, causing displacement of Mcl-1 from Bak, to selectively radiosensitize pancreatic cancer cells.

  7. Tissue inhibitor of matrix metalloproteinase-1 expression in colorectal cancer liver metastases is associated with vascular structures

    DEFF Research Database (Denmark)

    Illemann, Martin; Eefsen, Rikke Helene Løvendahl; Bird, Nigel Charles

    2016-01-01

    several proteases, involved in the degradation of extracellular matrix components, are up-regulated. In liver metastases, their expression is growth pattern dependent. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is a strong prognostic marker in plasma from colorectal cancer patients...

  8. Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors activate the aryl hydrocarbon receptor

    Energy Technology Data Exchange (ETDEWEB)

    Moyer, Benjamin J. [Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Rojas, Itzel Y. [Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Murray, Iain A. [Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA 16802 (United States); Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA 16802 (United States); Lee, Seokwon; Hazlett, Haley F. [Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Perdew, Gary H. [Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA 16802 (United States); Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA 16802 (United States); Tomlinson, Craig R., E-mail: Craig.R.Tomlinson@Dartmouth.edu [Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States)

    2017-05-15

    Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in the immune system by regulating tryptophan levels and T cell differentiation. Several tumor types overexpress IDO1 to avoid immune surveillance making IDO1 of interest as a target for therapeutic intervention. As a result, several IDO1 inhibitors are currently being tested in clinical trials for cancer treatment as well as several other diseases. Many of the IDO1 inhibitors in clinical trials naturally bear structural similarities to the IDO1 substrate tryptophan, as such, they fulfill many of the structural and functional criteria as potential AHR ligands. Using mouse and human cell-based luciferase gene reporter assays, qPCR confirmation experiments, and CYP1A1 enzyme activity assays, we report that some of the promising clinical IDO1 inhibitors also act as agonists for the aryl hydrocarbon receptor (AHR), best known for its roles in xenobiotic metabolism and as another key regulator of the immune response. The dual role as IDO antagonist and AHR agonist for many of these IDO target drugs should be considered for full interrogation of their biological mechanisms and clinical outcomes. - Highlights: • Indoleamine-2,3-dioxygenase 1 (IDO1) inhibitors are in cancer clinical trials. • Some IDO1 inhibitors also potently activate AHR signaling. • The dual role of the IDO1 inhibitors may explain some past paradoxical findings. • AHR induction studies must be included in assessing clinical suitability.

  9. Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors activate the aryl hydrocarbon receptor

    International Nuclear Information System (INIS)

    Moyer, Benjamin J.; Rojas, Itzel Y.; Murray, Iain A.; Lee, Seokwon; Hazlett, Haley F.; Perdew, Gary H.; Tomlinson, Craig R.

    2017-01-01

    Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in the immune system by regulating tryptophan levels and T cell differentiation. Several tumor types overexpress IDO1 to avoid immune surveillance making IDO1 of interest as a target for therapeutic intervention. As a result, several IDO1 inhibitors are currently being tested in clinical trials for cancer treatment as well as several other diseases. Many of the IDO1 inhibitors in clinical trials naturally bear structural similarities to the IDO1 substrate tryptophan, as such, they fulfill many of the structural and functional criteria as potential AHR ligands. Using mouse and human cell-based luciferase gene reporter assays, qPCR confirmation experiments, and CYP1A1 enzyme activity assays, we report that some of the promising clinical IDO1 inhibitors also act as agonists for the aryl hydrocarbon receptor (AHR), best known for its roles in xenobiotic metabolism and as another key regulator of the immune response. The dual role as IDO antagonist and AHR agonist for many of these IDO target drugs should be considered for full interrogation of their biological mechanisms and clinical outcomes. - Highlights: • Indoleamine-2,3-dioxygenase 1 (IDO1) inhibitors are in cancer clinical trials. • Some IDO1 inhibitors also potently activate AHR signaling. • The dual role of the IDO1 inhibitors may explain some past paradoxical findings. • AHR induction studies must be included in assessing clinical suitability.

  10. 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.

  11. Ribonuclease inhibitor 1 regulates erythropoiesis by controlling GATA1 translation.

    Science.gov (United States)

    Chennupati, Vijaykumar; Veiga, Diogo Ft; Maslowski, Kendle M; Andina, Nicola; Tardivel, Aubry; Yu, Eric Chi-Wang; Stilinovic, Martina; Simillion, Cedric; Duchosal, Michel A; Quadroni, Manfredo; Roberts, Irene; Sankaran, Vijay G; MacDonald, H Robson; Fasel, Nicolas; Angelillo-Scherrer, Anne; Schneider, Pascal; Hoang, Trang; Allam, Ramanjaneyulu

    2018-04-02

    Ribosomal proteins (RP) regulate specific gene expression by selectively translating subsets of mRNAs. Indeed, in Diamond-Blackfan anemia and 5q- syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anemia. Little is known about the molecular mechanisms of selective mRNA translation and involvement of ribosomal-associated factors in this process. Ribonuclease inhibitor 1 (RNH1) is a ubiquitously expressed protein that binds to and inhibits pancreatic-type ribonucleases. Here, we report that RNH1 binds to ribosomes and regulates erythropoiesis by controlling translation of the erythroid transcription factor GATA1. Rnh1-deficient mice die between embryonic days E8.5 and E10 due to impaired production of mature erythroid cells from progenitor cells. In Rnh1-deficient embryos, mRNA levels of Gata1 are normal, but GATA1 protein levels are decreased. At the molecular level, we found that RNH1 binds to the 40S subunit of ribosomes and facilitates polysome formation on Gata1 mRNA to confer transcript-specific translation. Further, RNH1 knockdown in human CD34+ progenitor cells decreased erythroid differentiation without affecting myelopoiesis. Our results reveal an unsuspected role for RNH1 in the control of GATA1 mRNA translation and erythropoiesis.

  12. Dynamin inhibitors induce caspase-mediated apoptosis following cytokinesis failure in human cancer cells and this is blocked by Bcl-2 overexpression

    Directory of Open Access Journals (Sweden)

    Braithwaite Antony W

    2011-06-01

    Full Text Available Abstract Background The aim of both classical (e.g. taxol and targeted anti-mitotic agents (e.g. Aurora kinase inhibitors is to disrupt the mitotic spindle. Such compounds are currently used in the clinic and/or are being tested in clinical trials for cancer treatment. We recently reported a new class of targeted anti-mitotic compounds that do not disrupt the mitotic spindle, but exclusively block completion of cytokinesis. This new class includes MiTMAB and OcTMAB (MiTMABs, which are potent inhibitors of the endocytic protein, dynamin. Like other anti-mitotics, MiTMABs are highly cytotoxic and possess anti-proliferative properties, which appear to be selective for cancer cells. The cellular response following cytokinesis failure and the mechanistic pathway involved is unknown. Results We show that MiTMABs induce cell death specifically following cytokinesis failure via the intrinsic apoptotic pathway. This involves cleavage of caspase-8, -9, -3 and PARP, DNA fragmentation and membrane blebbing. Apoptosis was blocked by the pan-caspase inhibitor, ZVAD, and in HeLa cells stably expressing the anti-apoptotic protein, Bcl-2. This resulted in an accumulation of polyploid cells. Caspases were not cleaved in MiTMAB-treated cells that did not enter mitosis. This is consistent with the model that apoptosis induced by MiTMABs occurs exclusively following cytokinesis failure. Cytokinesis failure induced by cytochalasin B also resulted in apoptosis, suggesting that disruption of this process is generally toxic to cells. Conclusion Collectively, these data indicate that MiTMAB-induced apoptosis is dependent on both polyploidization and specific intracellular signalling components. This suggests that dynamin and potentially other cytokinesis factors are novel targets for development of cancer therapeutics.

  13. Differential Role of Poly(ADP-ribose polymerase in D. discoideum growth and development

    Directory of Open Access Journals (Sweden)

    Begum Rasheedunnisa

    2011-03-01

    Full Text Available Abstract Background Poly(ADP-ribose polymerase is evolutionarily conserved as a responder to various forms of stress. Though PARP's role in cell death is well addressed, its role in development and multicellularity is still an enigma. We have previously reported the role of PARP in oxidative stress induced delayed development of D. discoideum. Results In the current study we highlight the involvement of PARP during D. discoideum development. Oxidative stress affects expression of aca and cAR1 thus affecting aggregation. Although parp expression is not affected during oxidative stress but it is involved during normal development as confirmed by our PARP down-regulation studies. Constitutive PARP down-regulation resulted in blocked development while no effect was observed on D. discoideum growth. Interestingly, stage specific PARP down-regulation arrested development at the slug stage. Conclusion These results emphasize that PARP is essential for complex differentiation and its function may be linked to multicellularity. This is the first report where the involvement of PARP during normal multicellular development in D. discoideum, an ancient eukaryote, is established which could be of evolutionary significance. Thus our study adds one more role to the multitasking function of PARP.

  14. 4-Hydroxynonenal activates Src through a non-canonical pathway that involves EGFR/PTP1B

    Science.gov (United States)

    Zhang, Hongqiao; Forman, Henry Jay

    2015-01-01

    Src, a non-receptor protein tyrosine kinase involved in many biological processes, can be activated through both redox-dependent and independent mechanisms. 4-Hydroxy-2-nonenal (HNE) is a lipid peroxidation product that is increased in pathophysiological conditions associated with Src activation. This study examined how HNE activates human c-Src. In the canonical pathway Src activation is initiated by dephosphorylation of pTyr530 followed by conformational change that causes Src auto-phosphorylation at Tyr419 and its activation. HNE increased Src activation in both dose- and time-dependent manner, while it also increased Src phosphorylation at Tyr530 (pTyr530 Src), suggesting that HNE activated Src via a non-canonical mechanism. Protein tyrosine phosphatase 1B inhibitor (539741), at concentrations that increased basal pTyr530 Src, also increased basal Src activity and significantly reduced HNE-mediated Src activation. The EGFR inhibitor, AG1478, and EGFR silencing, abrogated HNE-mediated EGFR activation and inhibited basal and HNE-induced Src activity. In addition, AG1478 also eliminated the increase of basal Src activation by a PTP1B inhibitor. Taken together these data suggest that HNE can activate Src partly through a non-canonical pathway involving activation of EGFR and inhibition of PTP1B. PMID:26453921

  15. 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.

  16. Ligand-based modeling of Akt3 lead to potent dual Akt1/Akt3 inhibitor.

    Science.gov (United States)

    Al-Sha'er, Mahmoud A; Taha, Mutasem O

    2018-02-13

    Akt1 and Akt3 are important serine/threonine-specific protein kinases involved in G2 phase required by cancer cells to maintain cell cycle and to prevent cell death. Accordingly, inhibitors of these kinases should have potent anti-cancer properties. This prompted us to use pharmacophore/QSAR modeling to identify optimal binding models and physicochemical descriptors that explain bioactivity variation within a set of 74 diverse Akt3 inhibitors. Two successful orthogonal pharmacophores were identified and further validated using receiver operating characteristic (ROC) curve analyses. The pharmacophoric models and associated QSAR equation were applied to screen the national cancer institute (NCI) list of compounds for new Akt3 inhibitors. Six hits showed significant experimental anti-Akt3 IC 50 values, out of which one compound exhibited dual low micromolar anti-Akt1 and anti-Akt3 inhibitory profiles. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. ABCB1, ABCG2, and PTEN determine the response of glioblastoma to temozolomide and ABT-888 therapy

    NARCIS (Netherlands)

    Lin, Fan; de Gooijer, Mark C; Roig, Eloy Moreno; Buil, Levi C M; Christner, Susan M; Beumer, Jan H; Würdinger, Thomas; Beijnen, Jos H|info:eu-repo/dai/nl/071919570; van Tellingen, Olaf

    2014-01-01

    PURPOSE: Little is known about the optimal clinical use of ABT-888 (veliparib) for treatment of glioblastoma. ABT-888 is a PARP inhibitor undergoing extensive clinical evaluation in glioblastoma, because it may synergize with the standard-of-care temozolomide (TMZ). We have elucidated important

  18. Novel orally bioavailable EZH1/2 dual inhibitors with greater antitumor efficacy than an EZH2 selective inhibitor.

    Science.gov (United States)

    Honma, Daisuke; Kanno, Osamu; Watanabe, Jun; Kinoshita, Junzo; Hirasawa, Makoto; Nosaka, Emi; Shiroishi, Machiko; Takizawa, Takeshi; Yasumatsu, Isao; Horiuchi, Takao; Nakao, Akira; Suzuki, Keisuke; Yamasaki, Tomonori; Nakajima, Katsuyoshi; Hayakawa, Miho; Yamazaki, Takanori; Yadav, Ajay Singh; Adachi, Nobuaki

    2017-10-01

    Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 and represses gene expression to regulate cell proliferation and differentiation. Enhancer of zeste homolog 2 (EZH2) or its close homolog EZH1 functions as a catalytic subunit of PRC2, so there are two PRC2 complexes containing either EZH2 or EZH1. Tumorigenic functions of EZH2 and its synthetic lethality with some subunits of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes have been observed. However, little is known about the function of EZH1 in tumorigenesis. Herein, we developed novel, orally bioavailable EZH1/2 dual inhibitors that strongly and selectively inhibited methyltransferase activity of both EZH2 and EZH1. EZH1/2 dual inhibitors suppressed trimethylation of histone H3 lysine 27 in cells more than EZH2 selective inhibitors. They also showed greater antitumor efficacy than EZH2 selective inhibitor in vitro and in vivo against diffuse large B-cell lymphoma cells harboring gain-of-function mutation in EZH2. A hematological cancer panel assay indicated that EZH1/2 dual inhibitor has efficacy against some lymphomas, multiple myeloma, and leukemia with fusion genes such as MLL-AF9, MLL-AF4, and AML1-ETO. A solid cancer panel assay demonstrated that some cancer cell lines are sensitive to EZH1/2 dual inhibitor in vitro and in vivo. No clear correlation was detected between sensitivity to EZH1/2 dual inhibitor and SWI/SNF mutations, with a few exceptions. Severe toxicity was not seen in rats treated with EZH1/2 dual inhibitor for 14 days at drug levels higher than those used in the antitumor study. Our results indicate the possibility of EZH1/2 dual inhibitors for clinical applications. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  19. Evaluation of DNA Repair Function as a Predictor of Response in a Clinical Trial of PARP Inhibitor Monotherapy for Recurrent Ovarian Carcinoma

    Science.gov (United States)

    2015-10-01

    tumors that occur in families with germline BRCA1/2 mutations, including pancreatic cancer, mela - noma, and prostate cancer, have also been reported.20...temozolomide in mela - noma, BC, glioblastoma, and acute leukemia, as well as with signal transduction inhibitors (eg, gefitinib in EGFR-mutant non–small

  20. 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.

  1. Glycation inhibitors extend yeast chronological lifespan by reducing advanced glycation end products and by back regulation of proteins involved in mitochondrial respiration.

    Science.gov (United States)

    Kazi, Rubina S; Banarjee, Reema M; Deshmukh, Arati B; Patil, Gouri V; Jagadeeshaprasad, Mashanipalya G; Kulkarni, Mahesh J

    2017-03-06

    Advanced Glycation End products (AGEs) are implicated in aging process. Thus, reducing AGEs by using glycation inhibitors may help in attenuating the aging process. In this study using Saccharomyces cerevisiae yeast system, we show that Aminoguanidine (AMG), a well-known glycation inhibitor, decreases the AGE modification of proteins in non-calorie restriction (NR) (2% glucose) and extends chronological lifespan (CLS) similar to that of calorie restriction (CR) condition (0.5% glucose). Proteomic analysis revealed that AMG back regulates the expression of differentially expressed proteins especially those involved in mitochondrial respiration in NR condition, suggesting that it switches metabolism from fermentation to respiration, mimicking CR. AMG induced back regulation of differentially expressed proteins could be possibly due to its chemical effect or indirectly by glycation inhibition. To delineate this, Metformin (MET), a structural analog of AMG and a mild glycation inhibitor and Hydralazine (HYD), another potent glycation inhibitor but not structural analog of AMG were used. HYD was more effective than MET in mimicking AMG suggesting that glycation inhibition was responsible for restoration of differentially expressed proteins. Thus glycation inhibitors particularly AMG, HYD and MET extend yeast CLS by reducing AGEs, modulating the expression of proteins involved in mitochondrial respiration and possibly by scavenging glucose. This study reports the role of glycation in aging process. In the non-caloric restriction condition, carbohydrates such as glucose promote protein glycation and reduce CLS. While, the inhibitors of glycation such as AMG, HYD, MET mimic the caloric restriction condition by back regulating deregulated proteins involved in mitochondrial respiration which could facilitate shift of metabolism from fermentation to respiration and extend yeast CLS. These findings suggest that glycation inhibitors can be potential molecules that can be used

  2. DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers.

    Directory of Open Access Journals (Sweden)

    Ana Osorio

    2014-04-01

    Full Text Available Single Nucleotide Polymorphisms (SNPs in genes involved in the DNA Base Excision Repair (BER pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase, and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2. Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2 gene (HR: 1.09, 95% CI (1.03-1.16, p = 2.7 × 10(-3 for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8 × 10(-3. DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied.

  3. Angiotensin-converting enzyme inhibitors delay the occurrence of renal involvement and are associated with a decreased risk of disease activity in patients with systemic lupus erythematosus--results from LUMINA (LIX): a multiethnic US cohort.

    Science.gov (United States)

    Durán-Barragán, S; McGwin, G; Vilá, L M; Reveille, J D; Alarcón, G S

    2008-07-01

    To examine if angiotensin-converting enzyme (ACE) inhibitor use delays the occurrence of renal involvement and decreases the risk of disease activity in SLE patients. SLE patients (Hispanics, African Americans and Caucasians) from the lupus in minorities: nature vs nurture (LUMINA) cohort were studied. Renal involvement was defined as ACR criterion and/or biopsy-proven lupus nephritis. Time-to-renal involvement was examined by univariable and multivariable Cox proportional hazards regression analyses. Disease activity was examined with a case-crossover design and a conditional logistic regression model; in the case intervals, a decrease in the SLAM-R score >or=4 points occurred but not in the control intervals. Eighty of 378 patients (21%) were ACE inhibitor users; 298 (79%) were not. The probability of renal involvement free-survival at 10 yrs was 88.1% for users and 75.4% for non-users (P = 0.0099, log rank test). Users developed persistent proteinuria and/or biopsy-proven lupus nephritis (7.1%) less frequently than non-users (22.9%), P = 0.016. By multivariable Cox proportional hazards regression analyses, ACE inhibitors use [hazard ratio (HR) 0.27; 95% CI 0.09, 0.78] was associated with a longer time-to-renal involvement occurrence whereas African American ethnicity (HR 3.31; 95% CI 1.44, 7.61) was with a shorter time. ACE inhibitor use (54/288 case and 254/1148 control intervals) was also associated with a decreased risk of disease activity (HR 0.56; 95% CI 0.34, 0.94). ACE inhibitor use delays the development of renal involvement and associates with a decreased risk of disease activity in SLE; corroboration of these findings in other lupus cohorts is desirable before practice recommendations are formulated.

  4. JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

    Directory of Open Access Journals (Sweden)

    Michael Van Meter

    2016-09-01

    Full Text Available The accumulation of damage caused by oxidative stress has been linked to aging and to the etiology of numerous age-related diseases. The longevity gene, sirtuin 6 (SIRT6, promotes genome stability by facilitating DNA repair, especially under oxidative stress conditions. Here we uncover the mechanism by which SIRT6 is activated by oxidative stress to promote DNA double-strand break (DSB repair. We show that the stress-activated protein kinase, c-Jun N-terminal kinase (JNK, phosphorylates SIRT6 on serine 10 in response to oxidative stress. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites and is required for efficient recruitment of poly (ADP-ribose polymerase 1 (PARP1 to DNA break sites and for efficient repair of DSBs. Our results demonstrate a post-translational mechanism regulating SIRT6, and they provide the link between oxidative stress signaling and DNA repair pathways that may be critical for hormetic response and longevity assurance.

  5. Neutralizing antibody and anti-retroviral drug sensitivities of HIV-1 isolates resistant to small molecule CCR5 inhibitors

    International Nuclear Information System (INIS)

    Pugach, Pavel; Ketas, Thomas J.; Michael, Elizabeth; Moore, John P.

    2008-01-01

    The small molecule CCR5 inhibitors are a new class of drugs for treating infection by human immunodeficiency virus type 1 (HIV-1). They act by binding to the CCR5 co-receptor and preventing its use during HIV-1-cell fusion. Escape mutants can be raised against CCR5 inhibitors in vitro and will arise when these drugs are used clinically. Here, we have assessed the responses of CCR5 inhibitor-resistant viruses to other anti-retroviral drugs that act by different mechanisms, and their sensitivities to neutralizing antibodies (NAbs). The rationale for the latter study is that the resistance pathway for CCR5 inhibitors involves changes in the HIV-1 envelope glycoproteins (Env), which are also targets for NAbs. The escape mutants CC101.19 and D1/85.16 were selected for resistance to AD101 and vicriviroc (VVC), respectively, from the primary R5 HIV-1 isolate CC1/85. Each escape mutant was cross-resistant to other small molecule CCR5 inhibitors (aplaviroc, maraviroc, VVC, AD101 and CMPD 167), but sensitive to protein ligands of CCR5: the modified chemokine PSC-RANTES and the humanized MAb PRO-140. The resistant viruses also retained wild-type sensitivity to the nucleoside reverse transcriptase inhibitor (RTI) zidovudine, the non-nucleoside RTI nevirapine, the protease inhibitor atazanavir and other attachment and fusion inhibitors that act independently of CCR5 (BMS-806, PRO-542 and enfuvirtide). Of note is that the escape mutants were more sensitive than the parental CC1/85 isolate to a subset of neutralizing monoclonal antibodies and to some sera from HIV-1-infected people, implying that sequence changes in Env that confer resistance to CCR5 inhibitors can increase the accessibility of some NAb epitopes. The need to preserve NAb resistance may therefore be a constraint upon how escape from CCR5 inhibitors occurs in vivo

  6. Poly(ADP-RibosePolymerase-1 in Lung Inflammatory Disorders: A Review

    Directory of Open Access Journals (Sweden)

    Gurupreet S. Sethi

    2017-09-01

    Full Text Available Asthma, acute lung injury (ALI, and chronic obstructive pulmonary disease (COPD are lung inflammatory disorders with a common outcome, that is, difficulty in breathing. Corticosteroids, a class of potent anti-inflammatory drugs, have shown less success in the treatment/management of these disorders, particularly ALI and COPD; thus, alternative therapies are needed. Poly(ADP-ribosepolymerases (PARPs are the post-translational modifying enzymes with a primary role in DNA repair. During the last two decades, several studies have reported the critical role played by PARPs in a good of inflammatory disorders. In the current review, the studies that address the role of PARPs in asthma, ALI, and COPD have been discussed. Among the different members of the family, PARP-1 emerges as a key player in the orchestration of lung inflammation in asthma and ALI. In addition, PARP activation seems to be associated with the progression of COPD. Furthermore, PARP-14 seems to play a crucial role in asthma. STAT-6 and GATA-3 are reported to be central players in PARP-1-mediated eosinophilic inflammation in asthma. Interestingly, oxidative stress–PARP-1–NF-κB axis appears to be tightly linked with inflammatory response in all three-lung diseases despite their distinct pathophysiologies. The present review sheds light on PARP-1-regulated factors, which may be common or differential players in asthma/ALI/COPD and put forward our prospective for future studies.

  7. Interdependence of Inhibitor Recognition in HIV-1 Protease.

    Science.gov (United States)

    Paulsen, Janet L; Leidner, Florian; Ragland, Debra A; Kurt Yilmaz, Nese; Schiffer, Celia A

    2017-05-09

    Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1' and S2' subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1' subsite highly influences other subsites: the extension of the hydrophobic P1' moiety results in 1) reduced van der Waals contacts in the P2' subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor.

  8. Type 1 plaminogen activator inhibitor gene: Functional analysis and glucocorticoid regulation of its promoter

    International Nuclear Information System (INIS)

    Van Zonneveld, A.J.; Curriden, S.A.; Loskutoff, D.J.

    1988-01-01

    Plasminogen activator inhibitor type 1 is an important component of the fibrinolytic system and its biosynthesis is subject to complex regulation. To study this regulation at the level of transcription, the authors have identified and sequenced the promoter of the human plasminogen activator inhibitor type 1 gene. Nuclease protection experiments were performed by using endothelial cell mRNA and the transcription initiation (cap) site was established. Sequence analysis of the 5' flanking region of the gene revealed a perfect TATA box at position -28 to position -23, the conserved distance from the cap site. Comparative functional studies with the firefly luciferase gene as a reporter gene showed that fragments derived from this 5' flanking region exhibited high promoter activity when transfected into bovine aortic endothelial cells and mouse Ltk - fibroblasts but were inactive when introduced into HeLa cells. These studies indicate that the fragments contain the plasminogen activator inhibitor type 1 promoter and that it is expressed in a tissue-specific manner. Although the fragments were also silent in rat FTO2B hepatoma cells, their promoter activity could be induced up to 40-fold with the synthetic glucocorticoid dexamethasone. Promoter deletion mapping experiments and studies involving the fusion of promoter fragments to a heterologous gene indicated that dexamethasone induction is mediated by a glucocorticoid responsive element with enhancer-like properties located within the region between nucleotides -305 and +75 of the plasminogen activator inhibitor type 1 gene

  9. Structural characterization of human heme oxygenase-1 in complex with azole-based inhibitors.

    Science.gov (United States)

    Rahman, Mona N; Vlahakis, Jason Z; Roman, Gheorghe; Vukomanovic, Dragic; Szarek, Walter A; Nakatsu, Kanji; Jia, Zongchao

    2010-03-01

    The development of inhibitors specific for heme oxygenases (HO) aims to provide powerful tools in understanding the HO system. Based on the lead structure (2S, 4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[((4-aminophenyl)thio)methyl]-1,3-dioxolane (azalanstat, QC-1) we have synthesized structural modifications to develop novel and selective HO inhibitors. The structural study of human HO-1 (hHO-1) in complex with a select group of the inhibitors was initiated using X-ray crystallographic techniques. Comparison of the structures of four such compounds each in complex with hHO-1 revealed a common binding mode, despite having different structural fragments. The compounds bind to the distal side of heme through an azole "anchor" which coordinates with the heme iron. An expansion of the distal pocket, mainly due to distal helix flexibility, allows accommodation of the compounds without displacing heme or the critical Asp140 residue. Rather, binding displaces a catalytically critical water molecule and disrupts an ordered hydrogen-bond network involving Asp140. The presence of a triazole "anchor" may provide further stability via a hydrogen bond with the protein. A hydrophobic pocket acts to stabilize the region occupied by the phenyl or adamantanyl moieties of these compounds. Further, a secondary hydrophobic pocket is formed via "induced fit" to accommodate bulky substituents at the 4-position of the dioxolane ring. Copyright 2009 Elsevier Inc. All rights reserved.

  10. [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.

  11. Molecular insights on analogs of HIV PR inhibitors toward HTLV-1 PR through QM/MM interactions and molecular dynamics studies: comparative structure analysis of wild and mutant HTLV-1 PR.

    Science.gov (United States)

    Selvaraj, Chandrabose; Singh, Poonam; Singh, Sanjeev Kumar

    2014-12-01

    Retroviruses HTLV-1 and HIV-1 are the primary causative agents of fatal adult T-cell leukemia and acquired immune deficiency syndrome (AIDS) disease. Both retroviruses are similar in characteristics mechanism, and it encodes for protease that mainly involved in the viral replication process. On the basis of the therapeutic success of HIV-1 PR inhibitors, the protease of HTLV-1 is mainly considered as a potential target for chemotherapy. At the same time, structural similarities in both enzymes that originate HIV PR inhibitors can also be an HTLV-1 PR inhibitor. But the expectations failed because of rejection of HIV PR inhibitors from the HTLV-1 PR binding pocket. In this present study, the reason for the HIV PR inhibitor rejection from the HTLV-1 binding site was identified through sequence analysis and molecular dynamics simulation method. Functional analysis of M37A mutation in HTLV PR clearly shows that the MET37 specificity and screening of potential inhibitors targeting MET37 is performed by using approved 90% similar HIV PR inhibitor compounds. From this approach, we report few compounds with a tendency to accept/donate electron specifically to an important site residue MET37 in HTLV-1 PR binding pocket. Copyright © 2014 John Wiley & Sons, Ltd.

  12. Discovery of novel selenium derivatives as Pin1 inhibitors by high-throughput screening

    International Nuclear Information System (INIS)

    Subedi, Amit; Shimizu, Takeshi; Ryo, Akihide; Sanada, Emiko; Watanabe, Nobumoto; Osada, Hiroyuki

    2016-01-01

    Peptidyl prolyl cis/trans isomerization by Pin1 regulates various oncogenic signals during cancer progression, and its inhibition through multiple approaches has established Pin1 as a therapeutic target. However, lack of simplified screening systems has limited the discovery of potent Pin1 inhibitors. We utilized phosphorylation-dependent binding of Pin1 to its specific substrate to develop a screening system for Pin1 inhibitors. Using this system, we screened a chemical library, and identified a novel selenium derivative as Pin1 inhibitor. Based on structure-activity guided chemical synthesis, we developed more potent Pin1 inhibitors that inhibited cancer cell proliferation. -- Highlights: •Novel screening for Pin1 inhibitors based on Pin1 binding is developed. •A novel selenium compound is discovered as Pin1 inhibitor. •Activity guided chemical synthesis of selenium derivatives resulted potent Pin1 inhibitors.

  13. Allosteric Mutant IDH1 Inhibitors Reveal Mechanisms for IDH1 Mutant and Isoform Selectivity

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Xiaoling; Baird, Daniel; Bowen, Kimberly; Capka, Vladimir; Chen, Jinyun; Chenail, Gregg; Cho, YoungShin; Dooley, Julia; Farsidjani, Ali; Fortin, Pascal; Kohls, Darcy; Kulathila, Raviraj; Lin, Fallon; McKay, Daniel; Rodrigues, Lindsey; Sage, David; Touré, B. Barry; van der Plas, Simon; Wright, Kirk; Xu, Ming; Yin, Hong; Levell, Julian; Pagliarini, Raymond A. (Novartis)

    2017-03-01

    Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 “regulatory segment,” which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

  14. Investigation of naphthofuran moiety as potential dual inhibitor against BACE-1 and GSK-3β: molecular dynamics simulations, binding energy, and network analysis to identify first-in-class dual inhibitors against Alzheimer's disease.

    Science.gov (United States)

    Kumar, Akhil; Srivastava, Gaurava; Srivastava, Swati; Verma, Seema; Negi, Arvind S; Sharma, Ashok

    2017-08-01

    BACE-1 and GSK-3β are potential therapeutic drug targets for Alzheimer's disease. Recently, both the targets received attention for designing dual inhibitors for Alzheimer's disease. Until now, only two-scaffold triazinone and curcumin have been reported as BACE-1 and GSK-3β dual inhibitors. Docking, molecular dynamics, clustering, binding energy, and network analysis of triazinone derivatives with BACE-1 and GSK-3β was performed to get molecular insight into the first reported dual inhibitor. Further, we designed and evaluated a naphthofuran series for its ability to inhibit BACE-1 and GSK-3β with the computational approaches. Docking study of naphthofuran series showed a good binding affinity towards both the targets. Molecular dynamics, binding energy, and network analysis were performed to compare their binding with the targets and amino acids responsible for binding. Naphthofuran series derivatives showed good interaction within the active site residues of both of the targets. Hydrogen bond occupancy and binding energy suggested strong binding with the targets. Dual-inhibitor binding was mostly governed by the hydrophobic interactions for both of the targets. Per residue energy decomposition and network analysis identified the key residues involved in the binding and inhibiting BACE-1 and GSK-3β. The results indicated that naphthofuran series derivative 11 may be a promising first-in-class dual inhibitor against BACE-1 and GSK-3β. This naphthofuran series may be further explored to design better dual inhibitors. Graphical abstract Naphthofuran derivative as a dual inhibitor for BACE-1 and GSK-3β.

  15. Isozyme-specific fluorescent inhibitor of glutathione s-transferase omega 1.

    Science.gov (United States)

    Son, Junghyun; Lee, Jae-Jung; Lee, Jun-Seok; Schüller, Andreas; Chang, Young-Tae

    2010-05-21

    Recently, the glutathione S-transferase omega 1 (GSTO1) is suspected to be involved in certain cancers and neurodegenerative diseases. However, profound investigation on the pathological roles of GSTO1 has been hampered by the lack of specific methods to determine or modulate its activity in biological systems containing other isoforms with similar catalytic function. Here, we report a fluorescent compound that is able to inhibit and monitor the activity of GSTO1. We screened 43 fluorescent chemicals and found a compound (6) that binds specifically to the active site of GSTO1. We observed that compound 6 inhibits GSTO1 by covalent modification but spares other isoforms in HEK293 cells and demonstrated that compound 6 could report the activity of GSTO1 in NIH/3T3 or HEK293 cells by measuring the fluorescence intensity of the labeled amount of GSTO1 in SDS-PAGE. Compound 6 is a useful tool to study GSTO1, applicable as a specific inhibitor and an activity reporter.

  16. PTP1B inhibitor promotes endothelial cell motility by activating the DOCK180/Rac1 pathway.

    Science.gov (United States)

    Wang, Yuan; Yan, Feng; Ye, Qing; Wu, Xiao; Jiang, Fan

    2016-04-07

    Promoting endothelial cell (EC) migration is important not only for therapeutic angiogenesis, but also for accelerating re-endothelialization after vessel injury. Several recent studies have shown that inhibition of protein tyrosine phosphatase 1B (PTP1B) may promote EC migration and angiogenesis by enhancing the vascular endothelial growth factor receptor-2 (VEGFR2) signalling. In the present study, we demonstrated that PTP1B inhibitor could promote EC adhesion, spreading and migration, which were abolished by the inhibitor of Rac1 but not RhoA GTPase. PTP1B inhibitor significantly increased phosphorylation of p130Cas, and the interactions among p130Cas, Crk and DOCK180; whereas the phosphorylation levels of focal adhesion kinase, Src, paxillin, or Vav2 were unchanged. Gene silencing of DOCK180, but not Vav2, abrogated the effects of PTP1B inhibitor on EC motility. The effects of PTP1B inhibitor on EC motility and p130Cas/DOCK180 activation persisted in the presence of the VEGFR2 antagonist. In conclusion, we suggest that stimulation of the DOCK180 pathway represents an alternative mechanism of PTP1B inhibitor-stimulated EC motility, which does not require concomitant VEGFR2 activation as a prerequisite. Therefore, PTP1B inhibitor may be a useful therapeutic strategy for promoting EC migration in cardiovascular patients in which the VEGF/VEGFR functions are compromised.

  17. Natural inhibitors of tumor-associated proteases

    International Nuclear Information System (INIS)

    Magdolen, U.; Krol, J.; Sato, S.; Schmitt, M.; Magdolen, V.; Krueger, A.; Mueller, M.M.; Sperl, S.

    2002-01-01

    The turnover and remodelling of extracellular matrix (ECM) is an essential part of many normal biological processes including development, morphogenesis, and wound healing. ECM turnover also occurs in severe pathological situations like artherosclerosis, fibrosis, tumor invasion and metastasis. The major proteases involved in this turnover are serine proteases (especially the urokinase-type plasminogen activator/plasmin system), matrix metalloproteases (a family of about 20 zinc-dependent endopeptidases including collagenases, gelatinases, stromelysins, and membrane-type metalloproteases), and cysteine proteases. In vivo, the activity of these proteases is tightly regulated in the extracellular space by zymogen activation and/or controlled inhibition. In the present review, we give an overview on the structure and biochemical properties of important tumor-associated protease inhibitors such as plasminogen activator inhibitor type 1 and type 2 (PAI-1, PAI-2), tissue inhibitors of metalloproteinases (TIMP-1, -2, -3, and -4), and the cysteine protease inhibitor cystatin C. Interestingly, some of these inhibitors of tumor-associated proteases display multiple functions which rather promote than inhibit tumor progression, when the presence of inhibitors in the tumor tissue is not balanced. (author)

  18. 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 ...

  19. Inhibitors of Histone Deacetylases Are Weak Activators of the FMR1 Gene in Fragile X Syndrome Cell Lines

    Directory of Open Access Journals (Sweden)

    Alexander A. Dolskiy

    2017-01-01

    Full Text Available Fragile X syndrome is the most common cause of inherited intellectual disability in humans. It is a result of CGG repeat expansion in the 5′ untranslated region (5′ UTR of the FMR1 gene. This gene encodes the FMRP protein that is involved in neuronal development. Repeat expansion leads to heterochromatinization of the promoter, gene silencing, and the subsequent absence of FMRP. To date, there is no specific therapy for the syndrome. All treatments in clinic practice provide symptomatic therapy. The development of drug therapy for Fragile X syndrome treatment is connected with the search for inhibitors of enzymes that are responsible for heterochromatinization. Here, we report a weak transcriptional activity of the FMR1 gene and the absence of FMRP protein after Fragile X syndrome cell lines treatment with two FDA approved inhibitors of histone deacetylases, romidepsin and vorinostat. We demonstrate that romidepsin, an inhibitor of class I histone deacetylases, does not activate FMR1 expression in patient cell cultures, whereas vorinostat, an inhibitor of classes I and II histone deacetylases, activates a low level of FMR1 expression in some patient cell lines.

  20. Targeting poly (ADP-ribose polymerase partially contributes to bufalin-induced cell death in multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    He Huang

    Full Text Available Despite recent pharmaceutical advancements in therapeutic drugs, multiple myeloma (MM remains an incurable disease. Recently, ploy(ADP-ribose polymerase 1 (PARP1 has been shown as a potentially promising target for MM therapy. A previous report suggested bufalin, a component of traditional Chinese medicine ("Chan Su", might target PARP1. However, this hypothesis has not been verified. We here showed that bufalin could inhibit PARP1 activity in vitro and reduce DNA-damage-induced poly(ADP-ribosylation in MM cells. Molecular docking analysis revealed that the active site of bufalin interaction is within the catalytic domain of PAPR1. Thus, PARP1 is a putative target of bufalin. Furthermore, we showed, for the first time that the proliferation of MM cell lines (NCI-H929, U266, RPMI8226 and MM.1S and primary CD138(+ MM cells could be inhibited by bufalin, mainly via apoptosis and G2-M phase cell cycle arrest. MM cell apoptosis was confirmed by apoptotic cell morphology, Annexin-V positive cells, and the caspase3 activation. We further evaluated the role of PARP1 in bufalin-induced apoptosis, discovering that PARP1 overexpression partially suppressed bufalin-induced cell death. Moreover, bufalin can act as chemosensitizer to enhance the cell growth-inhibitory effects of topotecan, camptothecin, etoposide and vorinostat in MM cells. Collectively, our data suggest that bufalin is a novel PARP1 inhibitor and a potentially promising therapeutic agent against MM alone or in combination with other drugs.

  1. The design strategy of selective PTP1B inhibitors over TCPTP.

    Science.gov (United States)

    Li, XiangQian; Wang, LiJun; Shi, DaYong

    2016-08-15

    Protein tyrosine phosphatase 1B (PTP1B) has already been well studied as a highly validated therapeutic target for diabetes and obesity. However, the lack of selectivity limited further studies and clinical applications of PTP1B inhibitors, especially over T-cell protein tyrosine phosphatase (TCPTP). In this review, we enumerate the published specific inhibitors of PTP1B, discuss the structure-activity relationships by analysis of their X-ray structures or docking results, and summarize the characteristic of selectivity related residues and groups. Furthermore, the design strategy of selective PTP1B inhibitors over TCPTP is also proposed. We hope our work could provide an effective way to gain specific PTP1B inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Sibiriline, a new small chemical inhibitor of receptor-interacting protein kinase 1, prevents immune-dependent hepatitis.

    Science.gov (United States)

    Le Cann, Fabienne; Delehouzé, Claire; Leverrier-Penna, Sabrina; Filliol, Aveline; Comte, Arnaud; Delalande, Olivier; Desban, Nathalie; Baratte, Blandine; Gallais, Isabelle; Piquet-Pellorce, Claire; Faurez, Florence; Bonnet, Marion; Mettey, Yvette; Goekjian, Peter; Samson, Michel; Vandenabeele, Peter; Bach, Stéphane; Dimanche-Boitrel, Marie-Thérèse

    2017-09-01

    Necroptosis is a regulated form of cell death involved in several disease models including in particular liver diseases. Receptor-interacting protein kinases, RIPK1 and RIPK3, are the main serine/threonine kinases driving this cell death pathway. We screened a noncommercial, kinase-focused chemical library which allowed us to identify Sibiriline as a new inhibitor of necroptosis induced by tumor necrosis factor (TNF) in Fas-associated protein with death domain (FADD)-deficient Jurkat cells. Moreover, Sib inhibits necroptotic cell death induced by various death ligands in human or mouse cells while not protecting from caspase-dependent apoptosis. By using competition binding assay and recombinant kinase assays, we demonstrated that Sib is a rather specific competitive RIPK1 inhibitor. Molecular docking analysis shows that Sib is trapped closed to human RIPK1 adenosine triphosphate-binding site in a relatively hydrophobic pocket locking RIPK1 in an inactive conformation. In agreement with its RIPK1 inhibitory property, Sib inhibits both TNF-induced RIPK1-dependent necroptosis and RIPK1-dependent apoptosis. Finally, Sib protects mice from concanavalin A-induced hepatitis. These results reveal the small-molecule Sib as a new RIPK1 inhibitor potentially of interest for the treatment of immune-dependent hepatitis. © 2017 Federation of European Biochemical Societies.

  3. Classification of Cytochrome P450 1A2 Inhibitors and Non-Inhibitors by Machine Learning Techniques

    DEFF Research Database (Denmark)

    Vasanthanathan, Poongavanam; Taboureau, Olivier; Oostenbrink, Chris

    2009-01-01

    of CYP1A2 inhibitors and non-inhibitors. Training and test sets consisted of about 400 and 7000 compounds, respectively. Various machine learning techniques, like binary QSAR, support vector machine (SVM), random forest, kappa nearest neighbors (kNN), and decision tree methods were used to develop...

  4. 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

  5. Structure-Based Design of Potent and Selective 3-Phosphoinositide-Dependent Kinase-1 (PDK1) Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Medina, Jesus R.; Becker, Christopher J.; Blackledge, Charles W.; Duquenne, Celine; Feng, Yanhong; Grant, Seth W.; Heerding, Dirk; Li, William H.; Miller, William H.; Romeril, Stuart P.; Scherzer, Daryl; Shu, Arthur; Bobko, Mark A.; Chadderton, Antony R.; Dumble, Melissa; Gardiner, Christine M.; Gilbert, Seth; Liu, Qi; Rabindran, Sridhar K.; Sudakin, Valery; Xiang, Hong; Brady, Pat G.; Campobasso, Nino; Ward, Paris; Axten, Jeffrey M. (GSKPA)

    2014-10-02

    Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.

  6. Resistance of a human immunodeficiency virus type 1 isolate to a small molecule CCR5 inhibitor can involve sequence changes in both gp120 and gp41

    International Nuclear Information System (INIS)

    Anastassopoulou, Cleo G.; Ketas, Thomas J.; Depetris, Rafael S.; Thomas, Antonia M.; Klasse, Per Johan; Moore, John P.

    2011-01-01

    Here, we describe the genetic pathways taken by a human immunodeficiency virus type 1 (HIV-1) isolate, D101.12, to become resistant to the small molecule CCR5 inhibitor, vicriviroc (VCV), in vitro. Resistant D101.12 variants contained at least one substitution in the gp120 V3 region (H308P), plus one of two patterns of gp41 sequence changes involving the fusion peptide (FP) and a downstream residue: G514V+V535M or M518V+F519L+V535M. Studies of Env-chimeric and point-substituted viruses in peripheral blood mononuclear cells (PBMC) and TZM-bl cells showed that resistance can arise from the cooperative action of gp120 and gp41 changes, while retaining CCR5 usage. Modeling the VCV inhibition data from the two cell types suggests that D101.12 discriminates between high- and low-VCV affinity forms of CCR5 less than D1/85.16, a resistant virus with three FP substitutions.

  7. FoxM1 is a general target for proteasome inhibitors.

    Directory of Open Access Journals (Sweden)

    Uppoor G Bhat

    2009-08-01

    Full Text Available Proteasome inhibitors are currently in the clinic or in clinical trials, but the mechanism of their anticancer activity is not completely understood. The oncogenic transcription factor FoxM1 is one of the most overexpressed genes in human tumors, while its expression is usually halted in normal non-proliferating cells. Previously, we established that thiazole antibiotics Siomycin A and thiostrepton inhibit FoxM1 and induce apoptosis in human cancer cells. Here, we report that Siomycin A and thiostrepton stabilize the expression of a variety of proteins, such as p21, Mcl-1, p53 and hdm-2 and also act as proteasome inhibitors in vitro. More importantly, we also found that well-known proteasome inhibitors such as MG115, MG132 and bortezomib inhibit FoxM1 transcriptional activity and FoxM1 expression. In addition, overexpression of FoxM1 specifically protects against bortezomib-, but not doxorubicin-induced apoptosis. These data suggest that negative regulation of FoxM1 by proteasome inhibitors is a general feature of these drugs and it may contribute to their anticancer properties.

  8. The effect of C1-esterase inhibitor in definite and suspected streptococcal toxic shock syndrome. Report of seven patients.

    Science.gov (United States)

    Fronhoffs, S; Luyken, J; Steuer, K; Hansis, M; Vetter, H; Walger, P

    2000-10-01

    To evaluate the effect of adjunctive C1-esterase inhibitor substitution therapy on clinical characteristics and outcome of patients with streptococcal toxic shock syndrome (TSS). Observational. Medizinische Poliklinik, University of Bonn, Germany. Seven patients with direct or indirect evidence of streptococcal TSS. In addition to conventional and supportive therapy, all patients received 2-3 single doses of C1-esterase inhibitor totaling 6,000-10,000 U within the first 24 h after admission. All patients developed fulminant septic shock, multiorgan failure and/or capillary leak syndrome and necrotizing fasciitis within 10-72 h following the onset of first symptoms. Between 1 and 4 days following administration of C1-esterase inhibitor, a marked shift of fluid from extravascular to intravascular compartments took place in all but one patient, accompanied by a transient intra-alveolar lung edema and rapidly decreasing need for adrenergic agents. Six of seven patients survived. These clinical observations in a small series of patients and the favorable outcome point towards a positive effect of early and high-dose administration of C1-esterase inhibitor as adjunctive therapy in streptococcal TSS. The possible mechanism involved may be the attenuation of capillary leak syndrome (CLS) via early inactivation of complement and contact systems. Controlled studies are needed to establish an improvement of the survival rates of patients with streptococcal TSS following administration of C1-esterase inhibitor.

  9. Identification of Leishmania donovani Topoisomerase 1 inhibitors via intuitive scaffold hopping and bioisosteric modification of known Top 1 inhibitors

    Science.gov (United States)

    Mamidala, Rajinikanth; Majumdar, Papiya; Jha, Kunal Kumar; Bathula, Chandramohan; Agarwal, Rahul; Chary, M. Thirumala; Mazumdar, H. K.; Munshi, Parthapratim; Sen, Subhabrata

    2016-05-01

    A library of arylidenefuropyridinediones was discovered as potent inhibitors of Leishmania donovani Topoisomerase 1 (LdTop1) where the active molecules displayed considerable inhibition with single digit micromolar EC50 values. This molecular library was designed via intuitive scaffold hopping and bioisosteric modification of known topoisomerase 1 inhibitors such as camptothecin, edotecarin and etc. The design was rationalized by molecular docking analysis of the compound prototype with human topoisomerase 1 (HTop1) and Leishmania donovani topoisomerase 1(LdTop1). The most active compound 4 displayed no cytotoxicity against normal mammalian COS7 cell line (~100 fold less inhibition at the EC50). Similar to camptothecin, 4 interacted with free LdTop1 as observed in the preincubation DNA relaxation inhibition experiment. It also displayed anti-protozoal activity against Leishmania donovani promastigote. Crystal structure investigation of 4 and its molecular modelling with LdTop1 revealed putative binding sites in the enzyme that could be harnessed to generate molecules with better potency.

  10. Involvement of a Lipoxygenase-Like Enzyme in Abscisic Acid Biosynthesis 1

    Science.gov (United States)

    Creelman, Robert A.; Bell, Erin; Mullet, John E.

    1992-01-01

    Several lines of evidence indicate that abscisic acid (ABA) is derived from 9′-cis-neoxanthin or 9′-cis-violaxanthin with xanthoxin as an intermediate. 18O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11′, 12′) double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties. PMID:16668998

  11. 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%.

  12. PD-1 Checkpoint Inhibitor Associated Autoimmune Encephalitis

    Directory of Open Access Journals (Sweden)

    Stephanie Schneider

    2017-05-01

    Full Text Available Objective: To report first-hand narrative experience of autoimmune encephalitis and to briefly review currently available evidence of autoimmune encephalitis in cancer patients treated with immune checkpoint inhibitors. Setting: A case study is presented on the management of a patient who developed autoimmune encephalitis during nivolumab monotherapy occurring after 28 weeks on anti-PD-1 monotherapy (nivolumab 3 mg/kg every 2 weeks for non-small cell lung cancer. Results: No substantial improvement was observed by antiepileptic treatment. After administration of 80 mg methylprednisolone, neurologic symptoms disappeared within 24 h and the patient fully recovered. Conclusions: Immune checkpoint inhibitor treatment can lead to autoimmune encephalitis. Clinical trial data indicate a frequency of autoimmune encephalitis of ≥0.1 to <1% with a higher probability during combined or sequential anti-CTLA-4/anti-PD-1 therapy than during anti-PD-1 or anti-PD-L1 monotherapy. Further collection of evidence and translational research is warranted.

  13. Apoptosis Signal-Regulating Kinase 1 Is Involved in Brain-Derived Neurotrophic Factor (BDNF)-Enhanced Cell Motility and Matrix Metalloproteinase 1 Expression in Human Chondrosarcoma Cells

    Science.gov (United States)

    Lin, Chih-Yang; Chang, Sunny Li-Yun; Fong, Yi-Chin; Hsu, Chin-Jung; Tang, Chih-Hsin

    2013-01-01

    Chondrosarcoma is the primary malignancy of bone that is characterized by a potent capacity to invade locally and cause distant metastasis, and is therefore associated with poor prognoses. Chondrosarcoma further shows a predilection for metastasis to the lungs. The brain-derived neurotrophic factor (BDNF) is a small molecule in the neurotrophin family of growth factors that is associated with the disease status and outcome of cancers. However, the effect of BDNF on cell motility in human chondrosarcoma cells is mostly unknown. Here, we found that human chondrosarcoma cell lines had significantly higher cell motility and BDNF expression compared to normal chondrocytes. We also found that BDNF increased cell motility and expression of matrix metalloproteinase-1 (MMP-1) in human chondrosarcoma cells. BDNF-mediated cell motility and MMP-1 up-regulation were attenuated by Trk inhibitor (K252a), ASK1 inhibitor (thioredoxin), JNK inhibitor (SP600125), and p38 inhibitor (SB203580). Furthermore, BDNF also promoted Sp1 activation. Our results indicate that BDNF enhances the migration and invasion activity of chondrosarcoma cells by increasing MMP-1 expression through a signal transduction pathway that involves the TrkB receptor, ASK1, JNK/p38, and Sp1. BDNF thus represents a promising new target for treating chondrosarcoma metastasis. PMID:23892595

  14. A novel small molecule inhibitor of hepatitis C virus entry.

    Directory of Open Access Journals (Sweden)

    Carl J Baldick

    Full Text Available Small molecule inhibitors of hepatitis C virus (HCV are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc, blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.

  15. 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...

  16. Identification of novel targets for PGC-1α and histone deacetylase inhibitors in neuroblastoma cells

    International Nuclear Information System (INIS)

    Cowell, Rita M.; Talati, Pratik; Blake, Kathryn R.; Meador-Woodruff, James H.; Russell, James W.

    2009-01-01

    Recent evidence suggests that the transcriptional coactivator peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α) is involved in the pathology of Huntington's Disease (HD). While animals lacking PGC-1α express lower levels of genes involved in antioxidant defense and oxidative phosphorylation in the brain, little is known about other targets for PGC-1α in neuronal cells and whether there are ways to pharmacologically target PGC-1α in neurons. Here, PGC-1α overexpression in SH-SY5Y neuroblastoma cells upregulated expression of genes involved in mitochondrial function, glucose transport, fatty acid metabolism, and synaptic function. Overexpression also decreased vulnerability to hydrogen peroxide-induced cell death and caspase 3 activation. Treatment of cells with the histone deacetylase inhibitors (HDACi's) trichostatin A and valproic acid upregulated PGC-1α and glucose transporter 4 (GLUT4). These results suggest that PGC-1α regulates multiple pathways in neurons and that HDACi's may be good candidates to target PGC-1α and GLUT4 in HD and other neurological disorders.

  17. Antimalarial activity of HIV-1 protease inhibitor in chromone series.

    Science.gov (United States)

    Lerdsirisuk, Pradith; Maicheen, Chirattikan; Ungwitayatorn, Jiraporn

    2014-12-01

    Increasing parasite resistance to nearly all available antimalarial drugs becomes a serious problem to human health and necessitates the need to continue the search for new effective drugs. Recent studies have shown that clinically utilized HIV-1 protease (HIV-1 PR) inhibitors can inhibit the in vitro and in vivo growth of Plasmodium falciparum. In this study, a series of chromone derivatives possessing HIV-1 PR inhibitory activity has been tested for antimalarial activity against P. falciparum (K1 multi-drug resistant strain). Chromone 15, the potent HIV-1 PR inhibitor (IC50=0.65μM), was found to be the most potent antimalarial compound with IC50=0.95μM while primaquine and tafenoquine showed IC50=2.41 and 1.95μM, respectively. Molecular docking study of chromone compounds against plasmepsin II, an aspartic protease enzyme important in hemoglobin degradation, revealed that chromone 15 exhibited the higher binding affinity (binding energy=-13.24kcal/mol) than the known PM II inhibitors. Thus, HIV-1 PR inhibitor in chromone series has the potential to be a new class of antimalarial agent. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Structure based design of 11β-HSD1 inhibitors.

    Science.gov (United States)

    Singh, Suresh; Tice, Colin

    2010-11-01

    Controlling elevated tissue-specific levels of cortisol may provide a novel therapeutic approach for treating metabolic syndrome. This concept has spurred large scale medicinal chemistry efforts in the pharmaceutical industry for the design of 11β-HSD1 inhibitors. High resolution X-ray crystal structures of inhibitors in complex with the enzyme have facilitated the structure-based design of diverse classes of molecules. A summary of binding modes, trends in structure-activity relationships, and the pharmacodynamic data of inhibitors from each class is presented.

  19. Inhibitors of nuclease and redox activity of apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1).

    Science.gov (United States)

    Laev, Sergey S; Salakhutdinov, Nariman F; Lavrik, Olga I

    2017-05-01

    Human apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) is a multifunctional protein which is essential in the base excision repair (BER) pathway of DNA lesions caused by oxidation and alkylation. This protein hydrolyzes DNA adjacent to the 5'-end of an apurinic/apyrimidinic (AP) site to produce a nick with a 3'-hydroxyl group and a 5'-deoxyribose phosphate moiety or activates the DNA-binding activity of certain transcription factors through its redox function. Studies have indicated a role for APE1/Ref-1 in the pathogenesis of cancer and in resistance to DNA-interactive drugs. Thus, this protein has potential as a target in cancer treatment. As a result, major efforts have been directed to identify small molecule inhibitors against APE1/Ref-1 activities. These agents have the potential to become anticancer drugs. The aim of this review is to present recent progress in studies of all published small molecule APE1/Ref-1 inhibitors. The structures and activities of APE1/Ref-1 inhibitors, that target both DNA repair and redox activities, are presented and discussed. To date, there is an urgent need for further development of the design and synthesis of APE1/Ref-1 inhibitors due to high importance of this protein target. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Sustained Release Talazoparib Implants for Localized Treatment of BRCA1-deficient Breast Cancer.

    Science.gov (United States)

    Belz, Jodi E; Kumar, Rajiv; Baldwin, Paige; Ojo, Noelle Castilla; Leal, Ana S; Royce, Darlene B; Zhang, Di; van de Ven, Anne L; Liby, Karen T; Sridhar, Srinivas

    2017-01-01

    Talazoparib, a potent PARP inhibitor, has shown promising clinical and pre-clinical activity by inducing synthetic lethality in cancers with germline Brca1/2 mutations. Conventional oral delivery of Talazoparib is associated with significant off-target effects, therefore we sought to develop new delivery systems in the form of an implant loaded with Talazoparib for localized, slow and sustained release of the drug at the tumor site in Brca1 -deficient breast cancer. Poly(lactic-co-glycolic acid) (PLGA) implants (0.8 mm diameter) loaded with subclinical dose (25 or 50 µg) Talazoparib were fabricated and characterized. In vitro studies with Brca1 -deficient W780 and W0069 breast cancer cells were conducted to test sensitivity to PARP inhibition. The in vivo therapeutic efficacy of Talazoparib implants was assessed following a one-time intratumoral injection in Brca1 Co/Co ;MMTV-Cre;p53 +/- mice and compared to drug-free implants and oral gavage. Immunohistochemistry studies were performed on tumor sections using PCNA and γ-H2AX staining. Sustained release of Talazoparib was observed over 28 days in vitro . Mice treated with Talazoparib implants showed statistically significant tumor growth inhibition compared to those receiving drug-free implants or free Talazoparib orally. Talazoparib implants were well-tolerated at both drug doses and resulted in less weight loss than oral gavage. PARP inhibition in mice treated with Talazoparib implants significantly increased double-stranded DNA damage and decreased tumor cell proliferation as shown by PCNA and γ-H2AX staining as compared to controls. These results demonstrate that localized and sustained delivery of Talazoparib via implants has potential to provide superior treatment outcomes at sub-clinical doses with minimal toxicity in patients with BRCA1 deficient tumors.

  1. Members of the bcl-2 and caspase families regulate nuclear degeneration during chick lens fibre differentiation.

    Science.gov (United States)

    Wride, M A; Parker, E; Sanders, E J

    1999-09-01

    The optical clarity of the lens is ensured by the programmed removal of nuclei and other organelles from the lens fibre cells during development. The morphology of the degenerating nuclei is similar to that observed during apoptosis and is accompanied by DNA fragmentation. Proteins encoded by the bcl-2 proto-oncogene family are important in either promoting or inhibiting apoptosis, and caspases are involved in downstream proteolytic events. Here, the expression of bcl-2 family members (bcl-2, bax, bad, and bcl-x(s/l)) and caspases-1, -2, -3, -4, and -6 was investigated through a range of stages of chick lens development using immunocytochemistry, Western blotting, and affinity labelling for caspases using biotinylated caspase inhibitors. Using differentiating lens epithelial cell cultures, it was demonstrated that the addition to cultures of synthetic peptide inhibitors of caspases -1, -2, -4, -6, and -9 brought about a 50-70% reduction in the number of degenerating nuclei per unit area of culture, as assessed by image analysis. These effects were comparable to those seen when general inhibitors of caspases were added to cultures. On the other hand, inhibitors of caspases-3 and -8 were not effective in significantly reducing the number of TUNEL-labelled nuclei. Expression of the caspase substrates poly(ADP-ribose) polymerase (PARP) and the 45-kDa subunit of DNA fragmentation factor (DFF 45) was also observed in the developing lens. Western blots of cultures to which caspase inhibitors were added revealed alterations in the PARP cleavage pattern, but not in that of DFF. These results demonstrate a role for members of the bcl-2 family and caspases in the degeneration of lens fibre cell nuclei during chick secondary lens fibre development and support the proposal that this process has many characteristics in common with apoptosis. Copyright 1999 Academic Press.

  2. Microglial involvement in neuroplastic changes following focal brain ischemia in rats.

    Directory of Open Access Journals (Sweden)

    Alexandre Madinier

    2009-12-01

    Full Text Available The pathogenesis of ischemic stroke is a complex sequence of events including inflammatory reaction, for which the microglia appears to be a major cellular contributor. However, whether post-ischemic activation of microglial cells has beneficial or detrimental effects remains to be elucidated, in particular on long term brain plasticity events. The objective of our study was to determine, through modulation of post-stroke inflammatory response, to what extent microglial cells are involved in some specific events of neuronal plasticity, neurite outgrowth and synaptogenesis. Since microglia is a source of neurotrophic factors, the identification of the brain-derived neurophic factor (BDNF as possible molecular actor involved in these events was also attempted. As a means of down-regulating the microglial response induced by ischemia, 3-aminobenzamide (3-AB, 90 mg/kg, i.p. was used to inhibit the poly(ADP-ribose polymerase-1 (PARP-1. Indeed, PARP-1 contributes to the activation of the transcription factor NF-kB, which is essential to the upregulation of proinflammatory genes, in particular responsible for microglial activation/proliferation. Experiments were conducted in rats subjected to photothrombotic ischemia which leads to a strong and early microglial cells activation/proliferation followed by an infiltration of macrophages within the cortical lesion, events evaluated at serial time points up to 1 month post-ictus by immunostaining for OX-42 and ED-1. Our most striking finding was that the decrease in acute microglial activation induced by 3-AB was associated with a long term down-regulation of two neuronal plasticity proteins expression, synaptophysin (marker of synaptogenesis and GAP-43 (marker of neuritogenesis as well as to a significant decrease in tissue BDNF production. Thus, our data argue in favour of a supportive role for microglia in brain neuroplasticity stimulation possibly through BDNF production, suggesting that a targeted

  3. A novel human AP endonuclease with conserved zinc-finger-like motifs involved in DNA strand break responses

    Science.gov (United States)

    Kanno, Shin-ichiro; Kuzuoka, Hiroyuki; Sasao, Shigeru; Hong, Zehui; Lan, Li; Nakajima, Satoshi; Yasui, Akira

    2007-01-01

    DNA damage causes genome instability and cell death, but many of the cellular responses to DNA damage still remain elusive. We here report a human protein, PALF (PNK and APTX-like FHA protein), with an FHA (forkhead-associated) domain and novel zinc-finger-like CYR (cysteine–tyrosine–arginine) motifs that are involved in responses to DNA damage. We found that the CYR motif is widely distributed among DNA repair proteins of higher eukaryotes, and that PALF, as well as a Drosophila protein with tandem CYR motifs, has endo- and exonuclease activities against abasic site and other types of base damage. PALF accumulates rapidly at single-strand breaks in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner in human cells. Indeed, PALF interacts directly with PARP1 and is required for its activation and for cellular resistance to methyl-methane sulfonate. PALF also interacts directly with KU86, LIGASEIV and phosphorylated XRCC4 proteins and possesses endo/exonuclease activity at protruding DNA ends. Various treatments that produce double-strand breaks induce formation of PALF foci, which fully coincide with γH2AX foci. Thus, PALF and the CYR motif may play important roles in DNA repair of higher eukaryotes. PMID:17396150

  4. ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors.

    Science.gov (United States)

    Halasi, Marianna; Wang, Ming; Chavan, Tanmay S; Gaponenko, Vadim; Hay, Nissim; Gartel, Andrei L

    2013-09-01

    NAC (N-acetyl-L-cysteine) is commonly used to identify and test ROS (reactive oxygen species) inducers, and to inhibit ROS. In the present study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H₂O₂. However, only NAC, and not catalase or another ROS scavenger Trolox, was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compound, piperlongumine, leading to its description as a ROS inducer. In contrast, our own experiments showed that this compound depicts features of proteasome inhibitors including suppression of FOXM1 (Forkhead box protein M1), stabilization of cellular proteins, induction of ROS-independent apoptosis and enhanced accumulation of ubiquitin conjugates. In addition, NAC, but not catalase or Trolox, interfered with the activity of piperlongumine, further supporting that piperlongumine is a proteasome inhibitor. Most importantly, we showed that NAC, but not other ROS scavengers, directly binds to proteasome inhibitors. To our knowledge, NAC is the first known compound that directly interacts with and antagonizes the activity of proteasome inhibitors. Taken together, the findings of the present study suggest that, as a result of the dual nature of NAC, data interpretation might not be straightforward when NAC is utilized as an antioxidant to demonstrate ROS involvement in drug-induced apoptosis.

  5. Developing HIV-1 Protease Inhibitors through Stereospecific Reactions in Protein Crystals.

    Science.gov (United States)

    Olajuyigbe, Folasade M; Demitri, Nicola; De Zorzi, Rita; Geremia, Silvano

    2016-10-31

    Protease inhibitors are key components in the chemotherapy of HIV infection. However, the appearance of viral mutants routinely compromises their clinical efficacy, creating a constant need for new and more potent inhibitors. Recently, a new class of epoxide-based inhibitors of HIV-1 protease was investigated and the configuration of the epoxide carbons was demonstrated to play a crucial role in determining the binding affinity. Here we report the comparison between three crystal structures at near-atomic resolution of HIV-1 protease in complex with the epoxide-based inhibitor, revealing an in-situ epoxide ring opening triggered by a pH change in the mother solution of the crystal. Increased pH in the crystal allows a stereospecific nucleophile attack of an ammonia molecule onto an epoxide carbon, with formation of a new inhibitor containing amino-alcohol functions. The described experiments open a pathway for the development of new stereospecific protease inhibitors from a reactive lead compound.

  6. Developing HIV-1 Protease Inhibitors through Stereospecific Reactions in Protein Crystals

    Directory of Open Access Journals (Sweden)

    Folasade M. Olajuyigbe

    2016-10-01

    Full Text Available Protease inhibitors are key components in the chemotherapy of HIV infection. However, the appearance of viral mutants routinely compromises their clinical efficacy, creating a constant need for new and more potent inhibitors. Recently, a new class of epoxide-based inhibitors of HIV-1 protease was investigated and the configuration of the epoxide carbons was demonstrated to play a crucial role in determining the binding affinity. Here we report the comparison between three crystal structures at near-atomic resolution of HIV-1 protease in complex with the epoxide-based inhibitor, revealing an in-situ epoxide ring opening triggered by a pH change in the mother solution of the crystal. Increased pH in the crystal allows a stereospecific nucleophile attack of an ammonia molecule onto an epoxide carbon, with formation of a new inhibitor containing amino-alcohol functions. The described experiments open a pathway for the development of new stereospecific protease inhibitors from a reactive lead compound.

  7. Timely activation of budding yeast APCCdh1 involves degradation of its inhibitor, Acm1, by an unconventional proteolytic mechanism.

    Directory of Open Access Journals (Sweden)

    Michael Melesse

    Full Text Available Regulated proteolysis mediated by the ubiquitin proteasome system is a fundamental and essential feature of the eukaryotic cell division cycle. Most proteins with cell cycle-regulated stability are targeted for degradation by one of two related ubiquitin ligases, the Skp1-cullin-F box protein (SCF complex or the anaphase-promoting complex (APC. Here we describe an unconventional cell cycle-regulated proteolytic mechanism that acts on the Acm1 protein, an inhibitor of the APC activator Cdh1 in budding yeast. Although Acm1 can be recognized as a substrate by the Cdc20-activated APC (APCCdc20 in anaphase, APCCdc20 is neither necessary nor sufficient for complete Acm1 degradation at the end of mitosis. An APC-independent, but 26S proteasome-dependent, mechanism is sufficient for complete Acm1 clearance from late mitotic and G1 cells. Surprisingly, this mechanism appears distinct from the canonical ubiquitin targeting pathway, exhibiting several features of ubiquitin-independent proteasomal degradation. For example, Acm1 degradation in G1 requires neither lysine residues in Acm1 nor assembly of polyubiquitin chains. Acm1 was stabilized though by conditional inactivation of the ubiquitin activating enzyme Uba1, implying some requirement for the ubiquitin pathway, either direct or indirect. We identified an amino terminal predicted disordered region in Acm1 that contributes to its proteolysis in G1. Although ubiquitin-independent proteasome substrates have been described, Acm1 appears unique in that its sensitivity to this mechanism is strictly cell cycle-regulated via cyclin-dependent kinase (Cdk phosphorylation. As a result, Acm1 expression is limited to the cell cycle window in which Cdk is active. We provide evidence that failure to eliminate Acm1 impairs activation of APCCdh1 at mitotic exit, justifying its strict regulation by cell cycle-dependent transcription and proteolytic mechanisms. Importantly, our results reveal that strict cell

  8. Timely Activation of Budding Yeast APCCdh1 Involves Degradation of Its Inhibitor, Acm1, by an Unconventional Proteolytic Mechanism

    Science.gov (United States)

    Melesse, Michael; Choi, Eunyoung; Hall, Hana; Walsh, Michael J.; Geer, M. Ariel; Hall, Mark C.

    2014-01-01

    Regulated proteolysis mediated by the ubiquitin proteasome system is a fundamental and essential feature of the eukaryotic cell division cycle. Most proteins with cell cycle-regulated stability are targeted for degradation by one of two related ubiquitin ligases, the Skp1-cullin-F box protein (SCF) complex or the anaphase-promoting complex (APC). Here we describe an unconventional cell cycle-regulated proteolytic mechanism that acts on the Acm1 protein, an inhibitor of the APC activator Cdh1 in budding yeast. Although Acm1 can be recognized as a substrate by the Cdc20-activated APC (APCCdc20) in anaphase, APCCdc20 is neither necessary nor sufficient for complete Acm1 degradation at the end of mitosis. An APC-independent, but 26S proteasome-dependent, mechanism is sufficient for complete Acm1 clearance from late mitotic and G1 cells. Surprisingly, this mechanism appears distinct from the canonical ubiquitin targeting pathway, exhibiting several features of ubiquitin-independent proteasomal degradation. For example, Acm1 degradation in G1 requires neither lysine residues in Acm1 nor assembly of polyubiquitin chains. Acm1 was stabilized though by conditional inactivation of the ubiquitin activating enzyme Uba1, implying some requirement for the ubiquitin pathway, either direct or indirect. We identified an amino terminal predicted disordered region in Acm1 that contributes to its proteolysis in G1. Although ubiquitin-independent proteasome substrates have been described, Acm1 appears unique in that its sensitivity to this mechanism is strictly cell cycle-regulated via cyclin-dependent kinase (Cdk) phosphorylation. As a result, Acm1 expression is limited to the cell cycle window in which Cdk is active. We provide evidence that failure to eliminate Acm1 impairs activation of APCCdh1 at mitotic exit, justifying its strict regulation by cell cycle-dependent transcription and proteolytic mechanisms. Importantly, our results reveal that strict cell-cycle expression profiles

  9. mTOR is involved in 17β-estradiol-induced, cultured immature boar Sertoli cell proliferation via regulating the expression of SKP2, CCND1, and CCNE1.

    Science.gov (United States)

    Yang, Wei-Rong; Wang, Yong; Wang, Yi; Zhang, Jiao-Jiao; Zhang, Jia-Hua; Lu, Cheng; Wang, Xian-Zhong

    2015-04-01

    Mammalian target of rapamycin (mTOR) is known to be involved in mammalian cell proliferation, while S-phase kinase-associated protein 2 (SKP2) plays a vital role in the cell cycle. Within the testis, estrogen also plays an important role in Sertoli cell proliferation, although it is not clear how. The present study asked if mTOR is involved in 17β-estradiol-dependent Sertoli cell proliferation. We specifically assessed if extracellular signal-regulated kinase 1/2 (ERK1/2) and/or phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) exert convergent effects toward the activation of mTOR signaling, and if this signaling regulates the expression of SKP2 through retinoblastoma (RB) and early mitotic inhibitor 1 (EMI1) protein and on CCNE1 and CCND1 mRNA levels. Treatment with 17β-estradiol for 15-90 min activated mTOR, with mTOR phosphorylation peaking after 30 min. U0126 (5 μM), a specific inhibitor of (MEK1/2), and 10-DEBC (2 μM), a selective inhibitor of AKT, both significantly reduced 17β-estradiol-induced phosphorylation of mTOR. Rapamycin suppressed 17β-estradiol-induced Sertoli cell proliferation, appearing to act by reducing the abundance of SKP2, CCND1, and CCNE1 mRNA as well as RB and EMI1 protein. These data indicated that 17β-estradiol enhances Sertoli cell proliferation via mTOR activation, which involves both ERK1/2 and PI3K/AKT signaling. Activated mTOR subsequently increases SKP2 mRNA and protein expression by enhancing the expression of CCND1 and CCNE1, and inhibits SKP2 protein degradation by increasing EMI1 abundance. © 2015 Wiley Periodicals, Inc.

  10. TNF-α inhibitors reduce the pathological Th1 -Th17 /Th2 imbalance in cutaneous mesenchymal stem cells of psoriasis patients.

    Science.gov (United States)

    Campanati, Anna; Orciani, Monia; Lazzarini, Raffaella; Ganzetti, Giulia; Consales, Veronica; Sorgentoni, Giulia; Di Primio, Roberto; Offidani, Annamaria

    2017-04-01

    Psoriasis is a disease characterized by an imbalance between Th 1 and Th 17 and Th 2 inflammatory axes, in which cutaneous mesenchymal stem cells (MSCs) are early involved, as they show a greater relative expression of several genes encoding for Th 1 and Th 17 cytokines. Therapeutic implications of TNF-α inhibitors on differentiated skin cells have been largely described in psoriasis; however, their effects on MSCs derived from patients with psoriasis have been only partially described. The aim of this work was to evaluate the effect of TNF-α inhibitors on cytokine milieu expressed by MSCs isolated from the skin of patients with psoriasis. Resident MSCs from skin of patients with psoriasis and healthy subjects have been isolated, characterized and profiled by PCR and ELISA for the expression of 22 cytokines involved in Th 1 , Th 2 and Th 17 pathways, both before and after 12 weeks therapy with TNF-α inhibitors. The administration of TNF-α inhibitors for 12-weeks acts on MSCs as follows: it reduces the expression of several Th 1 -Th 17 cytokines whose levels are elevated at baseline (IL-6, IL-8, IL-12, IL-23A, IFN-γ, TNF-α, CCL2, CCL20, CXCL2, CXCL5, IL-17A, IL-17C, IL-17F, IL-21, G-CSF). Similarly, it enhances the expression of several Th 2 cytokines which are underexpressed at baseline (IL-2, IL-4, IL-5), reducing the expression of those overexpressed at baseline (TGF-β and IL-13). TNF-α inhibitors could contribute to reduce the pathological imbalance between the Th 1 -Th 17 vs Th 2 axis in MSCs of patients with psoriasis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Involvement of HDAC1 and the PI3K/PKC signaling pathways in NF-κB activation by the HDAC inhibitor apicidin

    International Nuclear Information System (INIS)

    Kim, Yong Kee; Seo, Dong-Wan; Kang, Dong-Won; Lee, Hoi Young; Han, Jeung-Whan; Kim, Su-Nam

    2006-01-01

    Histone deacetylase (HDAC) inhibitors are appreciated as one of promising anticancer drugs, but they exert differential responses depending on the cell type. We recently reported the critical role of NF-κB as a modulator in determining cell fate for apoptosis in response to an HDAC inhibitor. In this study, we investigate a possible signaling pathway required for NF-κB activation in response to the HDAC inhibitor apicidin. Treatment of HeLa cells with apicidin leads to an increase in transcriptional activity of NF-κB and the expression of its target genes, IL-8 and TNF-α. TNF-α expression by apicidin is induced at earlier time points than NF-κB activation or IL-8 expression. In addition, our data show that the early expression of TNF-α does not lead to activation of NF-κB, because disruption of TNF-α activity by a neutralizing antibody does not affect nuclear translocation of NF-κB, IκBα degradation or reporter gene activation by apicidin. However, this activation of NF-κB requires the PI3K and PKC signaling pathways, but not ERK or JNK. Furthermore, apicidin activation of NF-κB seems to result from HDAC1 inhibition, as evidenced by the observation that overexpression of HDAC1, but not HDAC2, 3 or 4, dramatically inhibits NF-κB reporter gene activity. Collectively, our results suggest that activation of NF-κB signaling by apicidin requires both the PI3K/PKC signaling pathways and HDAC1, and functions as a critical modulator in determining the cellular effect of apicidin

  12. Curcumin derivatives as HIV-1 protease inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R. [Univ. of California, San Francisco, CA (United States)

    1993-12-31

    Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

  13. 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.

  14. Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

    Directory of Open Access Journals (Sweden)

    Jeon Hyejin

    2012-06-01

    Full Text Available Abstract Background Plasminogen activator inhibitor type 1 (PAI-1 is the primary inhibitor of urokinase type plasminogen activators (uPA and tissue type plasminogen activators (tPA, which mediate fibrinolysis. PAI-1 is also involved in the innate immunity by regulating cell migration and phagocytosis. However, little is known about the role of PAI-1 in the central nervous system. Methods In this study, we identified PAI-1 in the culture medium of mouse mixed glial cells by liquid chromatography and tandem mass spectrometry. Secretion of PAI-1 from glial cultures was detected by ELISA and western blotting analysis. Cell migration was evaluated by in vitro scratch-wound healing assay or Boyden chamber assay and an in vivo stab wound injury model. Phagocytic activity was measured by uptake of zymosan particles. Results The levels of PAI-1 mRNA and protein expression were increased by lipopolysaccharide and interferon-γ stimulation in both microglia and astrocytes. PAI-1 promoted the migration of microglial cells in culture via the low-density lipoprotein receptor-related protein (LRP 1/Janus kinase (JAK/signal transducer and activator of transcription (STAT1 axis. PAI-1 also increased microglial migration in vivo when injected into mouse brain. PAI-1-mediated microglial migration was independent of protease inhibition, because an R346A mutant of PAI-1 with impaired PA inhibitory activity also promoted microglial migration. Moreover, PAI-1 was able to modulate microglial phagocytic activity. PAI-1 inhibited microglial engulfment of zymosan particles in a vitronectin- and Toll-like receptor 2/6-dependent manner. Conclusion Our results indicate that glia-derived PAI-1 may regulate microglial migration and phagocytosis in an autocrine or paracrine manner. This may have important implications in the regulation of brain microglial activities in health and disease.

  15. Effects of sequence changes in the HIV-1 gp41 fusion peptide on CCR5 inhibitor resistance

    International Nuclear Information System (INIS)

    Anastassopoulou, Cleo G.; Ketas, Thomas J.; Sanders, Rogier W.; Johan Klasse, Per; Moore, John P.

    2012-01-01

    A rare pathway of HIV-1 resistance to small molecule CCR5 inhibitors such as Vicriviroc (VCV) involves changes solely in the gp41 fusion peptide (FP). Here, we show that the G516V change is critical to VCV resistance in PBMC and TZM-bl cells, although it must be accompanied by either M518V or F519I to have a substantial impact. Modeling VCV inhibition data from the two cell types indicated that G516V allows both double mutants to use VCV-CCR5 complexes for entry. The model further identified F519I as an independent determinant of preference for the unoccupied, high-VCV affinity form of CCR5. From inhibitor-free reversion cultures, we also identified a substitution in the inner domain of gp120, T244A, which appears to counter the resistance phenotype created by the FP substitutions. Examining the interplay of these changes will enhance our understanding of Env complex interactions that influence both HIV-1 entry and resistance to CCR5 inhibitors.

  16. TNF α is involved in neuropathic pain induced by nucleoside reverse transcriptase inhibitor in rats

    Science.gov (United States)

    Zheng, Xuexing; Ouyang, Handong; Liu, Shue; Mata, Marina; Fink, David J.; Hao, Shuanglin

    2011-01-01

    In patients with HIV/AIDS, neuropathic pain is a common neurological complication. Infection with the HIV itself may lead to neuropathic pain, and painful symptoms are enhanced when patients are treated with nucleoside reverse transcriptase inhibitors (NRTI). The mechanisms by which NRTIs contribute to the development of neuropathic pain are not known. In the current studies, we tested the role of TNFα in antiretroviral drug-induced neuropathic pain. We administered 2′,3′-dideoxycytidine (ddC, one of the NRTIs) systemically to induce mechanical allodynia. We found that ddC induced overexpression of both mRNA and proteins of GFAP and TNFα in the spinal dorsal horn. TNFα was colocalized with GFAP in the spinal dorsal horn and with NeuN in the DRG. Knockdown of TNFα with siRNA blocked the mechanical allodynia induced by ddC. Intrathecal administration of glial inhibitor or recombinant TNF soluble receptor, reversed mechanical allodynia induced by ddC. These results suggest that TNFα is involved in NRTI-induced neuropathic pain. PMID:21741472

  17. Indanones as high-potency reversible inhibitors of monoamine oxidase.

    Science.gov (United States)

    Mostert, Samantha; Petzer, Anél; Petzer, Jacobus P

    2015-05-01

    Recent reports document that α-tetralone (3,4-dihydro-2H-naphthalen-1-one) is an appropriate scaffold for the design of high-potency monoamine oxidase (MAO) inhibitors. Based on the structural similarity between α-tetralone and 1-indanone, the present study involved synthesis of 34 1-indanone and related indane derivatives as potential inhibitors of recombinant human MAO-A and MAO-B. The results show that C6-substituted indanones are particularly potent and selective MAO-B inhibitors, with IC50 values ranging from 0.001 to 0.030 μM. C5-Substituted indanone and indane derivatives are comparatively weaker MAO-B inhibitors. Although the 1-indanone and indane derivatives are selective inhibitors of the MAO-B isoform, a number of homologues are also potent MAO-A inhibitors, with three homologues possessing IC50 values 1-indanone as a reversible MAO inhibitor with a competitive mode of inhibition. It may be concluded that 1-indanones are promising leads for the design of therapies for neurodegenerative and neuropsychiatric disorders such as Parkinson's disease and depression. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. 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...

  19. 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.

  20. Ubiquitin ligase Cbl-b is involved in icotinib (BPI-2009H)-induced apoptosis and G1 phase arrest of EGFR mutation-positive non-small-cell lung cancer.

    Science.gov (United States)

    Mu, Xiaodong; Zhang, Ye; Qu, Xiujuan; Hou, Kezuo; Kang, Jian; Hu, Xuejun; Liu, Yunpeng

    2013-01-01

    Epidermal growth factor receptor (EGFR) is one of the most promising targets for non-small-cell lung cancer (NSCLC). Icotinib, a highly selective EGFR tyrosine kinase inhibitor (EGFR-TKI), has shown promising clinical efficacy and safety in patients with NSCLC. The exact molecular mechanism of icotinib remains unclear. In this study, we first investigated the antiproliferative effect of icotinib on NSCLC cells. Icotinib significantly inhibited proliferation of the EGFR-mutated lung cancer HCC827 cells. The IC50 values at 48 and 72 h were 0.67 and 0.07 μ M, respectively. Flow cytometric analysis showed that icotinib caused the G1 phase arrest and increased the rate of apoptosis in HCC827 cells. The levels of cyclin D1 and cyclin A2 were decreased. The apoptotic process was associated with activation of caspase-3, -8, and poly(ADP-ribose) polymerase (PARP). Further study revealed that icotinib inhibited phosphorylation of EGFR, Akt, and extracellular signal-regulated kinase. In addition, icotinib upregulated ubiquitin ligase Cbl-b expression. These observations suggest that icotinib-induced upregulation of Cbl-b is responsible, at least in part, for the antitumor effect of icotinib via the inhibition of phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways in EGFR-mutated NSCLC cells.

  1. PTP1B Inhibitors from the Entomogenous Fungi Isaria fumosorosea

    OpenAIRE

    Jun Zhang; Lin-Lin Meng; Jing-Jing Wei; Peng Fan; Sha-Sha Liu; Wei-Yu Yuan; You-Xing Zhao; Du-Qiang Luo

    2017-01-01

    Protein tyrosine phosphatase 1B (PTP1B) is implicated as a negative regulator of insulin receptor (IR) signaling and a potential drug target for the treatment of type II diabetes and other associated metabolic syndromes. Thus, small molecule inhibitors of PTP1B can be considered as an attractive approach for the design of new therapeutic agents of type II diabetes and cancer diseases. In a continuing search for new PTP1B inhibitors, a new tetramic acid possessing a rare pyrrolidinedione skele...

  2. Progress of PD-1/PD-L1 Inhibitors in Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Zhansheng JIANG

    2017-02-01

    Full Text Available Pembrolizumab, an inhibitor target programmed death 1 (PD-1, was approved into the first line therapy in advanced non-small cell lung cancer (NSCLC. It was a milestone that immune checkpoints drugs have played an important role in the treatment system of NSCLC. The results of clinical trials revealed the superiority of PD-1/programmed death ligand 1 (PD-L1 inhibitors compared with chemotherapy in first-line, second-line and multidrug resistance phase therapy. Objective response rate (ORR was up to 80% with pembrolizumab plus chemotherapy, and progression-free survival (PFS with single pembrolizumab in first line was nearly 1 year (10.3 months, the hazard ratio for death fell by 40%. Overall survival (OS was more or less 1 year with single drug pembrolizumab, nivolumab and atezolizumab for second line therapy. PD-L1 expression was a predictor of PD-1/PD-L1 inhibitors. The positive rate of PD-L1 (more than 1% in advanced NSCLC was about 60% with little difference between the tissue types. However, there was no gold standard test of PD-L1 expression.

  3. [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.

  4. 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

  5. Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1.

    Science.gov (United States)

    Scarlatti, Francesca; Bauvy, Chantal; Ventruti, Annamaria; Sala, Giusy; Cluzeaud, Françoise; Vandewalle, Alain; Ghidoni, Riccardo; Codogno, Patrice

    2004-04-30

    The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C(2)-ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B(1). Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C(2)-ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C(2)-ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifen-dependent up-regulation. Tamoxifen and C(2)-ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.

  6. TGF-β1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells

    International Nuclear Information System (INIS)

    Gomes, Luciana R; Terra, Letícia F; Wailemann, Rosângela AM; Labriola, Leticia; Sogayar, Mari C

    2012-01-01

    Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-β1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models. The mRNA expression levels of TGF-β isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-β1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays. In general, TGF-β2, TβRI and TβRII are over-expressed in more aggressive cells, except for TβRI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-β1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-β1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-β1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-β1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-β1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-β1-enhanced migration and invasion capacities were blocked by p

  7. 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.

  8. Ovarian Cancer and BRCA1/2 Testing: Opportunities to improve clinical care and disease prevention

    Directory of Open Access Journals (Sweden)

    Katherine eKarakasis

    2016-05-01

    Full Text Available Without prevention or screening options available, ovarian cancer is the most lethal malignancy of the female reproductive tract. High grade serous ovarian cancer (HGSOC is the most common histologic subtype, and the role of germline BRCA1/2 mutation in predisposition and prognosis is established. Given the targeted treatment opportunities with PARP inhibitors, a predictive role for BRCA1/2 mutation has emerged. Despite recommendations to provide BRCA1/2 testing to all women with histologically confirmed HGSOC, uniform implementation remains challenging. The opportunity to review and revise genetic screening and testing practices will identify opportunities where universal adoption of BRCA1/2 mutation testing will impact and improve treatment of women with ovarian cancer. Improving education and awareness of genetic testing for women with cancer, as well as the broader general community, will help focus much needed attention on opportunities to advance prevention and screening programs in ovarian cancer. This is imperative not only for women with cancer, those at risk of developing cancer, but also for their first-degree relatives. In addition, BRCA1/2 testing may have direct implications for patients with other types of cancers, many which are now being found to have BRCA1/2 involvement.

  9. Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

    DEFF Research Database (Denmark)

    Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter

    2003-01-01

    The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosyla......The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous...... with the glycosylation sites could be excluded as explanation for the differential reactivity. The latency transition of non-glycosylated, but not of glycosylated PAI-1, was strongly accelerated by a non-ionic detergent. The different biochemical properties of glycosylated and non-glycosylated PAI-1 depended...

  10. Saururus cernuus lignans-Potent small molecule inhibitors of hypoxia-inducible factor-1

    International Nuclear Information System (INIS)

    Hossain, Chowdhury Faiz; Kim, Yong-Pil; Baerson, Scott R.; Zhang Lei; Bruick, Richard K.; Mohammed, Kaleem A.; Agarwal, Ameeta K.; Nagle, Dale G.; Zhou Yudong

    2005-01-01

    Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B 1 , manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC 50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF, and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1α protein accumulation without affecting HIF-1α mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors

  11. Interaction of HIV-1 reverse transcriptase ribonuclease H with an acylhydrazone inhibitor.

    Science.gov (United States)

    Gong, Qingguo; Menon, Lakshmi; Ilina, Tatiana; Miller, Lena G; Ahn, Jinwoo; Parniak, Michael A; Ishima, Rieko

    2011-01-01

    HIV-1 reverse transcriptase is a bifunctional enzyme, having both DNA polymerase (RNA- and DNA-dependent) and ribonuclease H activities. HIV-1 reverse transcriptase has been an exceptionally important target for antiretroviral therapeutic development, and nearly half of the current clinically used antiretrovirals target reverse transcriptase DNA polymerase. However, no inhibitors of reverse transcriptase ribonuclease H are on the market or in preclinical development. Several drug-like small molecule inhibitors of reverse transcriptase ribonuclease H have been described, but little structural information is available about the interactions between reverse transcriptase ribonuclease H and inhibitors that exhibit antiviral activity. In this report, we describe NMR studies of the interaction of a new ribonuclease H inhibitor, BHMP07, with a catalytically active HIV-1 reverse transcriptase ribonuclease H domain fragment. We carried out solution NMR experiments to identify the interaction interface of BHMP07 with the ribonuclease H domain fragment. Chemical shift changes of backbone amide signals at different BHMP07 concentrations clearly demonstrate that BHMP07 mainly recognizes the substrate handle region in the ribonuclease H fragment. Using ribonuclease H inhibition assays and reverse transcriptase mutants, the binding specificity of BHMP07 was compared with another inhibitor, dihydroxy benzoyl naphthyl hydrazone. Our results provide a structural characterization of the ribonuclease H inhibitor interaction and are likely to be useful for further improvements of the inhibitors. © 2010 John Wiley & Sons A/S.

  12. Functional C1-inhibitor diagnostics in hereditary angioedema: assay evaluation and recommendations

    DEFF Research Database (Denmark)

    Wagenaar-Bos, Ineke G A; Drouet, Christian; Aygören-Pursun, Emel

    2008-01-01

    Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent episodes of potentially life-threatening angioedema. The most widespread underlying genetic deficiency is a heterozygous deficiency of the serine protease inhibitor C1 esterase inhibitor (C1-Inh). In addition ...

  13. Altered poly(ADP-ribose) metabolism impairs cellular responses to genotoxic stress in a hypomorphic mutant of poly(ADP-ribose) glycohydrolase

    International Nuclear Information System (INIS)

    Gao Hong; Coyle, Donna L.; Meyer-Ficca, Mirella L.; Meyer, Ralph G.; Jacobson, Elaine L.; Wang, Zhao-Qi; Jacobson, Myron K.

    2007-01-01

    Genotoxic stress activates nuclear poly(ADP-ribose) (PAR) metabolism leading to PAR synthesis catalyzed by DNA damage activated poly(ADP-ribose) polymerases (PARPs) and rapid PAR turnover by action of nuclear poly(ADP-ribose) glycohydrolase (PARG). The involvement of PARP-1 and PARP-2 in responses to DNA damage has been well studied but the involvement of nuclear PARG is less well understood. To gain insights into the function of nuclear PARG in DNA damage responses, we have quantitatively studied PAR metabolism in cells derived from a hypomorphic mutant mouse model in which exons 2 and 3 of the PARG gene have been deleted (PARG-Δ2,3 cells), resulting in a nuclear PARG containing a catalytic domain but lacking the N-terminal region (A domain) of the protein. Following DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we found that the activity of both PARG and PARPs in intact cells is increased in PARG-Δ2,3 cells. The increased PARG activity leads to decreased PARP-1 automodification with resulting increased PARP activity. The degree of PARG activation is greater than PARP, resulting in decreased PAR accumulation. Following MNNG treatment, PARG-Δ2,3 cells show reduced formation of XRCC1 foci, delayed H2AX phosphorylation, decreased DNA break intermediates during repair, and increased cell death. Our results show that a precise coordination of PARPs and PARG activities is important for normal cellular responses to DNA damage and that this coordination is defective in the absence of the PARG A domain

  14. Adventures in Scaffold Morphing: Discovery of Fused Ring Heterocyclic Checkpoint Kinase 1 (CHK1) Inhibitors.

    Science.gov (United States)

    Yang, Bin; Vasbinder, Melissa M; Hird, Alexander W; Su, Qibin; Wang, Haixia; Yu, Yan; Toader, Dorin; Lyne, Paul D; Read, Jon A; Breed, Jason; Ioannidis, Stephanos; Deng, Chun; Grondine, Michael; DeGrace, Nancy; Whitston, David; Brassil, Patrick; Janetka, James W

    2018-02-08

    Checkpoint kinase 1 (CHK1) inhibitors are potential cancer therapeutics that can be utilized for enhancing the efficacy of DNA damaging agents. Multiple small molecule CHK1 inhibitors from different chemical scaffolds have been developed and evaluated in clinical trials in combination with chemotherapeutics and radiation treatment. Scaffold morphing of thiophene carboxamide ureas (TCUs), such as AZD7762 (1) and a related series of triazoloquinolines (TZQs), led to the identification of fused-ring bicyclic CHK1 inhibitors, 7-carboxamide thienopyridines (7-CTPs), and 7-carboxamide indoles. X-ray crystal structures reveal a key intramolecular noncovalent sulfur-oxygen interaction in aligning the hinge-binding carboxamide group to the thienopyridine core in a coplanar fashion. An intramolecular hydrogen bond to an indole NH was also effective in locking the carboxamide in the preferred bound conformation to CHK1. Optimization on the 7-CTP series resulted in the identification of lead compound 44, which displayed respectable drug-like properties and good in vitro and in vivo potency.

  15. Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors: A template-based approach-Part 2

    Energy Technology Data Exchange (ETDEWEB)

    Labroli, Marc; Paruch, Kamil; Dwyer, Michael P.; Alvarez, Carmen; Keertikar, Kartik; Poker, Cory; Rossman, Randall; Duca, Jose S.; Fischmann, Thierry O.; Madison, Vincent; Parry, David; Davis, Nicole; Seghezzi, Wolfgang; Wiswell, Derek; Guzi, Timothy J. [Merck

    2013-11-20

    Previous efforts by our group have established pyrazolo[1,5-a]pyrimidine as a viable core for the development of potent and selective CDK inhibitors. As part of an effort to utilize the pyrazolo[1,5-a]pyrimidine core as a template for the design and synthesis of potent and selective kinase inhibitors, we focused on a key regulator in the cell cycle progression, CHK1. Continued SAR development of the pyrazolo[1,5-a]pyrimidine core at the C5 and C6 positions, in conjunction with previously disclosed SAR at the C3 and C7 positions, led to the discovery of potent and selective CHK1 inhibitors.

  16. Overexpression of BAX INHIBITOR-1 Links Plasma Membrane Microdomain Proteins to Stress.

    Science.gov (United States)

    Ishikawa, Toshiki; Aki, Toshihiko; Yanagisawa, Shuichi; Uchimiya, Hirofumi; Kawai-Yamada, Maki

    2015-10-01

    BAX INHIBITOR-1 (BI-1) is a cell death suppressor widely conserved in plants and animals. Overexpression of BI-1 enhances tolerance to stress-induced cell death in plant cells, although the molecular mechanism behind this enhancement is unclear. We recently found that Arabidopsis (Arabidopsis thaliana) BI-1 is involved in the metabolism of sphingolipids, such as the synthesis of 2-hydroxy fatty acids, suggesting the involvement of sphingolipids in the cell death regulatory mechanism downstream of BI-1. Here, we show that BI-1 affects cell death-associated components localized in sphingolipid-enriched microdomains of the plasma membrane in rice (Oryza sativa) cells. The amount of 2-hydroxy fatty acid-containing glucosylceramide increased in the detergent-resistant membrane (DRM; a biochemical counterpart of plasma membrane microdomains) fraction obtained from BI-1-overexpressing rice cells. Comparative proteomics analysis showed quantitative changes of DRM proteins in BI-1-overexpressing cells. In particular, the protein abundance of FLOTILLIN HOMOLOG (FLOT) and HYPERSENSITIVE-INDUCED REACTION PROTEIN3 (HIR3) markedly decreased in DRM of BI-1-overexpressing cells. Loss-of-function analysis demonstrated that FLOT and HIR3 are required for cell death by oxidative stress and salicylic acid, suggesting that the decreased levels of these proteins directly contribute to the stress-tolerant phenotypes in BI-1-overexpressing rice cells. These findings provide a novel biological implication of plant membrane microdomains in stress-induced cell death, which is negatively modulated by BI-1 overexpression via decreasing the abundance of a set of key proteins involved in cell death. © 2015 American Society of Plant Biologists. All Rights Reserved.

  17. Part 1: Notch-sparing γ-secretase inhibitors: The identification of novel naphthyl and benzofuranyl amide analogs.

    Science.gov (United States)

    Lu, Dai; Wei, Han-Xun; Zhang, Jing; Gu, Yongli; Osenkowski, Pamela; Ye, Wenjuan; Selkoe, Dennis J; Wolfe, Michael S; Augelli-Szafran, Corinne E

    2016-05-01

    γ-Secretase is one of two proteases directly involved in the production of the amyloid β-peptide (Aβ), which is pathogenic in Alzheimer's disease. Inhibition of γ-secretase to suppress the production of Aβ should not block processing of one of its alternative substrates, Notch1 receptors, as interference with Notch1 signaling leads to severe toxic effects. In the course of our studies to identify γ-secretase inhibitors with selectivity for APP over Notch, 1 [3-(benzyl(isopropyl)amino)-1-(naphthalen-2-yl)propan-1-one] was found to inhibit γ-secretase-mediated Aβ production without interfering with γ-secretase-mediated Notch processing in purified enzyme assays. As 1 is chemically unstable, efforts to increase the stability of this compound led to the identification of 2 [naphthalene-2-carboxylic acid benzyl-isopropyl-amide] which showed similar biological activity to compound 1. Synthesis and evaluation of a series of amide analogs resulted in benzofuranyl amide analogs that showed promising Notch-sparing γ-secretase inhibitory effects. This class of compounds may serve as a novel lead series for further study in the development of γ-secretase inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Structural investigation of HIV-1 nonnucleoside reverse transcriptase inhibitors: 2-Aryl-substituted benzimidazoles

    Science.gov (United States)

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

    2009-11-01

    Acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is one of the most destructive epidemics in history. Inhibitors of HIV enzymes are the main targets to develop drugs against that disease. Nonnucleoside reverse transcriptase inhibitors of HIV-1 (NNRTIs) are potentially effective and nontoxic. Structural studies provide information necessary to design more active compounds. The crystal structures of four NNRTI derivatives of 2-aryl-substituted N-benzyl-benzimidazole are presented here. Analysis of the geometrical parameters shows that the structures of the investigated inhibitors are rigid. The important geometrical parameter is the dihedral angle between the planes of the π-electron systems of the benzymidazole and benzyl moieties. The values of these dihedral angles are in a narrow range for all investigated inhibitors. There is no significant difference between the structure of the free inhibitor and the inhibitor in the complex with RT HIV-1. X-ray structures of the investigated inhibitors are a good basis for modeling enzyme-inhibitor interactions in rational drug design.

  19. Niraparib Maintenance Therapy in Platinum-Sensitive, Recurrent Ovarian Cancer

    NARCIS (Netherlands)

    Mirza, M. R.; Monk, B. J.; Herrstedt, J.; Oza, A. M.; Mahner, S.; Redondo, A.; Fabbro, M.; Ledermann, J. A.; Lorusso, D.; Vergote, I.; Ben-Baruch, N. E.; Marth, C.; Madry, R.; Christensen, R. D.; Berek, J. S.; Dorum, A.; Tinker, A. V.; du Bois, A.; Gonzalez-Martin, A.; Follana, P.; Benigno, B.; Rosenberg, P.; Gilbert, L.; Rimel, B. J.; Buscema, J.; Balser, J. P.; Agarwal, S.; Matulonis, U. A.; van der Zee, A.G.J.

    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,

  20. Discovery of inhibitors of Leishmania β-1,2-mannosyltransferases using a click-chemistry-derived guanosine monophosphate library.

    Directory of Open Access Journals (Sweden)

    Phillip van der Peet

    Full Text Available Leishmania spp. are a medically important group of protozoan parasites that synthesize a novel intracellular carbohydrate reserve polymer termed mannogen. Mannogen is a soluble homopolymer of β-1,2-linked mannose residues that accumulates in the major pathogenic stages in the sandfly vector and mammalian host. While several steps in mannogen biosynthesis have been defined, none of the enzymes have been isolated or characterized. We report the development of a simple assay for the GDP-mannose-dependent β-1,2-mannosyltransferases involved in mannogen synthesis. This assay utilizes octyl α-D-mannopyranoside to prime the formation of short mannogen oligomers up to 5 mannose residues. This assay was used to screen a focussed library of 44 GMP-triazole adducts for inhibitors. Several compounds provided effective inhibition of mannogen β-1,2-mannosyltransferases in a cell-free membrane preparation. This assay and inhibitor compounds will be useful for dissecting the role of different mannosyltransferases in regulating de novo biosynthesis and elongation reactions in mannogen metabolism.

  1. Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of ADAMTS1 Inhibitors

    Directory of Open Access Journals (Sweden)

    Guanhua Du

    2011-12-01

    Full Text Available A disintegrin and metalloprotease with thrombospondin type I motifs-1 (ADAMTS1 plays a crucial role in inflammatory joint diseases and its inhibitors are potential candidates for anti-arthritis drugs. For the purposes of drug discovery, we reported the development and validation of fluorescence resonance energy transfer (FRET assay for high-throughput screening (HTS of the ADAMTS1 inhibitors. A FRET substrate was designed for a quantitative assay of ADAMTS1 activity and enzyme kinetics studies. The assay was developed into a 50-µL, 384-well assay format for high throughput screening of ADAMTS1 inhibitors with an overall Z’ factor of 0.89. ADAMTS1 inhibitors were screened against a diverse library of 40,960 total compounds with the established HTS system. Four structurally related hits, naturally occurring compounds, kuwanon P, kuwanon X, albafuran C and mulberrofuran J, extracted from the Chinese herb Morus alba L., were identified for further investigation. The results suggest that this FRET assay is an excellent tool, not only for measurement of ADAMTS1 activity but also for discovery of novel ADAMTS1 inhibitors with HTS.

  2. Effects of cIAP-1, cIAP-2 and XIAP triple knockdown on prostate cancer cell susceptibility to apoptosis, cell survival and proliferation.

    LENUS (Irish Health Repository)

    Gill, Catherine

    2009-01-01

    BACKGROUND: Manipulating apoptotic resistance represents an important strategy for the treatment of hormone refractory prostate cancer. We hypothesised that the Inhibitor of Apoptosis (IAP) Proteins may be mediating this resistance and knockdown of cIAP-1, cIAP-2 and XIAP would increase sensitivity to apoptosis. METHODS: cIAP-1, cIAP-2 and XIAP where knocked down either individually or in combination using siRNA in androgen independent prostate cancer PC-3 cells as confirmed by real-time PCR and western blotting. Cells were then treated with TRAIL, Etoposide, or Tunicamycin, and apoptosis assessed by PI DNA staining. Apoptosis was confirmed with Annexin V labelling and measurement of PARP cleavage, and was inhibited using the pan-caspase inhibitor, zVAD.fmk. Clonogenic assays and assessment of ID-1 expression by western blotting were used to measure recovery and proliferation. RESULTS: PC-3 are resistant to TRAIL induced apoptosis and have elevated expression of cIAP-1, cIAP-2 and XIAP. Combined knockdown sensitised PC-3 to TRAIL induced apoptosis, but not to Etoposide or Tunicmycin, with corresponding increases in caspase activity and PARP cleavage which was inhibited by ZVAD.fmk. Triple knock down decreased proliferation which was confirmed by decreased ID-1 expression. CONCLUSION: Simultaneous knock down of the IAPs not only sensitised the PC-3 to TRAIL but also inhibited their proliferation rates and clonogenic survival. The inability to alter sensitivity to other triggers of apoptosis suggests that this effect is specific for death receptor pathways and knock down might facilitate immune-surveillance mechanisms to counter cancer progression and, in combination with therapeutic approaches using TRAIL, could represent an important treatment strategy.

  3. Effects of cIAP-1, cIAP-2 and XIAP triple knockdown on prostate cancer cell susceptibility to apoptosis, cell survival and proliferation

    Directory of Open Access Journals (Sweden)

    Dowling Catherine

    2009-06-01

    Full Text Available Abstract Background Manipulating apoptotic resistance represents an important strategy for the treatment of hormone refractory prostate cancer. We hypothesised that the Inhibitor of Apoptosis (IAP Proteins may be mediating this resistance and knockdown of cIAP-1, cIAP-2 and XIAP would increase sensitivity to apoptosis. Methods cIAP-1, cIAP-2 and XIAP where knocked down either individually or in combination using siRNA in androgen independent prostate cancer PC-3 cells as confirmed by real-time PCR and western blotting. Cells were then treated with TRAIL, Etoposide, or Tunicamycin, and apoptosis assessed by PI DNA staining. Apoptosis was confirmed with Annexin V labelling and measurement of PARP cleavage, and was inhibited using the pan-caspase inhibitor, zVAD.fmk. Clonogenic assays and assessment of ID-1 expression by western blotting were used to measure recovery and proliferation. Results PC-3 are resistant to TRAIL induced apoptosis and have elevated expression of cIAP-1, cIAP-2 and XIAP. Combined knockdown sensitised PC-3 to TRAIL induced apoptosis, but not to Etoposide or Tunicmycin, with corresponding increases in caspase activity and PARP cleavage which was inhibited by ZVAD.fmk. Triple knock down decreased proliferation which was confirmed by decreased ID-1 expression. Conclusion Simultaneous knock down of the IAPs not only sensitised the PC-3 to TRAIL but also inhibited their proliferation rates and clonogenic survival. The inability to alter sensitivity to other triggers of apoptosis suggests that this effect is specific for death receptor pathways and knock down might facilitate immune-surveillance mechanisms to counter cancer progression and, in combination with therapeutic approaches using TRAIL, could represent an important treatment strategy.

  4. DISTRIBUTION AND ELIMINATION OF THE GLYCOSIDASE INHIBITORS 1-DEOXYMANNOJIRIMYCIN AND N-METHYL-1-DEOXYNOJIRIMYCIN IN THE RAT INVIVO

    NARCIS (Netherlands)

    FABER, ED; NEEFJES, JJ; PLOEGH, HL; MEIJER, DKF

    1992-01-01

    We studied the pharmacokinetics of two synthetic derivatives of 1-deoxynojirimycin in the rat after intravenous administration. The mannosidase IA/B inhibitor 1-deoxymannojirimycin and the glucosidase inhibitor N-methyl-1-deoxynojirimycin exhibited minimal plasma protein binding and showed a rapid

  5. Acidic tumor microenvironment abrogates the efficacy of mTORC1 inhibitors.

    Science.gov (United States)

    Faes, Seraina; Duval, Adrian P; Planche, Anne; Uldry, Emilie; Santoro, Tania; Pythoud, Catherine; Stehle, Jean-Christophe; Horlbeck, Janine; Letovanec, Igor; Riggi, Nicolo; Demartines, Nicolas; Dormond, Olivier

    2016-12-05

    Blocking the mechanistic target of rapamycin complex-1 (mTORC1) with chemical inhibitors such as rapamycin has shown limited clinical efficacy in cancer. The tumor microenvironment is characterized by an acidic pH which interferes with cancer therapies. The consequences of acidity on the anti-cancer efficacy of mTORC1 inhibitors have not been characterized and are thus the focus of our study. Cancer cell lines were treated with rapamycin in acidic or physiological conditions and cell proliferation was investigated. The effect of acidity on mTORC1 activity was determined by Western blot. The anticancer efficacy of rapamycin in combination with sodium bicarbonate to increase the intratumoral pH was tested in two different mouse models and compared to rapamycin treatment alone. Histological analysis was performed on tumor samples to evaluate proliferation, apoptosis and necrosis. Exposing cancer cells to acidic pH in vitro significantly reduced the anti-proliferative effect of rapamycin. At the molecular level, acidity significantly decreased mTORC1 activity, suggesting that cancer cell proliferation is independent of mTORC1 in acidic conditions. In contrast, the activation of mitogen-activated protein kinase (MAPK) or AKT were not affected by acidity, and blocking MAPK or AKT with a chemical inhibitor maintained an anti-proliferative effect at low pH. In tumor mouse models, the use of sodium bicarbonate increased mTORC1 activity in cancer cells and potentiated the anti-cancer efficacy of rapamycin. Combining sodium bicarbonate with rapamycin resulted in increased tumor necrosis, increased cancer cell apoptosis and decreased cancer cell proliferation as compared to single treatment. Taken together, these results emphasize the inefficacy of mTORC1 inhibitors in acidic conditions. They further highlight the potential of combining sodium bicarbonate with mTORC1 inhibitors to improve their anti-tumoral efficacy.

  6. Identification of catechols as histone-lysine demethylase inhibitors

    DEFF Research Database (Denmark)

    Nielsen, Anders L; Kristensen, Line H; Stephansen, Karen B

    2012-01-01

    Identification of inhibitors of histone-lysine demethylase (HDM) enzymes is important because of their involvement in the development of cancer. An ELISA-based assay was developed for identification of inhibitors of the HDM KDM4C in a natural products library. Based on one of the hits with affinity...... in the low µM range (1, a catechol), a subset of structurally related compounds was selected and tested against a panel of HDMs. In this subset, two inhibitors (2 and 10) had comparable affinities towards KDM4C and KDM6A but no effect on PHF8. One inhibitor restored H3K9me3 levels in KDM4C transfected U2-OS...

  7. Multimerized CHR-derived peptides as HIV-1 fusion inhibitors.

    Science.gov (United States)

    Nomura, Wataru; Hashimoto, Chie; Suzuki, Takaharu; Ohashi, Nami; Fujino, Masayuki; Murakami, Tsutomu; Yamamoto, Naoki; Tamamura, Hirokazu

    2013-08-01

    To date, several HIV-1 fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of an HIV-1 envelope protein gp41 have been discovered. We have shown that a synthetic peptide mimetic of a trimer form of the CHR-derived peptide C34 has potent inhibitory activity against the HIV-1 fusion mechanism, compared to a monomer C34 peptide. The present study revealed that a dimeric form of C34 is evidently structurally critical for fusion inhibitors, and that the activity of multimerized CHR-derived peptides in fusion inhibition is affected by the properties of the unit peptides C34, SC34EK, and T20. The fluorescence-based study suggested that the N36-interactive sites of the C34 trimer, including hydrophobic residues, are exposed outside the trimer and that trimerization of C34 caused a remarkable increase in fusion inhibitory activity. The present results could be useful in the design of fusion inhibitors against viral infections which proceed via membrane fusion with host cells. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. Dynamin-Related Protein 1 Inhibitors Protect against Ischemic Toxicity through Attenuating Mitochondrial Ca2+ Uptake from Endoplasmic Reticulum Store in PC12 Cells

    Directory of Open Access Journals (Sweden)

    Ye Tian

    2014-02-01

    Full Text Available Intracellular calcium homeostasis disorder and mitochondrial dysfunction are involved in many acute and chronic brain diseases, including ischemic brain injury. An imbalance in mitochondrial fission and fusion is one of the most important structural abnormalities found in a large number of mitochondrial dysfunction related diseases. Here, we investigated the effects of mitochondrial division inhibitor A (mdivi A and mdivi B, two small molecule inhibitors of mitochondrial fission protein dunamin-related protein 1 (Drp-1, in neuronal injury induced by oxygen-glucose deprivation (OGD in PC12 cells. We found that mdivi A and mdivi B inhibited OGD-induced neuronal injury through attenuating apoptotic cell death. These two inhibitors also preserved mitochondrial function, as evidenced by reduced reactive oxygen species (ROS generation and cytochrome c release, as well as prevented loss of mitochondrial membrane potential (MMP. Moreover, mdivi A and mdivi B significantly suppressed mitochondrial Ca2+ uptake, but had no effect on cytoplasmic Ca2+ after OGD injury. The results of calcium imaging and immunofluorescence staining showed that Drp-1 inhibitors attenuated endoplasmic reticulum (ER Ca2+ release and prevented ER morphological changes induced by OGD. These results demonstrate that Drp-1 inhibitors protect against ischemic neuronal injury through inhibiting mitochondrial Ca2+ uptake from the ER store and attenuating mitochondrial dysfunction.

  9. PARP16/ARTD15 is a novel endoplasmic-reticulum-associated mono-ADP-ribosyltransferase that interacts with, and modifies karyopherin-ß1.

    Directory of Open Access Journals (Sweden)

    Simone Di Paola

    Full Text Available BACKGROUND: Protein mono-ADP-ribosylation is a reversible post-translational modification that modulates the function of target proteins. The enzymes that catalyze this reaction in mammalian cells are either bacterial pathogenic toxins or endogenous cellular ADP-ribosyltransferases. The latter include members of three different families of proteins: the well characterized arginine-specific ecto-enzymes ARTCs, two sirtuins and, more recently, novel members of the poly(ADP-ribose polymerase (PARP/ARTD family that have been suggested to act as cellular mono-ADP-ribosyltransferases. Here, we report on the characterisation of human ARTD15, the only known ARTD family member with a putative C-terminal transmembrane domain. METHODOLOGY/PRINCIPAL FINDINGS: Immunofluorescence and electron microscopy were performed to characterise the sub-cellular localisation of ARTD15, which was found to be associated with membranes of the nuclear envelope and endoplasmic reticulum. The orientation of ARTD15 was determined using protease protection assay, and is shown to be a tail-anchored protein with a cytosolic catalytic domain. Importantly, by combining immunoprecipitation with mass spectrometry and using cell lysates from cells over-expressing FLAG-ARTD15, we have identified karyopherin-ß1, a component of the nuclear trafficking machinery, as a molecular partner of ARTD15. Finally, we demonstrate that ARTD15 is a mono-ADP-ribosyltransferase able to induce the ADP-ribosylation of karyopherin-ß1, thus defining the first substrate for this enzyme. CONCLUSIONS/SIGNIFICANCE: Our data reveal that ARTD15 is a novel ADP-ribosyltransferase enzyme with a new intracellular location. Finally, the identification of karyopherin-ß1 as a target of ARTD15-mediated ADP-ribosylation, hints at a novel regulatory mechanism of karyopherin-ß1 functions.

  10. 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.

  11. 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.

  12. Design of novel HIV-1 protease inhibitors incorporating isophthalamide-derived P2-P3 ligands: Synthesis, biological evaluation and X-ray structural studies of inhibitor-HIV-1 protease complex

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Arun K.; Brindisi, Margherita; Nyalapatla, Prasanth R.; Takayama, Jun; Ella-Menye, Jean-Rene; Yashchuk, Sofiya; Agniswamy, Johnson; Wang, Yuan-Fang; Aoki, Manabu; Amano, Masayuki; Weber, Irene T.; Mitsuya, Hiroaki

    2017-10-01

    Based upon molecular insights from the X-ray structures of inhibitor-bound HIV-1 protease complexes, we have designed a series of isophthalamide-derived inhibitors incorporating substituted pyrrolidines, piperidines and thiazolidines as P2-P3 ligands for specific interactions in the S2-S3 extended site. Compound 4b has shown an enzyme Ki of 0.025 nM and antiviral IC50 of 69 nM. An X-ray crystal structure of inhibitor 4b-HIV-1 protease complex was determined at 1.33 Å resolution. We have also determined X-ray structure of 3b-bound HIV-1 protease at 1.27 Å resolution. These structures revealed important molecular insight into the inhibitor–HIV-1 protease interactions in the active site.

  13. Oxaliplatin triggers necrosis as well as apoptosis in gastric cancer SGC-7901 cells

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Ping; Zhu, Xueping [Department of Immunology, Anhui Medical University, Hefei 230032 (China); Jin, Wei [Department of Otolaryngology, Chaohu Hospital of Anhui Medical University, Chaohu 238000 (China); Hao, Shumei; Liu, Qi [Department of Immunology, Anhui Medical University, Hefei 230032 (China); Zhang, Linjie, E-mail: zlj33@ahmu.edu.cn [Department of Immunology, Anhui Medical University, Hefei 230032 (China)

    2015-05-01

    Intrinsic apoptotic pathway is considered to be responsible for cell death induced by platinum anticancer drugs. While in this study, we found that, necrosis is an indispensable pathway besides apoptosis in oxaliplatin-treated gastric cancer SGC-7901 cells. Upon exposure to oxaliplatin, both apoptotic and necrotic features were observed. The majority of dead cells were double positive for Annexin V and propidium iodide (PI). Moreover, mitochondrial membrane potential collapsed and caspase cascades were activated. However, ultrastructural changes under transmission electron microscope, coupled with the release of cellular contents, demonstrated the rupture of the plasma membrane. Oxaliplatin administration did not stimulate reactive oxygen species (ROS) production and autophagy, but elevated the protein level of Bmf. In addition, receptor interacting protein 1 (RIP1), but not receptor interacting protein 3 (RIP3) and its downstream components participated in this death process. Necrostatin-1 (Nec-1) blocked oxaliplatin-induced cell death nearly completely, whereas z-VAD-fmk could partially suppress cell death. Oxaliplatin treatment resulted in poly(ADP-ribose) polymerase-1 (PARP-1) overactivation, as indicated by the increase of poly(ADP-ribose) (PAR), which led to NAD{sup +} and ATP depletion. PARP-1 inhibitor, olaparib, could significantly block oxaliplatin-induced cell death, thus confirming that PARP-1 activation is mainly responsible for the cytotoxicity of oxaliplatin. Phosphorylation of H2AX at Ser139 and translocalization of apoptosis-inducing factor (AIF) are critical for this death process. Taken together, these results indicate that oxaliplatin can bypass canonical cell death pathways to kill gastric cancer cells, which may be of therapeutic advantage in the treatment of gastric cancer. - Highlights: • Oxaliplatin induces apoptotic and necrotic cell death. • Nec-1 can inhibit oxaliplatin-induced cell death nearly completely. • RIP3 and its

  14. Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

    DEFF Research Database (Denmark)

    Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter Durand

    2003-01-01

    The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosyla...

  15. Structure-Based Design of Novel HIV-1 Protease Inhibitors to Combat Drug Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh,A.; Sridhar, P.; Leshchenko, S.; Hussain, A.; Li, J.; Kovalevsky, A.; Walters, D.; Wedelind, J.; Grum-Tokars, V.; et al.

    2006-01-01

    Structure-based design and synthesis of novel HIV protease inhibitors are described. The inhibitors are designed specifically to interact with the backbone of HIV protease active site to combat drug resistance. Inhibitor 3 has exhibited exceedingly potent enzyme inhibitory and antiviral potency. Furthermore, this inhibitor maintains impressive potency against a wide spectrum of HIV including a variety of multi-PI-resistant clinical strains. The inhibitors incorporated a stereochemically defined 5-hexahydrocyclopenta[b]furanyl urethane as the P2-ligand into the (R)-(hydroxyethylamino)sulfonamide isostere. Optically active (3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[b]furan was prepared by an enzymatic asymmetrization of meso-diacetate with acetyl cholinesterase, radical cyclization, and Lewis acid-catalyzed anomeric reduction as the key steps. A protein-ligand X-ray crystal structure of inhibitor 3-bound HIV-1 protease (1.35 Angstroms resolution) revealed extensive interactions in the HIV protease active site including strong hydrogen bonding interactions with the backbone. This design strategy may lead to novel inhibitors that can combat drug resistance.

  16. Inhibitor discovery of full-length New Delhi metallo-β-lactamase-1 (NDM-1.

    Directory of Open Access Journals (Sweden)

    Bingzheng Shen

    Full Text Available New Delhi metallo-β-lactmase-1 (NDM-1 has recently attracted extensive attention for its biological activities to catalyze the hydrolysis of almost all of β-lactam antibiotics. To study the catalytic property of NDM-1, the steady-kinetic parameters of NDM-1 toward several kinds of β-lactam antibiotics have been detected. It could effectively hydrolyze most β-lactams (k cat/K m ratios between 0.03 to 1.28 µmol⁻¹.s⁻¹, except aztreonam. We also found that thiophene-carboxylic acid derivatives could inhibit NDM-1 and have shown synergistic antibacterial activity in combination with meropenem. Flexible docking and quantum mechanics (QM study revealed electrostatic interactions between the sulfur atom of thiophene-carboxylic acid derivatives and the zinc ion of NDM-1, along with hydrogen bond between inhibitor and His189 of NDM-1. The interaction models proposed here can be used in rational design of NDM-1 inhibitors.

  17. Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination.

    Science.gov (United States)

    Nakatsuka, Erika; Sawada, Kenjiro; Nakamura, Koji; Yoshimura, Akihito; Kinose, Yasuto; Kodama, Michiko; Hashimoto, Kae; Mabuchi, Seiji; Makino, Hiroshi; Morii, Eiichi; Yamaguchi, Yoichi; Yanase, Takeshi; Itai, Akiko; Morishige, Ken-Ichirou; Kimura, Tadashi

    2017-10-27

    In the present study, the therapeutic potential of targeting plasminogen activator inhibitor-1 (PAI-1) in ovarian cancer was tested. Tissues samples from 154 cases of ovarian carcinoma were immunostained with anti-PAI-1 antibody, and the prognostic value was analyzed. Among the samples, 67% (104/154) showed strong PAI-1 expression; this was significantly associated with poor prognosis (progression-free survival: 20 vs. 31 months, P = 0.0033). In particular, among patients with stage II-IV serous adenocarcinoma, PAI-1 expression was an independent prognostic factor. The effect of a novel PAI-1 inhibitor, IMD-4482, on ovarian cancer cell lines was assessed and its therapeutic potential was examined using a xenograft mouse model of ovarian cancer. IMD-4482 inhibited in vitro cell adhesion to vitronectin in PAI-1-positive ovarian cancer cells, followed by the inhibition of extracellular signal-regulated kinase and focal adhesion kinase phosphorylation through dissociation of the PAI-urokinase receptor complex from integrin αVβ3. IMD-4482 caused G0/G1 cell arrest and inhibited the proliferation of PAI-1-positive ovarian cancer cells. In the xenograft model, IMD-4482 significantly inhibited peritoneal dissemination with the reduction of PAI-1 expression and the inhibition of focal adhesion kinase phosphorylation. Collectively, the functional inhibition of PAI-1 significantly inhibited ovarian cancer progression, and targeting PAI-1 may be a potential therapeutic strategy in ovarian cancer.

  18. Imprinted CDKN1C is a tumor suppressor in rhabdoid tumor and activated by restoration of SMARCB1 and histone deacetylase inhibitors.

    Directory of Open Access Journals (Sweden)

    Elizabeth M Algar

    Full Text Available SMARCB1 is deleted in rhabdoid tumor, an aggressive paediatric malignancy affecting the kidney and CNS. We hypothesized that the oncogenic pathway in rhabdoid tumors involved epigenetic silencing of key cell cycle regulators as a consequence of altered chromatin-remodelling, attributable to loss of SMARCB1, and that this hypothesis if proven could provide a biological rationale for testing epigenetic therapies in this disease. We used an inducible expression system to show that the imprinted cell cycle inhibitor CDKN1C is a downstream target for SMARCB1 and is transcriptionally activated by increased histone H3 and H4 acetylation at the promoter. We also show that CDKN1C expression induces cell cycle arrest, CDKN1C knockdown with siRNA is associated with increased proliferation, and is able to compete against the anti-proliferative effect of restored SMARCB1 expression. The histone deacetylase inhibitor (HDACi, Romidepsin, specifically restored CDKN1C expression in rhabdoid tumor cells through promoter histone H3 and H4 acetylation, recapitulating the effect of SMARCB1 on CDKNIC allelic expression, and induced cell cycle arrest in G401 and STM91-01 rhabdoid tumor cell lines. CDKN1C expression was also shown to be generally absent in clinical specimens of rhabdoid tumor, however CDKN1A and CDKN1B expression persisted. Our observations suggest that maintenance of CDKN1C expression plays a critical role in preventing rhabdoid tumor growth. Significantly, we report for the first time, parallels between the molecular pathways of SMARCB1 restoration and Romidepsin treatment, and demonstrate a biological basis for the further exploration of histone deacetylase inhibitors as relevant therapeutic reagents in the treatment of rhabdoid tumor.

  19. 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.

  20. Aldehyde Dehydrogenase 1 and Raf Kinase Inhibitor Protein ...

    African Journals Online (AJOL)

    Aldehyde Dehydrogenase 1 and Raf Kinase Inhibitor Protein Expression Defines the Proliferative Nature of Cervical Cancer Stem Cells. ... of cervical cancer stem cells and also to validate them in initial and advanced stages of cervical cancer. Keywords: Cervical cancer, ALDH1, BALB/c-nu/nu, HeLa cells, RKIP, Sox2 ...

  1. Tissue inhibitor of matrix metalloproteinase-1 mediates erythropoietin-induced neuroprotection in hypoxia ischemia.

    Science.gov (United States)

    Souvenir, Rhonda; Fathali, Nancy; Ostrowski, Robert P; Lekic, Tim; Zhang, John H; Tang, Jiping

    2011-10-01

    Previous studies have shown that erythropoietin (EPO) is neuroprotective in both in vivo and in vitro models of hypoxia ischemia. However these studies hold limited clinical translations because the underlying mechanism remains unclear and the key molecules involved in EPO-induced neuroprotection are still to be determined. This study investigated if tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and its upstream regulator signaling molecule Janus kinase-2 (JAK-2) are critical in EPO-induced neuroprotection. Hypoxia ischemia (HI) was modeled in-vitro by oxygen and glucose deprivation (OGD) and in-vivo by a modified version of Rice-Vannucci model of HI in 10-day-old rat pups. EPO treated cells were exposed to AG490, an inhibitor of JAK-2 or TIMP-1 neutralizing antibody for 2h with OGD. Cell death, phosphorylation of JAK-2 and signal transducers and activators of transcription protein-3 (STAT-3), TIMP-1 expression, and matrix metalloproteinase-9 (MMP-9) activity were measured and compared with normoxic group. Hypoxic ischemic animals were treated one hour following HI and evaluated 48 h after. Our data showed that EPO significantly increased cell survival, associated with increased TIMP-1 activity, phosphorylation of JAK-2 and STAT-3, and decreased MMP-9 activity in vivo and in vitro. EPO's protective effects were reversed by inhibition of JAK-2 or TIMP-1 in both models. We concluded that JAK-2, STAT-3 and TIMP-1 are key mediators of EPO-induced neuroprotection during hypoxia ischemia injury. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Smoking History Predicts Sensitivity to PARP Inhibitor Veliparib in Patients with Advanced Non-Small Cell Lung Cancer.

    Science.gov (United States)

    Reck, Martin; Blais, Normand; Juhasz, Erzsebet; Gorbunova, Vera; Jones, C Michael; Urban, Laszlo; Orlov, Sergey; Barlesi, Fabrice; Kio, Ebenezer; Keilholz, Ulrich; Qin, Qin; Qian, Jiang; Nickner, Caroline; Dziubinski, Juliann; Xiong, Hao; Mittapalli, Rajendar K; Dunbar, Martin; Ansell, Peter; He, Lei; McKee, Mark; Giranda, Vincent; Ramalingam, Suresh S

    2017-07-01

    Tobacco-related NSCLC is associated with reduced survival and greater genomic instability. Veliparib, a potent poly(adenosine diphosphate-ribose) polymerase inhibitor, augments platinum-induced DNA damage. A phase 2 trial of untreated advanced NSCLC showed a trend for improved outcomes (hazard ratio [HR] = 0.80, 95% confidence interval: 0.54-1.18, p = 0.27 for overall survival and HR = 0.72, 95% CI: 0.45-1.15, p = 0.17 for progression-free survival) when veliparib was added to carboplatin/paclitaxel. Here we report an exploratory analysis by smoking history. Patients were randomized 2:1 to receive carboplatin/paclitaxel with veliparib, 120 mg (n = 105), or placebo (n = 53). Patients were stratified by histologic subtype and smoking history (recent smokers [n = 95], former smokers [n = 42], and never-smokers [n = 21]). Plasma cotinine level was measured as a chemical index of smoking. Mutation status was assessed by whole exome sequencing (n = 38). Smoking history, histologic subtype, age, Eastern Cooperative Oncology Group performance status, sex, and geographic region predicted veliparib benefit in univariate analyses. In multivariate analysis, history of recent smoking was most predictive for veliparib benefit. Recent smokers treated with veliparib derived significantly greater progression-free survival and overall survival benefits (HR = 0.38 [p Smoking history predicted for efficacy with a veliparib-chemotherapy combination; toxicity was acceptable regardless of smoking history. A prespecified analysis of recent smokers is planned for ongoing phase 3 studies of veliparib in NSCLC. Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

  3. Genetics Home Reference: complete plasminogen activator inhibitor 1 deficiency

    Science.gov (United States)

    ... well studied in a large family belonging to the Old Order Amish population of eastern and southern Indiana. Additional cases in North ... Human plasminogen activator inhibitor-1 (PAI-1) deficiency: characterization of a large kindred with a null mutation in the PAI-1 gene. Blood. 1997 Jul 1;90( ...

  4. 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.

  5. 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.

  6. Imidazopyridine- and purine-thioacetamide derivatives: potent inhibitors of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1).

    Science.gov (United States)

    Chang, Lei; Lee, Sang-Yong; Leonczak, Piotr; Rozenski, Jef; De Jonghe, Steven; Hanck, Theodor; Müller, Christa E; Herdewijn, Piet

    2014-12-11

    Nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) belongs to the family of ecto-nucleotidases, which control extracellular nucleotide, nucleoside, and (di)phosphate levels. To study the (patho)physiological roles of NPP1 potent and selective inhibitors with drug-like properties are required. Therefore, a compound library was screened for NPP1 inhibitors using a colorimetric assay with p-nitrophenyl 5'-thymidine monophosphate (p-Nph-5'-TMP) as an artificial substrate. This led to the discovery of 2-(3H-imidazo[4,5-b]pyridin-2-ylthio)-N-(3,4-dimethoxyphenyl)acetamide (5a) as a hit compound with a Ki value of 217 nM. Subsequent structure-activity relationship studies led to the development of purine and imidazo[4,5-b]pyridine analogues with high inhibitory potency (Ki values of 5.00 nM and 29.6 nM, respectively) when assayed with p-Nph-5'-TMP as a substrate. Surprisingly, the compounds were significantly less potent when tested versus ATP as a substrate, with Ki values in the low micromolar range. A prototypic inhibitor was investigated for its mechanism of inhibition and found to be competitive versus both substrates.

  7. Preparation and biological evaluation of conformationally constrained BACE1 inhibitors.

    Science.gov (United States)

    Winneroski, Leonard L; Schiffler, Matthew A; Erickson, Jon A; May, Patrick C; Monk, Scott A; Timm, David E; Audia, James E; Beck, James P; Boggs, Leonard N; Borders, Anthony R; Boyer, Robert D; Brier, Richard A; Hudziak, Kevin J; Klimkowski, Valentine J; Garcia Losada, Pablo; Mathes, Brian M; Stout, Stephanie L; Watson, Brian M; Mergott, Dustin J

    2015-07-01

    The BACE1 enzyme is a key target for Alzheimer's disease. During our BACE1 research efforts, fragment screening revealed that bicyclic thiazine 3 had low millimolar activity against BACE1. Analysis of the co-crystal structure of 3 suggested that potency could be increased through extension toward the S3 pocket and through conformational constraint of the thiazine core. Pursuit of S3-binding groups produced low micromolar inhibitor 6, which informed the S3-design for constrained analogs 7 and 8, themselves prepared via independent, multi-step synthetic routes. Biological characterization of BACE inhibitors 6-8 is described. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Ubiquitin Ligase Cbl-b Is Involved in Icotinib (BPI-2009H-Induced Apoptosis and G1 Phase Arrest of EGFR Mutation-Positive Non-Small-Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Xiaodong Mu

    2013-01-01

    Full Text Available Epidermal growth factor receptor (EGFR is one of the most promising targets for non-small-cell lung cancer (NSCLC. Icotinib, a highly selective EGFR tyrosine kinase inhibitor (EGFR-TKI, has shown promising clinical efficacy and safety in patients with NSCLC. The exact molecular mechanism of icotinib remains unclear. In this study, we first investigated the antiproliferative effect of icotinib on NSCLC cells. Icotinib significantly inhibited proliferation of the EGFR-mutated lung cancer HCC827 cells. The IC50 values at 48 and 72 h were 0.67 and 0.07 μM, respectively. Flow cytometric analysis showed that icotinib caused the G1 phase arrest and increased the rate of apoptosis in HCC827 cells. The levels of cyclin D1 and cyclin A2 were decreased. The apoptotic process was associated with activation of caspase-3, -8, and poly(ADP-ribose polymerase (PARP. Further study revealed that icotinib inhibited phosphorylation of EGFR, Akt, and extracellular signal-regulated kinase. In addition, icotinib upregulated ubiquitin ligase Cbl-b expression. These observations suggest that icotinib-induced upregulation of Cbl-b is responsible, at least in part, for the antitumor effect of icotinib via the inhibition of phosphoinositide 3-kinase (PI3K/Akt and mitogen-activated protein kinase pathways in EGFR-mutated NSCLC cells.

  9. Discovery of Selective Phosphodiesterase 1 Inhibitors with Memory Enhancing Properties.

    Science.gov (United States)

    Dyck, Brian; Branstetter, Bryan; Gharbaoui, Tawfik; Hudson, Andrew R; Breitenbucher, J Guy; Gomez, Laurent; Botrous, Iriny; Marrone, Tami; Barido, Richard; Allerston, Charles K; Cedervall, E Peder; Xu, Rui; Sridhar, Vandana; Barker, Ryan; Aertgeerts, Kathleen; Schmelzer, Kara; Neul, David; Lee, Dong; Massari, Mark Eben; Andersen, Carsten B; Sebring, Kristen; Zhou, Xianbo; Petroski, Robert; Limberis, James; Augustin, Martin; Chun, Lawrence E; Edwards, Thomas E; Peters, Marco; Tabatabaei, Ali

    2017-04-27

    A series of potent thienotriazolopyrimidinone-based PDE1 inhibitors was discovered. X-ray crystal structures of example compounds from this series in complex with the catalytic domain of PDE1B and PDE10A were determined, allowing optimization of PDE1B potency and PDE selectivity. Reduction of hERG affinity led to greater than a 3000-fold selectivity for PDE1B over hERG. 6-(4-Methoxybenzyl)-9-((tetrahydro-2H-pyran-4-yl)methyl)-8,9,10,11-tetrahydropyrido[4',3':4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one was identified as an orally bioavailable and brain penetrating PDE1B enzyme inhibitor with potent memory-enhancing effects in a rat model of object recognition memory.

  10. 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.

  11. Effects and Mechanisms of Checkpoint Inhibitors (CTLA-4, PD-1 and PD-L1 Inhibitors as New Immunotherapeutic Agents for Bladder Cancer

    Directory of Open Access Journals (Sweden)

    Serdar Çelik

    2018-04-01

    Full Text Available Since intravesical Bacillus Calmette-Guerin (BCG began to be used for bladder cancer, our understanding of the importance of immune mechanisms in bladder cancer has steadily grown. With developments in immunotherapy in recent years, the use of new immunotherapeutic agents for bladder cancer, especially chemotherapy-resistant invasive and metastatic cancers, has opened the way for research in this area. Of these new therapeutic agents, this article reviews studies published on PubMed or listed on the ClinicalTrials.gov website as of December 2017 regarding the effects and mechanisms of action of checkpoint inhibitors [cytotoxic t-lymphocyte associated protein-4, programmed cell death 1 receptor (PD-1 and PD-1 ligand inhibitors] on bladder cancer. Because checkpoint inhibitors were first used for chemotherapy-resistant bladder cancer after identification of positive expression in tumor cells and especially in tumor-infiltrating mononuclear cells, significant objective response rates and survival advantages have been reported. Research continues regarding the use of these agents as first- and second-line treatment for metastatic disease in combination with chemotherapy; their efficacy in neoadjuvant, adjuvant, and bladder-preserving approaches to muscle-invasive bladder cancer (MIBC disease, and their use in non-muscle-invasize bladder cancer (NMIBC, especially BCG-refractory disease. Depending on the results of these ongoing studies, immunotherapy may direct the treatment of bladder cancer in the future.

  12. Covalent Allosteric Inactivation of Protein Tyrosine Phosphatase 1B (PTP1B) by an Inhibitor-Electrophile Conjugate.

    Science.gov (United States)

    Punthasee, Puminan; Laciak, Adrian R; Cummings, Andrea H; Ruddraraju, Kasi Viswanatharaju; Lewis, Sarah M; Hillebrand, Roman; Singh, Harkewal; Tanner, John J; Gates, Kent S

    2017-04-11

    Protein tyrosine phosphatase 1B (PTP1B) is a validated drug target, but it has proven difficult to develop medicinally useful, reversible inhibitors of this enzyme. Here we explored covalent strategies for the inactivation of PTP1B using a conjugate composed of an active site-directed 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide inhibitor connected via a short linker to an electrophilic α-bromoacetamide moiety. Inhibitor-electrophile conjugate 5a caused time-dependent loss of PTP1B activity consistent with a covalent inactivation mechanism. The inactivation occurred with a second-order rate constant of (1.7 ± 0.3) × 10 2 M -1 min -1 . Mass spectrometric analysis of the inactivated enzyme indicated that the primary site of modification was C121, a residue distant from the active site. Previous work provided evidence that covalent modification of the allosteric residue C121 can cause inactivation of PTP1B [Hansen, S. K., Cancilla, M. T., Shiau, T. P., Kung, J., Chen, T., and Erlanson, D. A. (2005) Biochemistry 44, 7704-7712]. Overall, our results are consistent with an unusual enzyme inactivation process in which noncovalent binding of the inhibitor-electrophile conjugate to the active site of PTP1B protects the nucleophilic catalytic C215 residue from covalent modification, thus allowing inactivation of the enzyme via selective modification of allosteric residue C121.

  13. Structure-guided evolution of potent and selective CHK1 inhibitors through scaffold morphing.

    Science.gov (United States)

    Reader, John C; Matthews, Thomas P; Klair, Suki; Cheung, Kwai-Ming J; Scanlon, Jane; Proisy, Nicolas; Addison, Glynn; Ellard, John; Piton, Nelly; Taylor, Suzanne; Cherry, Michael; Fisher, Martin; Boxall, Kathy; Burns, Samantha; Walton, Michael I; Westwood, Isaac M; Hayes, Angela; Eve, Paul; Valenti, Melanie; de Haven Brandon, Alexis; Box, Gary; van Montfort, Rob L M; Williams, David H; Aherne, G Wynne; Raynaud, Florence I; Eccles, Suzanne A; Garrett, Michelle D; Collins, Ian

    2011-12-22

    Pyrazolopyridine inhibitors with low micromolar potency for CHK1 and good selectivity against CHK2 were previously identified by fragment-based screening. The optimization of the pyrazolopyridines to a series of potent and CHK1-selective isoquinolines demonstrates how fragment-growing and scaffold morphing strategies arising from a structure-based understanding of CHK1 inhibitor binding can be combined to successfully progress fragment-derived hit matter to compounds with activity in vivo. The challenges of improving CHK1 potency and selectivity, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps, which progressed through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and ultimately to imidazo[4,5-c]pyridines and isoquinolines. A potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, which potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts in nude mice.

  14. Antifungal drugs as corrosion inhibitors for aluminium in 0.1 M HCl

    Energy Technology Data Exchange (ETDEWEB)

    Obot, I.B. [Department of Chemistry, Faculty of Science, University of Uyo, Uyo (Nigeria)], E-mail: proffoime@yahoo.com; Obi-Egbedi, N.O. [Department of Chemistry, University of Ibadan, Ibadan (Nigeria); Umoren, S.A. [Department of Chemistry, Faculty of Science, University of Uyo, Uyo (Nigeria)

    2009-08-15

    The inhibitive capabilities of Clotrimazole (CTM) and Fluconazole (FLC), two antifungal drugs, on the electrochemical corrosion of aluminium in 0.1 M HCl solution has been studied using weight loss measurements at 30 and 50 deg. C. The results indicate that both compound act as inhibitors in the acidic corrodent. At constant acid concentration, the inhibition efficiency (%I) increased with increase in the concentration of the inhibitors. Increase in temperature increased the corrosion rate in the absence and presence of the inhibitors but decreased the inhibition efficiency. CTM and FLC adsorbed on the surface of aluminium according to the Langmuir adsorption isotherm model at all the concentrations and temperatures studied. Phenomenon of physical adsorption is proposed from the activation parameter obtained. Thermodynamic parameters reveal that the adsorption process is spontaneous. The reactivity of these compounds was analyzed through theoretical calculations based on AM1 semi-empirical method to explain the different efficiencies of these compounds as corrosion inhibitors. CTM was found to be a better inhibitor than FLC.

  15. Drug Discovery of Host CLK1 Inhibitors for Influenza Treatment

    Directory of Open Access Journals (Sweden)

    Mian Zu

    2015-11-01

    Full Text Available The rapid evolution of influenza virus makes antiviral drugs less effective, which is considered to be a major bottleneck in antiviral therapy. The key proteins in the host cells, which are related with the replication cycle of influenza virus, are regarded as potential drug targets due to their distinct advantage of lack of evolution and drug resistance. Cdc2-like kinase 1 (CLK1 in the host cells is responsible for alternative splicing of the M2 gene of influenza virus during influenza infection and replication. In this study, we carried out baculovirus-mediated expression and purification of CLK1 and established a reliable screening assay for CLK1 inhibitors. After a virtual screening of CLK1 inhibitors was performed, the activities of the selected compounds were evaluated. Finally, several compounds with strong inhibitory activity against CLK1 were discovered and their in vitro anti-influenza virus activities were validated using a cytopathic effect (CPE reduction assay. The assay results showed that clypearin, corilagin, and pinosylvine were the most potential anti-influenza virus compounds as CLK1 inhibitors among the compounds tested. These findings will provide important information for new drug design and development in influenza treatment, and CLK1 may be a potent drug target for anti-influenza drug screening and discovery.

  16. Inhibition of SIRT1 by a small molecule induces apoptosis in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Kalle, Arunasree M., E-mail: arunasreemk@ilsresearch.org [Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, AP 500 046 (India); Mallika, A. [Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, AP 500 046 (India); Badiger, Jayasree [HKE' s Smt. V.G. College for Women, Aiwan-E-Shahi Area, Gulbarga, KA 585 102 (India); Alinakhi [Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, AP 500 046 (India); Talukdar, Pinaki [Department of Chemistry, Indian Institute of Science Education and Research, First Floor, Central Tower, Sai Trinity Building Garware Circle, Sutarwadi, PashanPune, Maharashtra 411 021 (India); Sachchidanand [Lupin Research Park, 46/47, A, Village Nande, Taluka Mulshi, Dist. Pune 411 042 (India)

    2010-10-08

    Research highlights: {yields} Novel small molecule SIRT1 inhibitor better than sirtinol. {yields} IC{sub 50} 500 nM. {yields} Specific tumor cytotoxicity towards breast cancer cells. {yields} Restoration of H3K9 acetylation levels to baseline when co-treated with SIRT1 activator (Activator X) and inhibitor (ILS-JGB-1741). -- Abstract: Overexpression of SIRT1, a NAD{sup +}-dependent class III histone deacetylases (HDACs), is implicated in many cancers and therefore could become a promising antitumor target. Here we demonstrate a small molecule SIRT1 inhibitor, ILS-JGB-1741(JGB1741) with potent inhibitory effects on the proliferation of human metastatic breast cancer cells, MDA-MB 231. The molecule has been designed using medicinal chemistry approach based on known SIRT1 inhibitor, sirtinol. The molecule showed a significant inhibition of SIRT1 activity compared to sirtinol. Studies on the antitumor effects of JGB on three different cancer cell lines, K562, HepG2 and MDA-MB 231 showed an IC{sub 50} of 1, 10 and 0.5 {mu}M, respectively. Further studies on MDA-MB 231 cells showed a dose-dependent increase in K9 and K382 acetylation of H3 and p53, respectively. Results also demonstrated that JGB1741-induced apoptosis is associated with increase in cytochrome c release, modulation in Bax/Bcl2 ratio and cleavage of PARP. Flowcytometric analysis showed increased percentage of apoptotic cells, decrease in mitochondrial membrane potential and increase in multicaspase activation. In conclusion, the present study indicates the potent apoptotic effects of JGB1741 in MDA-MB 231 cells.

  17. Kinetic intermediates en route to the final serpin-protease complex: studies of complexes of α1-protease inhibitor with trypsin.

    Science.gov (United States)

    Maddur, Ashoka A; Swanson, Richard; Izaguirre, Gonzalo; Gettins, Peter G W; Olson, Steven T

    2013-11-01

    Serpin protein protease inhibitors inactivate their target proteases through a unique mechanism in which a major serpin conformational change, resulting in a 70-Å translocation of the protease from its initial reactive center loop docking site to the opposite pole of the serpin, kinetically traps the acyl-intermediate complex. Although the initial Michaelis and final trapped acyl-intermediate complexes have been well characterized structurally, the intermediate stages involved in this remarkable transformation are not well understood. To better characterize such intermediate steps, we undertook rapid kinetic studies of the FRET and fluorescence perturbation changes of site-specific fluorophore-labeled derivatives of the serpin, α1-protease inhibitor1PI), which report the serpin and protease conformational changes involved in transforming the Michaelis complex to the trapped acyl-intermediate complex in reactions with trypsin. Two kinetically resolvable conformational changes were observed in the reactions, ascribable to (i) serpin reactive center loop insertion into sheet A with full protease translocation but incomplete protease distortion followed by, (ii) full conformational distortion and movement of the protease and coupled serpin conformational changes involving the F helix-sheet A interface. Kinetic studies of calcium effects on the labeled α1PI-trypsin reactions demonstrated both inactive and low activity states of the distorted protease in the final complex that were distinct from the intermediate distorted state. These studies provide new insights into the nature of the serpin and protease conformational changes involved in trapping the acyl-intermediate complex in serpin-protease reactions and support a previously proposed role for helix F in the trapping mechanism.

  18. Functional C1-inhibitor diagnostics in hereditary angioedema: Assay evaluation and recommendations

    NARCIS (Netherlands)

    Wagenaar-Bos, Ineke G. A.; Drouet, Christian; Aygoeren-Pursun, Emel; Bork, Konrad; Bucher, Christoph; Bygum, Anette; Farkas, Henriette; Fust, George; Gregorek, Hanna; Hack, C. Erik; Hickey, Alaco; Joller-Jemelka, Helen I.; Kapusta, Maria; Kreuz, Wolfhart; Longhurst, Hilary; Lopez-Trascasa, Margarita; Madalinski, Kazimierz; Naskalski, Jerzy; Nieuwenhuys, Ed; Ponard, Denise; Truedsson, Lennart; Varga, Lilian; Nielsen, Erik Waage; Wagner, Eric; Zingale, Lorenza; Cicardi, Marco; van Ham, S. Marieke

    2008-01-01

    Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent episodes of potentially life-threatening angioedema. The most widespread underlying genetic deficiency is a heterozygous deficiency of the serine protease inhibitor Cl esterase inhibitor (C1-Inh). In addition to

  19. A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity.

    Science.gov (United States)

    Heijink, Anne Margriet; Blomen, Vincent A; Bisteau, Xavier; Degener, Fabian; Matsushita, Felipe Yu; Kaldis, Philipp; Foijer, Floris; van Vugt, Marcel A T M

    2015-12-08

    The Wee1 cell cycle checkpoint kinase prevents premature mitotic entry by inhibiting cyclin-dependent kinases. Chemical inhibitors of Wee1 are currently being tested clinically as targeted anticancer drugs. Wee1 inhibition is thought to be preferentially cytotoxic in p53-defective cancer cells. However, TP53 mutant cancers do not respond consistently to Wee1 inhibitor treatment, indicating the existence of genetic determinants of Wee1 inhibitor sensitivity other than TP53 status. To optimally facilitate patient selection for Wee1 inhibition and uncover potential resistance mechanisms, identification of these currently unknown genes is necessary. The aim of this study was therefore to identify gene mutations that determine Wee1 inhibitor sensitivity. We performed a genome-wide unbiased functional genetic screen in TP53 mutant near-haploid KBM-7 cells using gene-trap insertional mutagenesis. Insertion site mapping of cells that survived long-term Wee1 inhibition revealed enrichment of G1/S regulatory genes, including SKP2, CUL1, and CDK2. Stable depletion of SKP2, CUL1, or CDK2 or chemical Cdk2 inhibition rescued the γ-H2AX induction and abrogation of G2 phase as induced by Wee1 inhibition in breast and ovarian cancer cell lines. Remarkably, live cell imaging showed that depletion of SKP2, CUL1, or CDK2 did not rescue the Wee1 inhibition-induced karyokinesis and cytokinesis defects. These data indicate that the activity of the DNA replication machinery, beyond TP53 mutation status, determines Wee1 inhibitor sensitivity, and could serve as a selection criterion for Wee1-inhibitor eligible patients. Conversely, loss of the identified S-phase genes could serve as a mechanism of acquired resistance, which goes along with development of severe genomic instability.

  20. PTP1B Inhibitors from the Entomogenous Fungi Isaria fumosorosea.

    Science.gov (United States)

    Zhang, Jun; Meng, Lin-Lin; Wei, Jing-Jing; Fan, Peng; Liu, Sha-Sha; Yuan, Wei-Yu; Zhao, You-Xing; Luo, Du-Qiang

    2017-11-24

    Protein tyrosine phosphatase 1B (PTP1B) is implicated as a negative regulator of insulin receptor (IR) signaling and a potential drug target for the treatment of type II diabetes and other associated metabolic syndromes. Thus, small molecule inhibitors of PTP1B can be considered as an attractive approach for the design of new therapeutic agents of type II diabetes and cancer diseases. In a continuing search for new PTP1B inhibitors, a new tetramic acid possessing a rare pyrrolidinedione skeleton named fumosorinone A ( 1 ), together with five known ones 2 - 6 were isolated from the entomogenous fungus Isaria fumosorosea. The structures of 2 - 6 were elucidated by extensive spectroscopic analysis. Fumosorinone A ( 1 ) and beauvericin ( 6 ) showed significant PTP1B inhibitory activity with IC 50 value of 3.24 μM and 0.59 μM.

  1. Tissue inhibitor of metalloproteinase 1 (TIMP-1) as a biomarker in gastric cancer

    DEFF Research Database (Denmark)

    Grunnet, Mie; Mau-Sørensen, Morten; Brünner, Nils

    2013-01-01

    The value of Tissue Inhibitor of MetalloProteinase-1 (TIMP-1) as a biomarker in patients with gastric cancer (GC) is widely debated. The aim of this review is to evaluate available literature describing the association between levels of TIMP-1 in tumor tissue and/or blood and the prognosis...

  2. 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...

  3. Involvement of Prohibitin Upregulation in Abrin-Triggered Apoptosis

    Directory of Open Access Journals (Sweden)

    Yu-Huei Liu

    2012-01-01

    Full Text Available Abrin (ABR, a protein purified from the seeds of Abrus precatorius, induces apoptosis in various types of cancer cells. However, the detailed mechanism remains largely uncharacterized. By using a cDNA microarray platform, we determined that prohibitin (PHB, a tumor suppressor protein, is significantly upregulated in ABR-triggered apoptosis. ABR-induced upregulation of PHB is mediated by the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK pathway, as demonstrated by chemical inhibitors. In addition, ABR significantly induced the expression of Bax as well as the activation of caspase-3 and poly(ADP-ribose polymerase (PARP in Jurkat T cells, whereas the reduction of PHB by specific RNA interference delayed ABR-triggered apoptosis through the proapoptotic genes examined. Moreover, our results also indicated that nuclear translocation of the PHB-p53 complex may play a role in the transcription of Bax. Collectively, our data show that PHB plays a role in ABR-induced apoptosis, which may be helpful for the development of diagnostic or therapeutic agents.

  4. Predicting DPP-IV inhibitors with machine learning approaches

    Science.gov (United States)

    Cai, Jie; Li, Chanjuan; Liu, Zhihong; Du, Jiewen; Ye, Jiming; Gu, Qiong; Xu, Jun

    2017-04-01

    Dipeptidyl peptidase IV (DPP-IV) is a promising Type 2 diabetes mellitus (T2DM) drug target. DPP-IV inhibitors prolong the action of glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP), improve glucose homeostasis without weight gain, edema, and hypoglycemia. However, the marketed DPP-IV inhibitors have adverse effects such as nasopharyngitis, headache, nausea, hypersensitivity, skin reactions and pancreatitis. Therefore, it is still expected for novel DPP-IV inhibitors with minimal adverse effects. The scaffolds of existing DPP-IV inhibitors are structurally diversified. This makes it difficult to build virtual screening models based upon the known DPP-IV inhibitor libraries using conventional QSAR approaches. In this paper, we report a new strategy to predict DPP-IV inhibitors with machine learning approaches involving naïve Bayesian (NB) and recursive partitioning (RP) methods. We built 247 machine learning models based on 1307 known DPP-IV inhibitors with optimized molecular properties and topological fingerprints as descriptors. The overall predictive accuracies of the optimized models were greater than 80%. An external test set, composed of 65 recently reported compounds, was employed to validate the optimized models. The results demonstrated that both NB and RP models have a good predictive ability based on different combinations of descriptors. Twenty "good" and twenty "bad" structural fragments for DPP-IV inhibitors can also be derived from these models for inspiring the new DPP-IV inhibitor scaffold design.

  5. Molecular docking and 3D-QSAR studies on triazolinone and pyridazinone, non-nucleoside inhibitor of HIV-1 reverse transcriptase.

    Science.gov (United States)

    Sivan, Sree Kanth; Manga, Vijjulatha

    2010-06-01

    Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are allosteric inhibitors of the HIV-1 reverse transcriptase. Recently a series of Triazolinone and Pyridazinone were reported as potent inhibitors of HIV-1 wild type reverse transcriptase. In the present study, docking and 3D quantitative structure activity relationship (3D QSAR) studies involving comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed on 31 molecules. Ligands were built and minimized using Tripos force field and applying Gasteiger-Hückel charges. These ligands were docked into protein active site using GLIDE 4.0. The docked poses were analyzed; the best docked poses were selected and aligned. CoMFA and CoMSIA fields were calculated using SYBYL6.9. The molecules were divided into training set and test set, a PLS analysis was performed and QSAR models were generated. The model showed good statistical reliability which is evident from the r2 nv, q2 loo and r2 pred values. The CoMFA model provides the most significant correlation of steric and electrostatic fields with biological activities. The CoMSIA model provides a correlation of steric, electrostatic, acceptor and hydrophobic fields with biological activities. The information rendered by 3D QSAR model initiated us to optimize the lead and design new potential inhibitors.

  6. Metabolic responses in Candida tropicalis to complex inhibitors during xylitol bioconversion.

    Science.gov (United States)

    Wang, Shizeng; Li, Hao; Fan, Xiaoguang; Zhang, Jingkun; Tang, Pingwah; Yuan, Qipeng

    2015-09-01

    During xylitol fermentation, Candida tropicalis is often inhibited by inhibitors in hemicellulose hydrolysate. The mechanisms involved in the metabolic responses to inhibitor stress and the resistances to inhibitors are still not clear. To understand the inhibition mechanisms and the metabolic responses to inhibitors, a GC/MS-based metabolomics approach was performed on C. tropicalis treated with and without complex inhibitors (CI, including furfural, phenol and acetic acid). Partial least squares discriminant analysis was used to determine the metabolic variability between CI-treated groups and control groups, and 25 metabolites were identified as possible entities responsible for the discrimination caused by inhibitors. We found that xylose uptake rate and xylitol oxidation rate were promoted by CI treatment. Metabolomics analysis showed that the flux from xylulose to pentose phosphate pathway increased, and tricarboxylic acid cycle was disturbed by CI. Moreover, the changes in levels of 1,3-propanediol, trehalose, saturated fatty acids and amino acids showed different mechanisms involved in metabolic responses to inhibitor stress. The increase of 1,3-propanediol was considered to be correlated with regulating redox balance and osmoregulation. The increase of trehalose might play a role in protein stabilization and cellular membranes protection. Saturated fatty acids could cause the decrease of membrane fluidity and make the plasma membrane rigid to maintain the integrity of plasma membrane. The deeper understanding of the inhibition mechanisms and the metabolic responses to inhibitors will provide us with more information on the metabolism regulation during xylitol bioconversion and the construction of industrial strains with inhibitor tolerance for better utilization of bioresource. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Gardenia jasminoides Encodes an Inhibitor-2 Protein for Protein Phosphatase Type 1

    Science.gov (United States)

    Gao, Lan; Li, Hao-Ming

    2017-08-01

    Protein phosphatase-1 (PP1) regulates diverse, essential cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling. Inhibitor-2 (I-2) can inhibit the activity of PP1 and has been found in diverse organisms. In this work, a Gardenia jasminoides fruit cDNA library was constructed, and the GjI-2 cDNA was isolated from the cDNA library by sequencing method. The GjI-2 cDNA contains a predicted 543 bp open reading frame that encodes 180 amino acids. The bioinformatics analysis suggested that the GjI-2 has conserved PP1c binding motif, and contains a conserved phosphorylation site, which is important in regulation of its activity. The three-dimensional model structure of GjI-2 was buite, its similar with the structure of I-2 from mouse. The results suggest that GjI-2 has relatively conserved RVxF, FxxR/KxR/K and HYNE motif, and these motifs are involved in interaction with PP1.

  8. The Use of Plasma-Derived Complement C1-Esterase Inhibitor Concentrate (Berinert®) in the Treatment of Angiotensin Converting Enzyme-Inhibitor Related Angioedema

    DEFF Research Database (Denmark)

    Hermanrud, Thorbjørn; Duus, Nicolaj; Bygum, Anette

    2016-01-01

    Angioedema of the upper airways is a severe and potentially life-threatening condition. The incidence has been increasing in the past two decades, primarily due to pharmaceuticals influencing the generation or degradation of the vasoactive molecule bradykinin. Plasma-derived C1-esterase inhibitor...... concentrate is a well-established treatment option of hereditary and acquired complement C1-esterase inhibitor deficiency, which are also mediated by an increased level of bradykinin resulting in recurrent angioedema. We here present a case of severe angiotensin converting enzyme-inhibitor related angioedema...

  9. The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation

    International Nuclear Information System (INIS)

    Bolin, Celeste; Boudra, Mohammed-Tayyib; Fernet, Marie; Vaslin, Laurence; Pennaneach, Vincent; Zaremba, Tomasz; Favaudon, Vincent; Megnin-Chanet, Frederique; Hall, Janet; Biard, Denis; Cordelieres, Fabrice P.

    2012-01-01

    Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5KD cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro irradiated Cdk5KD cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5KD compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5- dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5KD cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5KD cells is due to a role of Cdk5 in other pathways or the altered polymer levels. (authors)

  10. Involvement of hypothalamic cyclooxygenase-2, interleukin-1β and melanocortin in the development of docetaxel-induced anorexia in rats.

    Science.gov (United States)

    Yamamoto, Kouichi; Asano, Keiko; Ito, Yui; Matsukawa, Naoki; Kim, Seikou; Yamatodani, Atsushi

    2012-12-16

    Docetaxel, a taxane derivative, is frequently used for the treatment of advanced breast cancer, non-small cell lung cancer, and metastatic prostate cancer. Clinical reports demonstrated that docetaxel-based chemotherapy often induces anorexia, but the etiology is not completely understood. To elucidate possible mechanisms, we investigated the involvement of central interleukin (IL)-1β, cyclooxygenase (COX)-2, and pro-opiomelanocortin (POMC) in the development of docetaxel-induced anorexia in rats. Rats received docetaxel (10mg/kg, i.p.) with or without pretreatment with selective COX-2 inhibitors, NS-398 (10 and 30 mg/kg, i.g.) or celecoxib (10 and 30 mg/kg, i.g.), and a non-selective COX inhibitor, indomethacin (10mg/kg, i.g.), then food intake was monitored for 24h after administration. We also examined expression of IL-1β, COX-2, and POMC mRNA in hypothalamus of docetaxel-treated rats and the effect of a COX-2 inhibitor on docetaxel-induced POMC mRNA expression. Food consumption in rats was significantly decreased 24h after administration of docetaxel and anorexia was partially reversed by all COX inhibitors. Administration of docetaxel increased IL-1β, COX-2, and POMC mRNA expression in the hypothalamus of rats. The time required to increase these gene expressions was comparable to the latency period of docetaxel-induced anorexia in rats. In addition, pretreatment with COX-2 inhibitors suppressed docetaxel-induced expression of POMC mRNA. These results suggest that IL-1β and COX-2 mRNA expression and subsequent activation of POMC in the hypothalamus may contribute to the development of docetaxel-induced anorexia in rats. Copyright © 2012. Published by Elsevier Ireland Ltd.

  11. A universal genetic testing initiative for patients with high-grade, non-mucinous epithelial ovarian cancer and the implications for cancer treatment.

    Science.gov (United States)

    Bednar, Erica M; Oakley, Holly D; Sun, Charlotte C; Burke, Catherine C; Munsell, Mark F; Westin, Shannon N; Lu, Karen H

    2017-08-01

    Genetic counseling (GC) and germline genetic testing (GT) for BRCA1 and BRCA2 are considered standard of care for patients with high-grade, non-mucinous epithelial ovarian, fallopian tube, and primary peritoneal cancers (HGOC). We describe a universal genetic testing initiative to increase the rates of recommendation and acceptance of GC and GT to >80% for patients with HGOC at our institution. Data from a consecutive cohort of patients seen in our gynecologic oncology clinics between 9/1/2012 and 8/31/2015 for evaluation of HGOC were retrospectively analyzed. Data were abstracted from the tumor registry, medical records, and research databases. Descriptive statistics were used to evaluate patient characteristics and GC, GT, and PARP inhibitor use. Various clinic interventions were developed, influenced by the Plan-Do-Study-Act cycle method, which included physician-coordinated GT, integrated GC, and assisted GC referrals. A cohort of 1636 patients presented to the gynecologic oncology clinics for evaluation of HGOC during our study period, and 1423 (87.0%) were recommended to have GC and GT. Of these, 1214 (85.3%) completed GT and 217 (17.9%) were found to have a BRCA1 or BRCA2 mutation. Among BRCA-positive patients, 167 had recurrent or progressive disease, and 56 of those received PARP inhibitor therapy. The rates of GC and GT recommendation and completion among patients with HGOC at our institution exceeded 80% following the implementation of a universal genetic testing initiative. Universal genetic testing of patients with HGOC is one strategy to identify those who may benefit from PARP inhibitor therapy. Copyright © 2017. Published by Elsevier Inc.

  12. A novel compound derived from danshensu inhibits apoptosis via upregulation of heme oxygenase-1 expression in SH-SY5Y cells.

    Science.gov (United States)

    Pan, Li-Long; Liu, Xin-Hua; Jia, Yao-Ling; Wu, Dan; Xiong, Qing-Hui; Gong, Qi-Hai; Wang, Yang; Zhu, Yi-Zhun

    2013-04-01

    Heme oxygenase-1 (HO-1) has potential anti-apoptotic properties. A novel compound [4-(2-acetoxy-3-((R)-3-(benzylthio)-1-methoxy-1-oxopropan-2- ylamino)-3-oxopropyl)-1,2-phenylene diacetate (DSC)] was synthesized by joining danshensu and cysteine through an appropriate linker. This study investigated if the cytoprotective properties of DSC involved the induction of HO-1. We evaluated the cytoprotective effects of DSC on H2O2-induced cell damage, apoptosis, intracellular and mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm) loss, and apoptosis-related proteins expression and its underlying mechanisms. DSC concentration-dependently attenuated cell death, lactate dehydrogenase release, intracellular and mitochondrial ROS production, and ΔΨm collapse, modulated apoptosis-related proteins (Bcl-2, Bax, caspase-3, p53, and cleaved PARP) expression, and inhibited phosphorylation of extracellular signal-regulated kinase 1/2 in SH-SY5Y cells induced by H2O2. In addition, DSC concentration-dependently induced HO-1 expression associated with nuclear translocation of nuclear factor-erythroid 2 related factor 2 (Nrf-2), while the effect of DSC was inhibited by a phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Furthermore, the protective effect of DSC on H2O2-induced cell death was abolished by HO-1 inhibitor ZnPP, but was mimicked by carbon monoxide-releasing moiety CORM-3 or HO-1 by-product bilirubin. Finally, DSC inhibited H2O2-induced changes of Bcl-2, Bax, and caspase-3 expression, and all of these effects were reversed by HO-1 silencing. Induction of HO-1 may be, at least in part, responsible for the anti-apoptotic property of DSC, an effect that involved the activation of PI3K/Akt/Nrf-2 axis. DSC might have the potential for beneficial therapeutic interventions for neurodegenerative diseases. Copyright © 2013. Published by Elsevier B.V.

  13. Plasma tissue inhibitor of metalloproteinases-1 as a biological marker?

    DEFF Research Database (Denmark)

    Lomholt, Anne F.; Frederiksen, Camilla B.; Christensen, Ib J.

    2007-01-01

    Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) may be a valuable biological marker in Colorectal Cancer (CRC). However, prospective validation of TIMP-1 as a biological marker should include a series of pre-analytical considerations. TIMP-1 is stored in platelets, which may degranulate during...

  14. PTP1B Inhibitors from the Entomogenous Fungi Isaria fumosorosea

    Directory of Open Access Journals (Sweden)

    Jun Zhang

    2017-11-01

    Full Text Available Protein tyrosine phosphatase 1B (PTP1B is implicated as a negative regulator of insulin receptor (IR signaling and a potential drug target for the treatment of type II diabetes and other associated metabolic syndromes. Thus, small molecule inhibitors of PTP1B can be considered as an attractive approach for the design of new therapeutic agents of type II diabetes and cancer diseases. In a continuing search for new PTP1B inhibitors, a new tetramic acid possessing a rare pyrrolidinedione skeleton named fumosorinone A (1, together with five known ones 2–6 were isolated from the entomogenous fungus Isaria fumosorosea. The structures of 2–6 were elucidated by extensive spectroscopic analysis. Fumosorinone A (1 and beauvericin (6 showed significant PTP1B inhibitory activity with IC50 value of 3.24 μM and 0.59 μM.

  15. Inhibitors of histone demethylases

    DEFF Research Database (Denmark)

    Lohse, Brian; Kristensen, Jesper L; Kristensen, Line H

    2011-01-01

    Methylated lysines are important epigenetic marks. The enzymes involved in demethylation have recently been discovered and found to be involved in cancer development and progression. Despite the relative recent discovery of these enzymes a number of inhibitors have already appeared. Most of the i...

  16. Effects of protease inhibitors on radiation transformation in vitro

    International Nuclear Information System (INIS)

    Kennedy, A.R.; Little, J.B.

    1981-01-01

    We have investigated the effects of three protease inhibitors, antipain, leupeptin, and soybean trypsin inhibitor, on the induction of oncogenic transformation in mouse C3H10T 1/2 cells by X-rays. The patterns of inhibition by the three protease inhibitors were different. Antipain was the most effective, having the ability to suppress completely radiation transformation as well as radiation transformation enhanced by the phorbol ester promoting agent 12-O-tetradecanoylphorbol-13-acetate. The fact that antipain could suppress transformation when present for only 1 day following irradiation suggests that an effect on a DNA repair process might be important in its action. Leupeptin was less effective than antipain in its inhibition of radiation transformation. Soybean trypsin inhibitor suppressed only the promotional effects of 12-O-tetradecanoylphorbol-13-acetate on transformation. Our results suggest that there may be more than one protease involved in carcinogenesis

  17. ERK mutations confer resistance to mitogen-activated protein kinase pathway inhibitors.

    Science.gov (United States)

    Goetz, Eva M; Ghandi, Mahmoud; Treacy, Daniel J; Wagle, Nikhil; Garraway, Levi A

    2014-12-01

    The use of targeted therapeutics directed against BRAF(V600)-mutant metastatic melanoma improves progression-free survival in many patients; however, acquired drug resistance remains a major medical challenge. By far, the most common clinical resistance mechanism involves reactivation of the MAPK (RAF/MEK/ERK) pathway by a variety of mechanisms. Thus, targeting ERK itself has emerged as an attractive therapeutic concept, and several ERK inhibitors have entered clinical trials. We sought to preemptively determine mutations in ERK1/2 that confer resistance to either ERK inhibitors or combined RAF/MEK inhibition in BRAF(V600)-mutant melanoma. Using a random mutagenesis screen, we identified multiple point mutations in ERK1 (MAPK3) and ERK2 (MAPK1) that could confer resistance to ERK or RAF/MEK inhibitors. ERK inhibitor-resistant alleles were sensitive to RAF/MEK inhibitors and vice versa, suggesting that the future development of alternating RAF/MEK and ERK inhibitor regimens might help circumvent resistance to these agents. ©2014 American Association for Cancer Research.

  18. Immune checkpoint inhibitors for metastatic bladder cancer.

    Science.gov (United States)

    Massari, Francesco; Di Nunno, Vincenzo; Cubelli, Marta; Santoni, Matteo; Fiorentino, Michelangelo; Montironi, Rodolfo; Cheng, Liang; Lopez-Beltran, Anto; Battelli, Nicola; Ardizzoni, Andrea

    2018-03-01

    Chemotherapy has represented the standard therapy for unresectable or metastatic urothelial carcinoma for more than 20 years. The growing knowledge of the interaction between tumour and immune system has led to the advent of new classes of drugs, the immune-checkpoints inhibitors, which are intended to change the current scenario. To date, immunotherapy is able to improve the overall responses and survival. Moreover, thanks to its safety profile immune-checkpoint inhibitors could be proposed also to patients unfit for standard chemotherapy. No doubts that these agents have started a revolution expected for years, but despite this encouraging results it appears clear that not all subjects respond to these agents and requiring the development of reliable predictive response factors able to isolate patients who can more benefit from these treatments as well as new strategies aimed to improve immunotherapy clinical outcome. In this review we describe the active or ongoing clinical trials involving Programmed Death Ligand 1 (PD-L1), Programmed Death receptor 1 (PD-1) and Cytotoxic-T Lymphocyte Antigen 4 (CTLA 4) inhibitors in urothelial carcinoma focusing our attention on the developing new immune-agents and combination strategies with immune-checkpoint inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Anti-inflammatory effect of a selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor via the stimulation of heme oxygenase-1 in LPS-activated mice and J774.1 murine macrophages

    Directory of Open Access Journals (Sweden)

    Sung Bum Park

    2016-08-01

    Full Text Available 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1 converts inactive cortisone to the active cortisol. 11β-HSD1 may be involved in the resolution of inflammation. In the present study, we investigate the anti-inflammatory effects of 2-(3-benzoyl-4-hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone (KR-66344, a selective 11β-HSD1 inhibitor, in lipopolysaccharide (LPS-activated C57BL/6J mice and macrophages. LPS increased 11β-HSD1 activity and expression in macrophages, which was inhibited by KR-66344. In addition, KR-66344 increased survival rate in LPS treated C57BL/6J mice. HO-1 mRNA expression level was increased by KR-66344, and this effect was reversed by the HO competitive inhibitor, ZnPP, in macrophages. Moreover, ZnPP reversed the suppression of ROS formation and cell death induced by KR-66344. ZnPP also suppressed animal survival rate in LPS plus KR-66344 treated C57BL/6J mice. In the spleen of LPS-treated mice, KR-66344 prevented cell death via suppression of inflammation, followed by inhibition of ROS, iNOS and COX-2 expression. Furthermore, LPS increased NFκB-p65 and MAPK phosphorylation, and these effects were abolished by pretreatment with KR-66344. Taken together, KR-66344 protects against LPS-induced animal death and spleen injury by inhibition of inflammation via induction of HO-1 and inhibition of 11β-HSD1 activity. Thus, we concluded that the selective 11β-HSD1 inhibitor may provide a novel strategy in the prevention/treatment of inflammatory disorders in patients.

  20. Interleukin-1β-induced iNOS expression in human lung carcinoma A549 cells: involvement of STAT and MAPK pathways

    International Nuclear Information System (INIS)

    Ravichandran, Kameswaran; Tyagi, Alpna; Deep, Gagan; Agarwal, Chapla; Agarwal, Rajesh

    2011-01-01

    For understanding of signaling molecules important in lung cancer growth and progression, IL-1β effect was analyzed on iNOS expression and key signaling molecules in human lung carcinoma A549 cells and established the role of specific signaling molecules by using specific chemical inhibitors. IL-1β exposure (10 ng/ml) induced strong iNOS expression in serum starved A549 cells. Detailed molecular analyses showed that IL-1β increased expression of phosphorylated STAT1 (Tyr701 and Ser727) and STAT3 (Tyr705 and Ser727) both in total cell lysates and nuclear lysates. Further, IL-1β exposure strongly activated MAPKs (ERK1/2, JNK1/2 and p38) and Akt as well as increased nuclear levels of NF-κB and HIF-1α in A549 cells. Use of specific chemical inhibitors for JAK1 kinase (piceatannol), JAK2 kinase (AG-490), MEK1/2 (PD98059) and JNK1/2 (SP600125) revealed that IL-1β-induced iNOS expression involved signaling pathways in addition to JAKSTAT and ERK1/2-JNK1/2 activation. Overall, these results suggested that instead of specific pharmacological inhibitors, use of chemopreventive agents with broad spectrum efficacy to inhibit IL-1β-induced signaling cascades and iNOS expression would be a better strategy towards lung cancer prevention and/or treatment. (author)

  1. R-268712, an orally active transforming growth factor-β type I receptor inhibitor, prevents glomerular sclerosis in a Thy1 nephritis model.

    Science.gov (United States)

    Terashima, Hideki; Kato, Mikio; Ebisawa, Masayuki; Kobayashi, Hideki; Suzuki, Kanae; Nezu, Yoshikazu; Sada, Toshio

    2014-07-05

    R-268712 is a novel and specific inhibitor of activin receptor-like kinase 5 (ALK5), a transforming growth factor β (TGF-β) type I receptor. Evaluation of in vitro inhibition indicated that R-268712 is a potent and selective inhibitor of ALK5 with an IC50 of 2.5nM, an approximately 5000-fold more selectivity for ALK5 than p38 mitogen-activated protein kinase (MAPK). Oral administration of R-268712 at doses of 1, 3 and 10mg/kg also inhibited the development of renal fibrosis in a dose-dependent manner in a unilateral ureteral obstruction (UUO) model. Additionally, we evaluated the efficacy of R-268712 in a heminephrectomized anti-Thy1 glomerulonephritis model at doses of 0.3 and 1mg/kg. R-268712 reduced proteinuria and glomerulosclerosis significantly with improvement of renal function. Collectively, these results suggested that R-268712 and other ALK5 inhibitors could suppress glomerulonephritis as well as glomerulosclerosis by an inhibitory mechanism that involves suppression of TGF-β signaling. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Dipicolinic Acid Derivatives as Inhibitors of New Delhi Metallo-β-lactamase-1.

    Science.gov (United States)

    Chen, Allie Y; Thomas, Pei W; Stewart, Alesha C; Bergstrom, Alexander; Cheng, Zishuo; Miller, Callie; Bethel, Christopher R; Marshall, Steven H; Credille, Cy V; Riley, Christopher L; Page, Richard C; Bonomo, Robert A; Crowder, Michael W; Tierney, David L; Fast, Walter; Cohen, Seth M

    2017-09-14

    The efficacy of β-lactam antibiotics is threatened by the emergence and global spread of metallo-β-lactamase (MBL) mediated resistance, specifically New Delhi metallo-β-lactamase-1 (NDM-1). By utilization of fragment-based drug discovery (FBDD), a new class of inhibitors for NDM-1 and two related β-lactamases, IMP-1 and VIM-2, was identified. On the basis of 2,6-dipicolinic acid (DPA), several libraries were synthesized for structure-activity relationship (SAR) analysis. Inhibitor 36 (IC 50 = 80 nM) was identified to be highly selective for MBLs when compared to other Zn(II) metalloenzymes. While DPA displayed a propensity to chelate metal ions from NDM-1, 36 formed a stable NDM-1:Zn(II):inhibitor ternary complex, as demonstrated by 1 H NMR, electron paramagnetic resonance (EPR) spectroscopy, equilibrium dialysis, intrinsic tryptophan fluorescence emission, and UV-vis spectroscopy. When coadministered with 36 (at concentrations nontoxic to mammalian cells), the minimum inhibitory concentrations (MICs) of imipenem against clinical isolates of Eschericia coli and Klebsiella pneumoniae harboring NDM-1 were reduced to susceptible levels.

  3. Carboxamide SIRT1 inhibitors block DBC1 binding via an acetylation-independent mechanism

    Science.gov (United States)

    Hubbard, Basil P; Loh, Christine; Gomes, Ana P; Li, Jun; Lu, Quinn; Doyle, Taylor LG; Disch, Jeremy S; Armour, Sean M; Ellis, James L; Vlasuk, George P; Sinclair, David A

    2013-01-01

    SIRT1 is an NAD+-dependent deacetylase that counteracts multiple disease states associated with aging and may underlie some of the health benefits of calorie restriction. Understanding how SIRT1 is regulated in vivo could therefore lead to new strategies to treat age-related diseases. SIRT1 forms a stable complex with DBC1, an endogenous inhibitor. Little is known regarding the biochemical nature of SIRT1-DBC1 complex formation, how it is regulated and whether or not it is possible to block this interaction pharmacologically. In this study, we show that critical residues within the catalytic core of SIRT1 mediate binding to DBC1 via its N-terminal region, and that several carboxamide SIRT1 inhibitors, including EX-527, can completely block this interaction. We identify two acetylation sites on DBC1 that regulate its ability to bind SIRT1 and suppress its activity. Furthermore, we show that DBC1 itself is a substrate for SIRT1. Surprisingly, the effect of EX-527 on SIRT1-DBC1 binding is independent of DBC1 acetylation. Together, these data show that protein acetylation serves as an endogenous regulatory mechanism for SIRT1-DBC1 binding and illuminate a new path to developing small-molecule modulators of SIRT1. PMID:23892437

  4. Radioprotection of IDH1-Mutated Cancer Cells by the IDH1-Mutant Inhibitor AGI-5198.

    Science.gov (United States)

    Molenaar, Remco J; Botman, Dennis; Smits, Myrthe A; Hira, Vashendriya V; van Lith, Sanne A; Stap, Jan; Henneman, Peter; Khurshed, Mohammed; Lenting, Krissie; Mul, Adri N; Dimitrakopoulou, Dionysia; van Drunen, Cornelis M; Hoebe, Ron A; Radivoyevitch, Tomas; Wilmink, Johanna W; Maciejewski, Jaroslaw P; Vandertop, W Peter; Leenders, William P; Bleeker, Fonnet E; van Noorden, Cornelis J

    2015-11-15

    Isocitrate dehydrogenase 1 (IDH1) is mutated in various types of human cancer to IDH1(R132H), a structural alteration that leads to catalysis of α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate. In this study, we present evidence that small-molecule inhibitors of IDH1(R132H) that are being developed for cancer therapy may pose risks with coadministration of radiotherapy. Cancer cells heterozygous for the IDH1(R132H) mutation exhibited less IDH-mediated production of NADPH, such that after exposure to ionizing radiation (IR), there were higher levels of reactive oxygen species, DNA double-strand breaks, and cell death compared with IDH1 wild-type cells. These effects were reversed by the IDH1(R132H) inhibitor AGI-5198. Exposure of IDH1 wild-type cells to D-2-hydroxyglutarate was sufficient to reduce IDH-mediated NADPH production and increase IR sensitivity. Mechanistic investigations revealed that the radiosensitivity of heterozygous cells was independent of the well-described DNA hypermethylation phenotype in IDH1-mutated cancers. Thus, our results argue that altered oxidative stress responses are a plausible mechanism to understand the radiosensitivity of IDH1-mutated cancer cells. Further, they offer an explanation for the relatively longer survival of patients with IDH1-mutated tumors, and they imply that administration of IDH1(R132H) inhibitors in these patients may limit irradiation efficacy in this setting. ©2015 American Association for Cancer Research.

  5. Erdheim Chester disease with appendicular skeletal, renal and pleural involvement responding to Zelboraf (BRAF inhibitor) treatment: case report

    International Nuclear Information System (INIS)

    Borys, Dariusz; Nystrom, Lucas; Song, Albert; Lomasney, Laurie M.

    2016-01-01

    Erdheim Chester disease is a rare non-Langerhans cell histiocytosis which may involve multiple organs including bone, soft tissue, lungs, cardiovascular system, kidneys (retroperitoneum), skin, and central nervous system. Bone involvement is most common followed by other organs. This case report describes a 58-year-old man who presented with progressive renal dysfunction presumed due to obstruction. The patient failed multiple urinary tract interventions, and clinical course was complicated by recurrent low-grade fevers, and bilateral knee pain. Advanced imaging and histopathological features on bone biopsy were consistent with Erdheim Chester disease. Molecular studies of tissue showed BRAF V600 mutation. This patient was treated with Zelboraf (vemurafenib) BRAF inhibitor with subsequent improvement in renal and pleural dysfunction as well as decreased histiocytic soft tissue masses on CT. (orig.)

  6. Erdheim Chester disease with appendicular skeletal, renal and pleural involvement responding to Zelboraf (BRAF inhibitor) treatment: case report

    Energy Technology Data Exchange (ETDEWEB)

    Borys, Dariusz [Loyola University Medical Center Chicago, Department of Pathology, Maywood, IL (United States); Loyola University Medical Center Chicago, Departmet of Orthopaedic Surgery, Maywood, IL (United States); Loyola University Medical Center, Maywood, IL (United States); Nystrom, Lucas [Loyola University Medical Center Chicago, Departmet of Orthopaedic Surgery, Maywood, IL (United States); Song, Albert [Loyola University Medical Center Chicago, Department of Radiology, Maywood, IL (United States); Lomasney, Laurie M. [Loyola University Medical Center Chicago, Departmet of Orthopaedic Surgery, Maywood, IL (United States); Loyola University Medical Center Chicago, Department of Radiology, Maywood, IL (United States)

    2016-10-15

    Erdheim Chester disease is a rare non-Langerhans cell histiocytosis which may involve multiple organs including bone, soft tissue, lungs, cardiovascular system, kidneys (retroperitoneum), skin, and central nervous system. Bone involvement is most common followed by other organs. This case report describes a 58-year-old man who presented with progressive renal dysfunction presumed due to obstruction. The patient failed multiple urinary tract interventions, and clinical course was complicated by recurrent low-grade fevers, and bilateral knee pain. Advanced imaging and histopathological features on bone biopsy were consistent with Erdheim Chester disease. Molecular studies of tissue showed BRAF V600 mutation. This patient was treated with Zelboraf (vemurafenib) BRAF inhibitor with subsequent improvement in renal and pleural dysfunction as well as decreased histiocytic soft tissue masses on CT. (orig.)

  7. SGLT-2 Inhibitors: Is There a Role in Type 1 Diabetes Mellitus Management?

    Science.gov (United States)

    Ahmed-Sarwar, Nabila; Nagel, Angela K; Leistman, Samantha; Heacock, Kevin

    2017-09-01

    The purpose of this review is to identify and evaluate disease management of patients with type 1 diabetes mellitus (T1DM) who were treated with a sodium-glucose cotransporter 2 (SGLT-2) inhibitor as an adjunct to insulin therapy. A PubMed (1969 to March 2017) and Ovid (1946 to March 2017) search was performed for articles published utilizing the following MESH terms: canagliflozin, empagliflozin, dapagliflozin, type 1 diabetes mellitus, insulin dependent diabetes, insulin, sodium-glucose transporter 2. There were no limitations placed on publication type. All English-language articles were evaluated for association of SGLT-2 inhibitors and type 1 diabetes. Further studies were identified by review of pertinent manuscript bibliographies. All 3 SGLT-2 inhibitors, when combined with insulin, resulted in an overall reduction of hemoglobin A1C (up to 0.49%), lower total daily insulin doses, and a reduction in weight (up to 2.7 kg). The combination therapy of insulin and SGLT-2 inhibitors also resulted in a lower incidence of hypoglycemia. Study duration varied from 2 to 18 weeks. A review of the identified literature indicated that there is a potential role for the combination of SGLT-2 inhibitors with insulin in T1DM for improving glycemic control without increasing the risk of hypoglycemia. The short duration and small sample sizes limit the ability to fully evaluate the incidences of diabetic ketoacidosis and urogenital infections. The risks associated with this combination of medications require further evaluation.

  8. Lack of association between level of Plasminogen Activator Inhibitor-1 and estimates of tumor angiogenesis in early breast cancer

    DEFF Research Database (Denmark)

    Offersen, Birgitte Vrou; Riisbro, Rikke; Knoop, Ann

    2007-01-01

    Plasminogen Activator Inhibitor type-1 (PAI-1) is involved in tumor invasion and progression. High levels of PAI-1 are associated with poor prognosis in breast cancer, and PAI-1 has been shown to play a role in angiogenic processes. Since estimates of tumor angiogenesis may predict poor prognosis...... we studied the relationship between PAI-1 and estimates of angiogenesis in breast cancer. Tumor tissue specimens from 438 breast cancer patients were included. Median follow-up was 10.3 years. Protein levels of PAI-1 were measured using an ELISA. Angiogenesis scores were performed using a Chalkley.......009) were independent markers of death from breast cancer. This study confirms high PAI-1 or high Chalkley counts as markers of poor prognosis in breast cancer patients, and suggests that the prognostic impact of PAI-1 is independent of its supposed involvement in tumor angiogenesis. Udgivelsesdato: 2007...

  9. Identification of human flap endonuclease 1 (FEN1) inhibitors using a machine learning based consensus virtual screening.

    Science.gov (United States)

    Deshmukh, Amit Laxmikant; Chandra, Sharat; Singh, Deependra Kumar; Siddiqi, Mohammad Imran; Banerjee, Dibyendu

    2017-07-25

    Human Flap endonuclease1 (FEN1) is an enzyme that is indispensable for DNA replication and repair processes and inhibition of its Flap cleavage activity results in increased cellular sensitivity to DNA damaging agents (cisplatin, temozolomide, MMS, etc.), with the potential to improve cancer prognosis. Reports of the high expression levels of FEN1 in several cancer cells support the idea that FEN1 inhibitors may target cancer cells with minimum side effects to normal cells. In this study, we used large publicly available, high-throughput screening data of small molecule compounds targeted against FEN1. Two machine learning algorithms, Support Vector Machine (SVM) and Random Forest (RF), were utilized to generate four classification models from huge PubChem bioassay data containing probable FEN1 inhibitors and non-inhibitors. We also investigated the influence of randomly selected Zinc-database compounds as negative data on the outcome of classification modelling. The results show that the SVM model with inactive compounds was superior to RF with Matthews's correlation coefficient (MCC) of 0.67 for the test set. A Maybridge database containing approximately 53 000 compounds was screened and top ranking 5 compounds were selected for enzyme and cell-based in vitro screening. The compound JFD00950 was identified as a novel FEN1 inhibitor with in vitro inhibition of flap cleavage activity as well as cytotoxic activity against a colon cancer cell line, DLD-1.

  10. Dual mTORC1/C2 inhibitors suppress cellular geroconversion (a senescence program).

    Science.gov (United States)

    Leontieva, Olga V; Demidenko, Zoya N; Blagosklonny, Mikhail V

    2015-09-15

    In proliferating cells, mTOR is active and promotes cell growth. When the cell cycle is arrested, then mTOR converts reversible arrest to senescence (geroconversion). Rapamycin and other rapalogs suppress geroconversion, maintaining quiescence instead. Here we showed that ATP-competitive kinase inhibitors (Torin1 and PP242), which inhibit both mTORC1 and TORC2, also suppressed geroconversion. Despite inhibition of proliferation (in proliferating cells), mTOR inhibitors preserved re-proliferative potential (RP) in arrested cells. In p21-arrested cells, Torin 1 and PP242 detectably suppressed geroconversion at concentrations as low as 1-3 nM and 10-30 nM, reaching maximal gerosuppression at 30 nM and 300 nM, respectively. Near-maximal gerosuppression coincided with inhibition of p-S6K(T389) and p-S6(S235/236). Dual mTOR inhibitors prevented senescent morphology and hypertrophy. Our study warrants investigation into whether low doses of dual mTOR inhibitors will prolong animal life span and delay age-related diseases. A new class of potential anti-aging drugs can be envisioned.

  11. Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair.

    Science.gov (United States)

    Robu, Mihaela; Shah, Rashmi G; Purohit, Nupur K; Zhou, Pengbo; Naegeli, Hanspeter; Shah, Girish M

    2017-08-15

    Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV-DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1-XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV-DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins.

  12. Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication.

    Science.gov (United States)

    Thenin-Houssier, Suzie; de Vera, Ian Mitchelle S; Pedro-Rosa, Laura; Brady, Angela; Richard, Audrey; Konnick, Briana; Opp, Silvana; Buffone, Cindy; Fuhrmann, Jakob; Kota, Smitha; Billack, Blase; Pietka-Ottlik, Magdalena; Tellinghuisen, Timothy; Choe, Hyeryun; Spicer, Timothy; Scampavia, Louis; Diaz-Griffero, Felipe; Kojetin, Douglas J; Valente, Susana T

    2016-04-01

    The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  13. Combination of HDAC inhibitor TSA and silibinin induces cell cycle arrest and apoptosis by targeting survivin and cyclinB1/Cdk1 in pancreatic cancer cells.

    Science.gov (United States)

    Feng, Wan; Cai, Dawei; Zhang, Bin; Lou, Guochun; Zou, Xiaoping

    2015-08-01

    Histone deacetylases (HDAC) are involved in diverse biological processes and therefore emerge as potential targets for pancreatic cancer. Silibinin, an active component of silymarin, is known to inhibit growth of pancreatic cancer in vivo and in vitro. Herein, we examined the cytotoxic effects of TSA in combination with silibinin and investigated the possible mechanism in two pancreatic cancer cell lines (Panc1 and Capan2). Our study found that combination treatment of HDAC inhibitor and silibinin exerted additive growth inhibitory effect on pancreatic cancer cell. Annexin V-FITC/PI staining and flow cytometry analysis demonstrated that combination therapy induced G2/M cell cycle arrest and apoptosis in Panc1and Capan2 cells. The induction of apoptosis was further confirmed by evaluating the activation of caspases. Moreover, treatment with TSA and silibinin resulted in a profound reduction in the expression of cyclinA2, cyclinB1/Cdk1 and survivin. Taken together, our study might indicate that the novel combination of HDAC inhibitor and silibinin could offer therapeutic potential against pancreatic cancer. Copyright © 2015. Published by Elsevier Masson SAS.

  14. Role of EGFR transactivation in preventing apoptosis in Pseudomonas aeruginosa-infected human corneal epithelial cells.

    Science.gov (United States)

    Zhang, Jing; Li, Hui; Wang, Jinzhao; Dong, Zheng; Mian, Shahzad; Yu, Fu-Shin X

    2004-08-01

    To determine the role of epidermal growth factor (EGF) receptor (EGFR)-mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa-infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase-mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis. Bacterial infection of HCECs induces

  15. Role of EGFR Transactivation in Preventing Apoptosis in Pseudomonas aeruginosa–Infected Human Corneal Epithelial Cells

    Science.gov (United States)

    Zhang, Jing; Li, Hui; Wang, Jinzhao; Dong, Zheng; Mian, Shahzad; Yu, Fu-Shin X.

    2009-01-01

    PURPOSE To determine the role of epidermal growth factor (EGF) receptor (EGFR)–mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). METHODS Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. RESULTS P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa–infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase–mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis

  16. 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

  17. 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.

  18. Insights into the molecular evolution of peptidase inhibitors in arthropods.

    Science.gov (United States)

    Alonso, Joaquin; Martinez, Manuel

    2017-01-01

    Peptidase inhibitors are key proteins involved in the control of peptidases. In arthropods, peptidase inhibitors modulate the activity of peptidases involved in endogenous physiological processes and peptidases of the organisms with which they interact. Exploring available arthropod genomic sequences is a powerful way to obtain the repertoire of peptidase inhibitors in every arthropod species and to understand the evolutionary mechanisms involved in the diversification of this kind of proteins. A genomic comparative analysis of peptidase inhibitors in species belonging to different arthropod taxonomic groups was performed. The results point out: i) species or clade-specific presence is shown for several families of peptidase inhibitors; ii) multidomain peptidase inhibitors are commonly found in many peptidase inhibitor families; iii) several families have a wide range of members in different arthropod species; iv) several peptidase inhibitor families show species-specific (or clade-specific) gene family expansions; v) functional divergence may be assumed for particular clades; vi) passive expansions may be used by natural selection to fix adaptations. In conclusion, conservation and divergence of duplicated genes and the potential recruitment as peptidase inhibitors of proteins from other families are the main mechanisms used by arthropods to fix diversity. This diversity would be associated to the control of target peptidases and, as consequence, to adapt to specific environments.

  19. Bullatacin Triggered ABCB1-Overexpressing Cell Apoptosis via the Mitochondrial-Dependent Pathway

    Directory of Open Access Journals (Sweden)

    Yong-Ju Liang

    2009-01-01

    Full Text Available This paper was to explore bullatacin-mediated multidrug-resistant cell apoptosis at extremely low concentration. To investigate its precise mechanisms, the pathway of cell apoptosis induced by bullatacin was examined. Bullatacin causes an upregulation of ROS and a downregulation of ΔΨm in a concentration-dependent manner in ABCB1-overexpressing KBv200 cells. In addition, cleavers of caspase-9, caspase-3, and PARP were observed following the release of cytochrome c from mitochondria after bullatacin treatment. However, neither cleavage of caspase-8 nor change of expression level of bcl-2, bax and Fas was observed by the same treatment. Pretreating KBv200 cells with N-acetylcysteine, an antioxidant modulator, resulted in a significant reduction of ROS generation and cell apoptosis induced by bullatacin. Bullatacin-induced apoptosis was antagonized by z-LEHD-fmk, a caspase-9 inhibitor, but not by z-IETD-fmk, a caspase-8 inhibitor. These implied that apoptosis of KBv200 cells induced by bullatacin was associated with the mitochondria-dependent pathway that was limited to activation of apical caspase-9.

  20. Increased DNA damage in progression of COPD: a response by poly(ADP-ribose polymerase-1.

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    Ingrid Oit-Wiscombe

    Full Text Available Chronic oxidative stress (OS, a major mechanism of chronic obstructive pulmonary disease (COPD, may cause significant damage to DNA. Poly(ADP-ribose polymerase (PARP-1 is rapidly activated by OS-induced DNA lesions. However, the degree of DNA damage along with the evolution of COPD is unclear. In peripheral blood mononuclear cells of non-smoking individuals, non-obstructive smokers, patients with COPD of all stages and those with COPD exacerbation, we evaluated DNA damage, PARP activity and PARP-1 mRNA expression using Comet Assay IV, biotinylated-NAD incorporation assay and qRT-PCR, respectively and subjected results to ordinal logistic regression modelling. Adjusted for demographics, smoking-related parameters and lung function, novel comet parameters, tail length/cell length ratio and tail migration/cell length ratio, showed the greatest increase along the study groups corresponding to the evolution of COPD [odds ratio (OR 7.88, 95% CI 4.26-14.57; p<0.001 and OR 3.91, 95% CI 2.69-5.66; p<0.001, respectively]. Analogously, PARP activity increased significantly over the groups (OR = 1.01; 95%; p<0.001. An antioxidant tetrapeptide UPF17 significantly reduced the PARP-1 mRNA expression in COPD, compared to that in non-obstructive individuals (p = 0.040. Tail length/cell length and tail migration/cell length ratios provide novel progression-sensitive tools for assessment of DNA damage. However, it remains to be elucidated whether inhibition of an elevated PARP-1 activity has a safe enough potential to break the vicious cycle of the development and progression of COPD.

  1. Finding Potent Sirt Inhibitor in Coffee: Isolation, Confirmation and Synthesis of Javamide-II (N-Caffeoyltryptophan as Sirt1/2 Inhibitor.

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    Jae B Park

    Full Text Available Recent studies suggest that Sirt inhibition may have beneficial effects on several human diseases such as neurodegenerative diseases and cancer. Coffee is one of most popular beverages with several positive health effects. Therefore, in this paper, potential Sirt inhibitors were screened using coffee extract. First, HPLC was utilized to fractionate coffee extract, then screened using a Sirt1/2 inhibition assay. The screening led to the isolation of a potent Sirt1/2 inhibitor, whose structure was determined as javamide-II (N-caffeoyltryptophan by NMR. For confirmation, the amide was chemically synthesized and its capacity of inhibiting Sirt1/2 was also compared with the isolated amide. Javamide-II inhibited Sirt2 (IC50; 8.7 μM better than Sirt1(IC50; 34μM. Since javamide-II is a stronger inhibitor for Sirt2 than Sirt1. The kinetic study was performed against Sirt2. The amide exhibited noncompetitive Sirt2 inhibition against the NAD+ (Ki = 9.8 μM and showed competitive inhibition against the peptide substrate (Ki = 5.3 μM. Also, a docking simulation showed stronger binding pose of javamide-II to Sirt2 than AGK2. In cellular levels, javamide-II was able to increase the acetylation of total lysine, cortactin and histone H3 in neuronal NG108-15 cells. In the same cells, the amide also increased the acetylation of lysine (K382 in p53, but not (K305. This study suggests that Javamide-II found in coffee may be a potent Sirt1/2 inhibitor, probably with potential use in some conditions of human diseases.

  2. Penta-acetyl geniposide-induced apoptosis involving transcription of NGF/p75 via MAPK-mediated AP-1 activation in C6 glioma cells

    International Nuclear Information System (INIS)

    Peng, C.-H.; Huang, C.-N.; Hsu, S.-P.; Wang, C.-J.

    2007-01-01

    We have demonstrated the herbal derivative penta-acetyl geniposide ((Ac) 5 GP) induces C6 glioma cell apoptosis through the critical sphingomyelinase (SMase)/nerve growth factor (NGF)/p75 and its downstream signals. It has been reported mitogen-activated protein kinase (MAPK) mediates NGF synthesis induced by SMase activation. In this study, ERK, p38 and JNK are shown to mediate (Ac) 5 GP-induced glioma cell apoptosis and elevation of NGF and p75. Treatment of PD98059 (ERK-specific inhibitor), SB203580 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) decreases the elevation of NGF and p75 mRNA induced by (Ac) 5 GP, indicating possible transcription regulation via MAPKs. The results of nuclear extract blotting and EMSA further confirm (Ac) 5 GP maximally increases AP-1 and NF-κB DNA binding at 6 h. Inhibition of ERK, p38 and JNK block the activation of AP-1 and NF-κB, suggesting these MAPKs are involved in (Ac) 5 GP-induced transcription regulation. We thereby used RT-PCR to analyze cells treated with (Ac) 5 GP, with or without AP-1 or NF-κB inhibitors. AP-1 inhibitor NDGA decreases NGF/p75 and expression of FasL and caspase 3 induced by (Ac) 5 GP, suggesting the importance of AP-1 in mediating NGF/p75 and their downstream apoptotic signals. However, FasL and caspase 3 do not change with the NF-κB inhibitor PDTC; NF-κB might be linked to other cellular events. Overall, we demonstrate that MAPK mediates (Ac) 5 GP-induced activation of AP-1, promoting the transcription of NGF/p75 and downstream apoptotic signals. These results further highlight the potential therapeutic effects of (Ac) 5 GP in chemoprevention or as an anti-tumor agent

  3. 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.

  4. Valproic Acid as a Potential Inhibitor of Plasmodium falciparum Histone Deacetylase 1 (PfHDAC1: An in Silico Approach

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    Mohamed A. Abdallah Elbadawi

    2015-02-01

    Full Text Available A new Plasmodium falciparum histone deacetylase1 (PfHDAC1 homology model was built based on the highest sequence identity available template human histone deacetylase 2 structure. The generated model was carefully evaluated for stereochemical accuracy, folding correctness and overall structure quality. All evaluations were acceptable and consistent. Docking a group of hydroxamic acid histone deacetylase inhibitors and valproic acid has shown binding poses that agree well with inhibitor-bound histone deacetylase-solved structural interactions. Docking affinity dG scores were in agreement with available experimental binding affinities. Further, enzyme-ligand complex stability and reliability were investigated by running 5-nanosecond molecular dynamics simulations. Thorough analysis of the simulation trajectories has shown that enzyme-ligand complexes were stable during the simulation period. Interestingly, the calculated theoretical binding energies of the docked hydroxamic acid inhibitors have shown that the model can discriminate between strong and weaker inhibitors and agrees well with the experimental affinities reported in the literature. The model and the docking methodology can be used in screening virtual libraries for PfHDAC1 inhibitors, since the docking scores have ranked ligands in accordance with experimental binding affinities. Valproic acid calculated theoretical binding energy suggests that it may inhibit PfHDAC1.

  5. Drugs against avian influenza a virus: design of novel sulfonate inhibitors of neuraminidase N1.

    Science.gov (United States)

    Udommaneethanakit, Thanyarat; Rungrotmongkol, Thanyada; Frecer, Vladimir; Seneci, Pierfausto; Miertus, Stanislav; Bren, Urban

    2014-01-01

    The outbreak of avian influenza A (H5N1) virus has raised a global concern for both the animal as well as human health. Besides vaccination, that may not achieve full protection in certain groups of patients, inhibiting neuraminidase or the transmembrane protein M2 represents the main measure of controlling the disease. Due to alarming emergence of influenza virus strains resistant to the currently available drugs, development of new neuraminidase N1 inhibitors is of utmost importance. The present paper provides an overview of the recent advances in the design of new antiviral drugs against avian influenza. It also reports findings in binding free energy calculations for nine neuraminidase N1 inhibitors (oseltamivir, zanamivir, and peramivir -carboxylate, -phosphonate, and -sulfonate) using the Linear Interaction Energy method. Molecular dynamics simulations of these inhibitors were performed in a free and two bound states - the so called open and closed conformations of neuraminidase N1. Obtained results successfully reproduce the experimental binding affinities of the already known neuraminidase N1 inhibitors, i.e. peramivir being a stronger binder than zanamivir that is in turn stronger binder than oseltamivir, or phosphonate inhibitors being stronger binders than their carboxylate analogues. In addition, the newly proposed sulfonate inhibitors are predicted to be the strongest binders - a fact to be confirmed by their chemical synthesis and a subsequent test of their biological activity. Finally, contributions of individual inhibitor moieties to the overall binding affinity are explicitly evaluated to assist further drug development towards inhibition of the H5N1 avian influenza A virus.

  6. Tackling Cancer Resistance by Immunotherapy: Updated Clinical Impact and Safety of PD-1/PD-L1 Inhibitors

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    Shifaa M. Abdin

    2018-01-01

    Full Text Available Cancer therapy has been constantly evolving with the hope of finding the most effective agents with the least toxic effects to eradicate tumors. Cancer immunotherapy is currently among the most promising options, fulfilling this hope in a wide range of tumors. Immunotherapy aims to activate immunity to fight cancer in a very specific and targeted manner; however, some abnormal immune reactions known as immune-related adverse events (IRAEs might occur. Therefore, many researchers are aiming to define the most proper protocols for managing these complications without interfering with the anticancer effect. One of these targeted approaches is the inhibition of the interaction between the checkpoint protein, programmed death-receptor 1 (PD-1, and its ligand, programmed death-ligand 1 (PD-L1, via a class of antibodies known as PD-1/PD-L1 inhibitors. These antibodies achieved prodigious success in a wide range of malignancies, including those where optimal treatment is not yet fully identified. In this review, we have critically explored and discussed the outcome of the latest PD-1 and PD-L1 inhibitor studies in different malignancies compared to standard chemotherapeutic alternatives with a special focus on the clinical efficacy and safety. The approval of the clinical applications of nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab in the last few years clearly highlights the hopeful future of PD-1/PD-L1 inhibitors for cancer patients. These promising results of PD-1/PD-L1 inhibitors have encouraged many ongoing preclinical and clinical trials to explore the extent of antitumor activity, clinical efficacy and safety as well as to extend their applications.

  7. Tackling Cancer Resistance by Immunotherapy: Updated Clinical Impact and Safety of PD-1/PD-L1 Inhibitors.

    Science.gov (United States)

    Abdin, Shifaa M; Zaher, Dana M; Arafa, El-Shaimaa A; Omar, Hany A

    2018-01-25

    Cancer therapy has been constantly evolving with the hope of finding the most effective agents with the least toxic effects to eradicate tumors. Cancer immunotherapy is currently among the most promising options, fulfilling this hope in a wide range of tumors. Immunotherapy aims to activate immunity to fight cancer in a very specific and targeted manner; however, some abnormal immune reactions known as immune-related adverse events (IRAEs) might occur. Therefore, many researchers are aiming to define the most proper protocols for managing these complications without interfering with the anticancer effect. One of these targeted approaches is the inhibition of the interaction between the checkpoint protein, programmed death-receptor 1 (PD-1), and its ligand, programmed death-ligand 1 (PD-L1), via a class of antibodies known as PD-1/PD-L1 inhibitors. These antibodies achieved prodigious success in a wide range of malignancies, including those where optimal treatment is not yet fully identified. In this review, we have critically explored and discussed the outcome of the latest PD-1 and PD-L1 inhibitor studies in different malignancies compared to standard chemotherapeutic alternatives with a special focus on the clinical efficacy and safety. The approval of the clinical applications of nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab in the last few years clearly highlights the hopeful future of PD-1/PD-L1 inhibitors for cancer patients. These promising results of PD-1/PD-L1 inhibitors have encouraged many ongoing preclinical and clinical trials to explore the extent of antitumor activity, clinical efficacy and safety as well as to extend their applications.

  8. Induction of intrachromosomal homologous recombination in whole plants

    International Nuclear Information System (INIS)

    Puchta, H.; Swoboda, P.; Hohn, B.

    1995-01-01

    The influence of different factors on frequencies of intrachromosomal homologous recombination in whole Arabidopsis thaliana and tobacco plants was analyzed using a disrupted β-glucuronidase marker gene. Recombination frequencies were enhanced several fold by DNA damaging agents like UV-light or MMS (methyl methanesulfonate). Applying 3-methoxybenzamide (3-MB), an inhibitor of poly(ADP)ribose polymerase (PARP), an enzyme that is postulated to be involved in DNA repair, enhanced homologous recombination frequencies strongly. These findings indicate that homologous recombination is involved in DNA repair and can (at least partially) compensate for other DNA repair pathways. Indications that recombination in plants can be induced by environmental stress factors that are not likely to be involved in DNA metabolism were also found; Arabidopsis plants growing in a medium containing 0.1 M NaCl exhibited elevated recombination frequencies. The possible general effects of ‘environmental’ challenges on genome flexibility are discussed. (author)

  9. VERO cells harbor a poly-ADP-ribose belt partnering their epithelial adhesion belt

    Directory of Open Access Journals (Sweden)

    Laura Lafon-Hughes

    2014-10-01

    Full Text Available Poly-ADP-ribose (PAR is a polymer of up to 400 ADP-ribose units synthesized by poly-ADP-ribose-polymerases (PARPs and degraded by poly-ADP-ribose-glycohydrolase (PARG. Nuclear PAR modulates chromatin compaction, affecting nuclear functions (gene expression, DNA repair. Diverse defined PARP cytoplasmic allocation patterns contrast with the yet still imprecise PAR distribution and still unclear functions. Based on previous evidence from other models, we hypothesized that PAR could be present in epithelial cells where cadherin-based adherens junctions are linked with the actin cytoskeleton (constituting the adhesion belt. In the present work, we have examined through immunofluorescence and confocal microscopy, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO. PAR was distinguished colocalizing with actin and vinculin in the epithelial belt, a location that has not been previously reported. Actin filaments disruption with cytochalasin D was paralleled by PAR belt disruption. Conversely, PARP inhibitors 3-aminobenzamide, PJ34 or XAV 939, affected PAR belt synthesis, actin distribution, cell shape and adhesion. Extracellular calcium chelation displayed similar effects. Our results demonstrate the existence of PAR in a novel subcellular localization. An initial interpretation of all the available evidence points towards TNKS-1 as the most probable PAR belt architect, although TNKS-2 involvement cannot be discarded. Forthcoming research will test this hypothesis as well as explore the existence of the PAR belt in other epithelial cells and deepen into its functional implications.

  10. Tissue inhibitor of metalloproteinase-2 (TIMP-2) regulates myogenesis and β1 integrin expression in vitro

    International Nuclear Information System (INIS)

    Lluri, Gentian; Langlois, Garret D.; Soloway, Paul D.; Jaworski, Diane M.

    2008-01-01

    Myogenesis in vitro involves myoblast cell cycle arrest, migration, and fusion to form multinucleated myotubes. Extracellular matrix (ECM) integrity during these processes is maintained by the opposing actions of matrix metalloproteinase (MMP) proteases and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs). Here, we report that TIMP-2, MMP-2, and MT1-MMP are differentially expressed during mouse myoblast differentiation in vitro. A specific role for TIMP-2 in myogenesis is demonstrated by altered TIMP-2 -/- myotube formation. When differentiated in horse serum-containing medium, TIMP-2 -/- myotubes are larger than wild-type myotubes. In contrast, when serum-free medium is used, TIMP-2 -/- myotubes are smaller than wild-type myotubes. Regardless of culture condition, myotube size is directly correlated with MMP activity and inversely correlated with β1 integrin expression. Treatment with recombinant TIMP-2 rescues reduced TIMP-2 -/- myotube size and induces increased MMP-9 activation and decreased β1 integrin expression. Treatment with either MMP-2 or MMP-9 similarly rescues reduced myotube size, but has no effect on β1 integrin expression. These data suggest a specific regulatory relationship between TIMP-2 and β1 integrin during myogenesis. Elucidating the role of TIMP-2 in myogenesis in vitro may lead to new therapeutic options for the use of TIMP-2 in myopathies and muscular dystrophies in vivo

  11. IGF-1 and PDGF-bb Suppress IL-1β-Induced Cartilage Degradation through Down-Regulation of NF-κB Signaling: Involvement of Src/PI-3K/AKT Pathway

    Science.gov (United States)

    Mobasheri, Ali; Buhrmann, Constanze; Aldinger, Constance; Rad, Jafar Soleimani; Shakibaei, Mehdi

    2011-01-01

    Objective Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that plays a key role in the pathogenesis of osteoarthritis (OA). Growth factors (GFs) capable of antagonizing the catabolic actions of cytokines may have therapeutic potential in the treatment of OA. Herein, we investigated the potential synergistic effects of insulin-like growth factor (IGF-1) and platelet-derived growth factor (PDGF-bb) on different mechanisms participating in IL-1β-induced activation of nuclear transcription factor-κB (NF-κB) and apoptosis in chondrocytes. Methods Primary chondrocytes were treated with IL-1β to induce dedifferentiation and co-treated with either IGF-1 or/and PDGF-bb and evaluated by immunoblotting and electron microscopy. Results Pretreatment of chondrocytes with IGF-1 or/and PDGF-bb suppressed IL-1β-induced NF-κB activation via inhibition of IκB-α kinase. Inhibition of IκB-α kinase by GFs led to the suppression of IκB-α phosphorylation and degradation, p65 nuclear translocation and NF-κB-regulated gene products involved in inflammation and cartilage degradation (COX-2, MMPs) and apoptosis (caspase-3). GFs or BMS-345541 (specific inhibitor of the IKK) reversed the IL-1β-induced down-regulation of collagen type II, cartilage specific proteoglycans, β1-integrin, Shc, activated MAPKinase, Sox-9 and up-regulation of active caspase-3. Furthermore, the inhibitory effects of IGF-1 or/and PDGF-bb on IL-1β-induced NF-κB activation were sensitive to inhibitors of Src (PP1), PI-3K (wortmannin) and Akt (SH-5), suggesting that the pathway consisting of non-receptor tyrosine kinase (Src), phosphatidylinositol 3-kinase and protein kinase B must be involved in IL-1β signaling. Conclusion The results presented suggest that IGF-1 and PDGF-bb are potent inhibitors of IL-1β-mediated activation of NF-κB and apoptosis in chondrocytes, may be mediated in part through suppression of Src/PI-3K/AKT pathway, which may contribute to their anti-inflammatory effects. PMID

  12. Subnanomolar Inhibitor of Cytochrome bc1 Complex Designed via Optimizing Interaction with Conformationally Flexible Residues

    Science.gov (United States)

    Zhao, Pei-Liang; Wang, Le; Zhu, Xiao-Lei; Huang, Xiaoqin; Zhan, Chang-Guo; Wu, Jia-Wei; Yang, Guang-Fu

    2009-01-01

    Cytochrome bc1 complex (EC 1.10.2.2, bc1), an essential component of the cellular respiratory chain and the photosynthetic apparatus in photosynthetic bacteria, has been identified as a promising target for new drugs and agricultural fungicides. X-ray diffraction structures of the free bc1 complex and its complexes with various inhibitors revealed that the phenyl group of Phe274 in the binding pocket exhibited significant conformational flexibility upon different inhibitors binding to optimize respective π-π interactions, whereas the side chains of other hydrophobic residues showed conformational stability. Therefore, in the present study, a strategy of optimizing the π-π interaction with conformationally flexible residues was proposed to design and discover new bc1 inhibitors with a higher potency. Eight new compounds were designed and synthesized, among which compound 5c with a Ki value of 570 pM was identified as the most promising drug or fungicide candidate, significantly more potent than the commercially available bc1 inhibitors including azoxystrobin (AZ), kresoxim-methyl (KM), and pyraclostrobin (PY). To our knowledge, this is the first bc1 inhibitor discovered from structure-based design with a potency of subnanomolar Ki value. For all of the compounds synthesized and assayed, the calculated binding free energies correlated reasonably well with the binding free energies derived from the experimental Ki values with a correlation coefficient of r2 = 0.89. The further inhibitory kinetics studies revealed that compound 5c is a non-competitive inhibitor with respect to substrate cytochrome c, but is a competitive inhibitor with respect to substrate ubiquinol. Due to its subnanomolar Ki potency and slow dissociation rate constant (k−0 = 0.00358 s−1), compound 5c could be used as a specific probe for further elucidation of the mechanism of bc1 function and as a new lead compound for future drug discovery. PMID:19928849

  13. Recent Advances of Colony-Stimulating Factor-1 Receptor (CSF-1R) Kinase and Its Inhibitors.

    Science.gov (United States)

    El-Gamal, Mohammed I; Al-Ameen, Shahad K; Al-Koumi, Dania M; Hamad, Mawadda G; Jalal, Nouran A; Oh, Chang-Hyun

    2018-01-17

    Colony stimulation factor-1 receptor (CSF-1R), which is also known as FMS kinase, plays an important role in initiating inflammatory, cancer, and bone disorders when it is overstimulated by its ligand, CSF-1. Innate immunity, as well as macrophage differentiation and survival, are regulated by the stimulation of the CSF-1R. Another ligand, interlukin-34 (IL-34), was recently reported to activate the CSF-1R receptor in a different manner. The relationship between CSF-1R and microglia has been reviewed. Both CSF-1 antibodies and small molecule CSF-1R kinase inhibitors have now been tested in animal models and in humans. In this Perspective, we discuss the role of CSF-1 and IL-34 in producing cancer, bone disorders, and inflammation. We also review the newly discovered and improved small molecule kinase inhibitors and monoclonal antibodies that have shown potent activity toward CSF-1R, reported from 2012 until 2017.

  14. A historical sketch of the discovery and development of HIV-1 integrase inhibitors.

    Science.gov (United States)

    Savarino, Andrea

    2006-12-01

    The long process of HIV-1 integrase inhibitor discovery and development can be attributed to both the complexity of HIV-1 integration and poor 'integration' of these researches into mainstream investigations on antiretroviral therapy in the mid-1990s. Of note, some fungal extracts investigated during this period contain the beta-hydroxyketo group, later recognised to be a key structural requirement for keto-enol acids (also referred to as diketo acids) and other integrase inhibitors. This review reconstructs (in the general context of the history of AIDS research) the principal steps that led to the integrase inhibitors currently in clinical trials, and discusses possible future directions.

  15. Efficacy of ATR inhibitors as single agents in Ewing sarcoma

    DEFF Research Database (Denmark)

    Nieto-Soler, Maria; Morgado-Palacin, Isabel; Lafarga, Vanesa

    2016-01-01

    Ewing sarcomas (ES) are pediatric bone tumors that arise from a driver translocation, most frequently EWS/FLI1. Current ES treatment involves DNA damaging agents, yet the basis for the sensitivity to these therapies remains unknown. Oncogene-induced replication stress (RS) is a known source...... efficacy in ES xenografts as single agents. Expression of EWS/FLI1 or EWS/ERG oncogenic translocations sensitizes non-ES cells to ATR inhibitors. Our data shed light onto the sensitivity of ES to genotoxic agents, and identify ATR inhibitors as a potential therapy for Ewing Sarcomas....

  16. 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.

  17. Inhibition of herpes simplex virus type 1 entry by chloride channel inhibitors tamoxifen and NPPB

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Kai [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of Life Science and Technology, Jinan University, Guangzhou (China); Chen, Maoyun [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of pharmacy, Jinan University, Guangzhou (China); Xiang, Yangfei; Ma, Kaiqi [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); Jin, Fujun [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of pharmacy, Jinan University, Guangzhou (China); Wang, Xiao [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Wang, Xiaoyan; Wang, Shaoxiang [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); Wang, Yifei, E-mail: twang-yf@163.com [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China)

    2014-04-18

    Highlights: • We analyze the anti-HSV potential of chloride channel inhibitors. • Tamoxifen and NPPB show anti-HSV-1 and anti-ACV-resistant HSV-1 activities. • HSV-1 infection induces intracellular chloride concentration increasing. • Tamoxifen and NPPB inhibit HSV-1 early infection. • Tamoxifen and NPPB prevent the fusion process of HSV-1. - Abstract: Herpes simplex virus type 1 (HSV-1) infection is very common worldwide and can cause significant health problems from periodic skin and corneal lesions to encephalitis. Appearance of drug-resistant viruses in clinical therapy has made exploring novel antiviral agents emergent. Here we show that chloride channel inhibitors, including tamoxifen and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB), exhibited extensive antiviral activities toward HSV-1 and ACV-resistant HSV viruses. HSV-1 infection induced chloride ion influx while treatment with inhibitors reduced the increase of intracellular chloride ion concentration. Pretreatment or treatment of inhibitors at different time points during HSV-1 infection all suppressed viral RNA synthesis, protein expression and virus production. More detailed studies demonstrated that tamoxifen and NPPB acted as potent inhibitors of HSV-1 early entry step by preventing viral binding, penetration and nuclear translocation. Specifically the compounds appeared to affect viral fusion process by inhibiting virus binding to lipid rafts and interrupting calcium homeostasis. Taken together, the observation that tamoxifen and NPPB can block viral entry suggests a stronger potential for these compounds as well as other ion channel inhibitors in antiviral therapy against HSV-1, especially the compound tamoxifen is an immediately actionable drug that can be reused for treatment of HSV-1 infections.

  18. Inhibition of herpes simplex virus type 1 entry by chloride channel inhibitors tamoxifen and NPPB

    International Nuclear Information System (INIS)

    Zheng, Kai; Chen, Maoyun; Xiang, Yangfei; Ma, Kaiqi; Jin, Fujun; Wang, Xiao; Wang, Xiaoyan; Wang, Shaoxiang; Wang, Yifei

    2014-01-01

    Highlights: • We analyze the anti-HSV potential of chloride channel inhibitors. • Tamoxifen and NPPB show anti-HSV-1 and anti-ACV-resistant HSV-1 activities. • HSV-1 infection induces intracellular chloride concentration increasing. • Tamoxifen and NPPB inhibit HSV-1 early infection. • Tamoxifen and NPPB prevent the fusion process of HSV-1. - Abstract: Herpes simplex virus type 1 (HSV-1) infection is very common worldwide and can cause significant health problems from periodic skin and corneal lesions to encephalitis. Appearance of drug-resistant viruses in clinical therapy has made exploring novel antiviral agents emergent. Here we show that chloride channel inhibitors, including tamoxifen and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB), exhibited extensive antiviral activities toward HSV-1 and ACV-resistant HSV viruses. HSV-1 infection induced chloride ion influx while treatment with inhibitors reduced the increase of intracellular chloride ion concentration. Pretreatment or treatment of inhibitors at different time points during HSV-1 infection all suppressed viral RNA synthesis, protein expression and virus production. More detailed studies demonstrated that tamoxifen and NPPB acted as potent inhibitors of HSV-1 early entry step by preventing viral binding, penetration and nuclear translocation. Specifically the compounds appeared to affect viral fusion process by inhibiting virus binding to lipid rafts and interrupting calcium homeostasis. Taken together, the observation that tamoxifen and NPPB can block viral entry suggests a stronger potential for these compounds as well as other ion channel inhibitors in antiviral therapy against HSV-1, especially the compound tamoxifen is an immediately actionable drug that can be reused for treatment of HSV-1 infections

  19. SP-transcription factors are involved in basal MVP promoter activity and its stimulation by HDAC inhibitors.

    Science.gov (United States)

    Steiner, Elisabeth; Holzmann, Klaus; Pirker, Christine; Elbling, Leonilla; Micksche, Michael; Berger, Walter

    2004-04-23

    The major vault protein (MVP) has been implicated in multidrug resistance, cellular transport, and malignant transformation. In this study we aimed to identify crucial MVP promoter elements that regulate MVP expression. By mutation as well as deletion analysis a conserved proximal GC-box element was demonstrated to be essential for basal human MVP promoter transactivation. Binding of Sp-family transcription factors but not AP2 to this element in vitro and in vivo was shown by EMSA and ChIP assays, respectively. Inhibition of GC-box binding by a dominant-negative Sp1-variant and by mithramycin A distinctly attenuated MVP promoter activity. In Sp-null Drosophila cells, the silent human MVP promoter was transactivated by several human Sp-family members. In human cells the MVP promoter was potently stimulated by the histone deacetylase (HDAC) inhibitors butyrate (NaB) and trichostatin A (TSA), resulting in enhanced MVP expression. This stimulation was substantially decreased by mutation of the single GC-box and by application of mithramycin A. Treatment with HDAC inhibitors led to a distinct decrease of Sp1 but increase of Sp3 binding in vivo to the respective promoter sequence as demonstrated by ChIP assays. Summarising, this study identifies variations in Sp-transcription factor binding to a single proximal GC-box element as critical for basal MVP promoter activation and its stimulation by HDAC inhibitors.

  20. Binding of the respiratory chain inhibitor ametoctradin to the mitochondrial bc1 complex.

    Science.gov (United States)

    Fehr, Marcus; Wolf, Antje; Stammler, Gerd

    2016-03-01

    Ametoctradin is an agricultural fungicide that inhibits the mitochondrial bc1 complex of oomycetes. The bc1 complex has two quinone binding sites that can be addressed by inhibitors. Depending on their binding sites and binding modes, the inhibitors show different degrees of cross-resistance that need to be considered when designing spray programmes for agricultural fungicides. The binding site of ametoctradin was unknown. Cross-resistance analyses, the reduction of isolated Pythium sp. bc1 complex in the presence of different inhibitors and molecular modelling studies were used to analyse the binding site and binding mode of ametoctradin. All three approaches provide data supporting the argument that ametoctradin binds to the Pythium bc1 complex similarly to stigmatellin. The binding mode of ametoctradin differs from other agricultural fungicides such as cyazofamid and the strobilurins. This explains the lack of cross-resistance with strobilurins and related inhibitors, where resistance is mainly caused by G143A amino acid exchange. Accordingly, mixtures or alternating applications of these fungicides and ametoctradin can help to minimise the risk of the emergence of new resistant isolates. © 2015 Society of Chemical Industry.

  1. Targeted Morphoproteomic Profiling of Ewing's Sarcoma Treated with Insulin-Like Growth Factor 1 Receptor (IGF1R) Inhibitors: Response/Resistance Signatures

    Science.gov (United States)

    Subbiah, Vivek; Naing, Aung; Brown, Robert E.; Chen, Helen; Doyle, Laurence; LoRusso, Patricia; Benjamin, Robert; Anderson, Pete; Kurzrock, Razelle

    2011-01-01

    Background Insulin-like growth factor 1 receptor (IGF1R) targeted therapies have resulted in responses in a small number of patients with advanced metastatic Ewing's sarcoma. We performed morphoproteomic profiling to better understand response/resistance mechanisms of Ewing's sarcoma to IGF1R inhibitor-based therapy. Methodology/Principal Findings This pilot study assessed two patients with advanced Ewing's sarcoma treated with IGF1R antibody alone followed by combined IGF1R inhibitor plus mammalian target of rapamycin (mTOR) inhibitor treatment once resistance to single-agent IGF1R inhibitor developed. Immunohistochemical probes were applied to detect p-mTOR (Ser2448), p-Akt (Ser473), p-ERK1/2 (Thr202/Tyr204), nestin, and p-STAT3 (Tyr 705) in the original and recurrent tumor. The initial remarkable radiographic responses to IGF1R-antibody therapy was followed by resistance and then response to combined IGF1R plus mTOR inhibitor therapy in both patients, and then resistance to the combination regimen in one patient. In patient 1, upregulation of p-Akt and p-mTOR in the tumor that relapsed after initial response to IGF1R antibody might explain the resistance that developed, and the subsequent response to combined IGF1R plus mTOR inhibitor therapy. In patient 2, upregulation of mTOR was seen in the primary tumor, perhaps explaining the initial response to the IGF1R and mTOR inhibitor combination, while the resistant tumor that emerged showed activation of the ERK pathway as well. Conclusion/Significance Morphoproteomic analysis revealed that the mTOR pathway was activated in these two patients with advanced Ewing's sarcoma who showed response to combined IGF1R and mTOR inhibition, and the ERK pathway in the patient in whom resistance to this combination emerged. Our pilot results suggests that morphoproteomic assessment of signaling pathway activation in Ewing's sarcoma merits further investigation as a guide to understanding response and resistance signatures. PMID

  2. Structure-Based Drug Design of Novel Potent and Selective Tetrahydropyrazolo[1,5- a ]pyrazines as ATR Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Barsanti, Paul A; Aversa, Robert J.; Jin, Xianming; Pan, Yue; Lu, Yipin; Elling, Robert; Jain, Rama; Knapp, Mark; Lan, Jiong; Lin, Xiaodong; Rudewicz, Patrick; Sim, Janet; Taricani, Lorena; Thomas, George; Xiao, Linda; Yue, Qin (Novartis)

    2015-01-08

    A saturation strategy focused on improving the selectivity and physicochemical properties of ATR inhibitor HTS hit 1 led to a novel series of highly potent and selective tetrahydropyrazolo[1,5-a]pyrazines. Use of PI3Kα mutants as ATR crystal structure surrogates was instrumental in providing cocrystal structures to guide the medicinal chemistry designs. Detailed DMPK studies involving cyanide and GSH as trapping agents during microsomal incubations, in addition to deuterium-labeled compounds as mechanistic probes uncovered the molecular basis for the observed CYP3A4 TDI in the series.

  3. Discovery and characterization of small molecule Rac1 inhibitors.

    Science.gov (United States)

    Arnst, Jamie L; Hein, Ashley L; Taylor, Margaret A; Palermo, Nick Y; Contreras, Jacob I; Sonawane, Yogesh A; Wahl, Andrew O; Ouellette, Michel M; Natarajan, Amarnath; Yan, Ying

    2017-05-23

    Aberrant activation of Rho GTPase Rac1 has been observed in various tumor types, including pancreatic cancer. Rac1 activates multiple signaling pathways that lead to uncontrolled proliferation, invasion and metastasis. Thus, inhibition of Rac1 activity is a viable therapeutic strategy for proliferative disorders such as cancer. Here we identified small molecule inhibitors that target the nucleotide-binding site of Rac1 through in silico screening. Follow up in vitro studies demonstrated that two compounds blocked active Rac1 from binding to its effector PAK1. Fluorescence polarization studies indicate that these compounds target the nucleotide-binding site of Rac1. In cells, both compounds blocked Rac1 binding to its effector PAK1 following EGF-induced Rac1 activation in a dose-dependent manner, while showing no inhibition of the closely related Cdc42 and RhoA activity. Furthermore, functional studies indicate that both compounds reduced cell proliferation and migration in a dose-dependent manner in multiple pancreatic cancer cell lines. Additionally, the two compounds suppressed the clonogenic survival of pancreatic cancer cells, while they had no effect on the survival of normal pancreatic ductal cells. These compounds do not share the core structure of the known Rac1 inhibitors and could serve as additional lead compounds to target pancreatic cancers with high Rac1 activity.

  4. Zinc promotes the death of hypoxic astrocytes by upregulating hypoxia-induced hypoxia-inducible factor-1alpha expression via poly(ADP-ribose) polymerase-1.

    Science.gov (United States)

    Pan, Rong; Chen, Chen; Liu, Wen-Lan; Liu, Ke-Jian

    2013-07-01

    Pathological release of excess zinc ions has been implicated in ischemic brain cell death. However, the underlying mechanisms remain to be elucidated. In stroke, ischemia-induced zinc release and hypoxia-inducible factor-1 (HIF-1) accumulation concurrently occur in the ischemic tissue. The present study tests the hypothesis that the presence of high intracellular zinc concentration is a major cause of modifications to PARP-1 and HIF-1α during hypoxia, which significantly contributes to cell death during ischemia. Primary cortical astrocytes and C8-D1A cells were exposed to different concentrations of zinc chloride. Cell death rate and protein expression of HIF-1 and Poly(ADP-ribose) polymerase (PARP)-1 were examined after 3-h hypoxic treatment. Although 3-h hypoxia or 100 μM of zinc alone did not induce noticeable cytotoxicity, their combination led to a dramatic increase in astrocytic cell death in a zinc-concentration-dependent manner. Exposure of astrocytes to hypoxia for 3 h remarkably increased the levels of intracellular zinc and HIF-1α protein, which was further augmented by added exogenous zinc. Notably, HIF-1α knockdown blocked zinc-induced astrocyte death. Moreover, knockdown of PARP-1, another important protein in the response of hypoxia, attenuated the overexpression of HIF-1α and reduced the cell death rate. Our studies show that zinc promotes hypoxic cell death through overexpression of the hypoxia response factor HIF-1α via the cell fate determine factor PARP-1 modification, which provides a novel mechanism for zinc-mediated ischemic brain injury. © 2013 John Wiley & Sons Ltd.

  5. JAK inhibitors for the treatment of myeloproliferative neoplasms and other disorders [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    William Vainchenker

    2018-01-01

    Full Text Available JAK inhibitors have been developed following the discovery of the JAK2V617F in 2005 as the driver mutation of the majority of non-BCR-ABL1 myeloproliferative neoplasms (MPNs. Subsequently, the search for JAK2 inhibitors continued with the discovery that the other driver mutations (CALR and MPL also exhibited persistent JAK2 activation. Several type I ATP-competitive JAK inhibitors with different specificities were assessed in clinical trials and exhibited minimal hematologic toxicity. Interestingly, these JAK inhibitors display potent anti-inflammatory activity. Thus, JAK inhibitors targeting preferentially JAK1 and JAK3 have been developed to treat inflammation, autoimmune diseases, and graft-versus-host disease. Ten years after the beginning of clinical trials, only two drugs have been approved by the US Food and Drug Administration: one JAK2/JAK1 inhibitor (ruxolitinib in intermediate-2 and high-risk myelofibrosis and hydroxyurea-resistant or -intolerant polycythemia vera and one JAK1/JAK3 inhibitor (tofacitinib in methotrexate-resistant rheumatoid arthritis. The non-approved compounds exhibited many off-target effects leading to neurological and gastrointestinal toxicities, as seen in clinical trials for MPNs. Ruxolitinib is a well-tolerated drug with mostly anti-inflammatory properties. Despite a weak effect on the cause of the disease itself in MPNs, it improves the clinical state of patients and increases survival in myelofibrosis. This limited effect is related to the fact that ruxolitinib, like the other type I JAK2 inhibitors, inhibits equally mutated and wild-type JAK2 (JAK2WT and also the JAK2 oncogenic activation. Thus, other approaches need to be developed and could be based on either (1 the development of new inhibitors specifically targeting JAK2V617F or (2 the combination of the actual JAK2 inhibitors with other therapies, in particular with molecules targeting pathways downstream of JAK2 activation or the stability of JAK2

  6. Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding.

    Science.gov (United States)

    Skagseth, Susann; Christopeit, Tony; Akhter, Sundus; Bayer, Annette; Samuelsen, Ørjan; Leiros, Hanna-Kirsti S

    2017-08-01

    Metallo-β-lactamases (MBLs) threaten the effectiveness of β-lactam antibiotics, including carbapenems, and are a concern for global public health. β-Lactam/β-lactamase inhibitor combinations active against class A and class D carbapenemases are used, but no clinically useful MBL inhibitor is currently available. Tripoli metallo-β-lactamase-1 (TMB-1) and TMB-2 are members of MBL subclass B1a, where TMB-2 is an S228P variant of TMB-1. The role of S228P was studied by comparisons of TMB-1 and TMB-2, and E119 was investigated through the construction of site-directed mutants of TMB-1, E119Q, E119S, and E119A (E119Q/S/A). All TMB variants were characterized through enzyme kinetic studies. Thermostability and crystallization analyses of TMB-1 were performed. Thiol-based inhibitors were investigated by determining the 50% inhibitory concentrations (IC 50 ) and binding using surface plasmon resonance (SPR) for analysis of TMB-1. Thermostability measurements found TMB-1 to be stabilized by high NaCl concentrations. Steady-state enzyme kinetics analyses found substitutions of E119, in particular, substitutions associated with the penicillins, to affect hydrolysis to some extent. TMB-2 with S228P showed slightly reduced catalytic efficiency compared to TMB-1. The IC 50 levels of the new thiol-based inhibitors were 0.66 μM (inhibitor 2a) and 0.62 μM (inhibitor 2b), and the equilibrium dissociation constant ( K D ) of inhibitor 2a was 1.6 μM; thus, both were more potent inhibitors than l-captopril (IC 50 = 47 μM; K D = 25 μM). The crystal structure of TMB-1 was resolved to 1.75 Å. Modeling of inhibitor 2b in the TMB-1 active site suggested that the presence of the W64 residue results in T-shaped π-π stacking and R224 cation-π interactions with the phenyl ring of the inhibitor. In sum, the results suggest that residues 119 and 228 affect the catalytic efficiency of TMB-1 and that inhibitors 2a and 2b are more potent inhibitors for TMB-1 than l-captopril. Copyright

  7. Electrochemical studies of novel corrosion inhibitor for mild steel in 1 M hydrochloric acid

    Directory of Open Access Journals (Sweden)

    Ahmed A. Al-Amiery

    2018-06-01

    Full Text Available The electrochemical performance of a novel organic corrosion inhibitor 6-(4-hydroxyphenyl-3-mercapto-7,8-dihydro-[1,2,4]triazolo[4,3-b][1,2,4,5]tetrazine [HT3], for mild steel in 1 M hydrochloric acid is evaluated by potentiodynamic curves. The experimental results show that the investigated inhibitor [HT3], which can effectively retard the corrosion process that occurs to mild steel with a hydrochloric acid solution by providing a protective coating for the mild steel that, can be weakened by increasing the temperature. Furthermore, the inhibition efficiency of [HT3] increased with increasing the concentrations of the inhibitors and decreased with increasing temperature. Keywords: Corrosion, Inhibitor, Mild steel, Potentiodynamic polarization, HT3, NMR, FT-IR

  8. Identification of a peptide inhibitor for the histone methyltransferase WHSC1.

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    Michael J Morrison

    Full Text Available WHSC1 is a histone methyltransferase that is responsible for mono- and dimethylation of lysine 36 on histone H3 and has been implicated as a driver in a variety of hematological and solid tumors. Currently, there is a complete lack of validated chemical matter for this important drug discovery target. Herein we report on the first fully validated WHSC1 inhibitor, PTD2, a norleucine-containing peptide derived from the histone H4 sequence. This peptide exhibits micromolar affinity towards WHSC1 in biochemical and biophysical assays. Furthermore, a crystal structure was solved with the peptide in complex with SAM and the SET domain of WHSC1L1. This inhibitor is an important first step in creating potent, selective WHSC1 tool compounds for the purposes of understanding the complex biology in relation to human disease.

  9. Potent and Selective BACE-1 Peptide Inhibitors Lower Brain Aβ Levels Mediated by Brain Shuttle Transport

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    Nadine Ruderisch

    2017-10-01

    Full Text Available Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidβ (Aβ antibodies and secretase inhibitors. However, the blood-brain barrier (BBB limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aβ levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aβ. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance.

  10. Structural insights into the binding mechanism of IDO1 with hydroxylamidine based inhibitor INCB14943

    International Nuclear Information System (INIS)

    Wu, You; Xu, Tingting; Liu, Jinsong; Ding, Ke; Xu, Jinxin

    2017-01-01

    IDO1 (indoleamine 2, 3-dioxygenase 1), a well characterized immunosuppressive enzyme, has attracted growing attention as a potential target for cancer immunotherapy. Hydroxylamidine compounds INCB024360 and INCB14943 (INCB024360 analogue) are highly effective IDO1 inhibitors. INCB024360 is undergoing clinical trials for treatment of various types of human cancer. Here, we determined the co-crystal structure of IDO1 and INCB14943, and elucidate the detailed binding mode. INCB14943 binds to heme iron in IDO1 protein through the oxime nitrogen. Further analysis also reveals that a halogen bonding interaction between the chlorine atom (3-Cl) of INCB14943 and the sulphur atom of C129 significantly improves the inhibition activity against IDO1. Comparing with the other reported inhibitors, the oxime nitrogen and halogen bond interaction are identified as the unique features of INCB14943 among the IDO1 inhibitors. Thus, our study provides novel insights into the interaction between a small molecule inhibitor INCB14943 and IDO1 protein. The structural information will facilitate future IDO1 inhibitor design. - Highlights: • This is the first co-crystal structure of IDO1 with hydroxylamidine compound. • INCB14943 binds to heme iron through oxime nitrogen instead of imidazole nitrogen. • Halogen bond interaction with C129 is another unique feature of INCB14943.

  11. Marjoram Extract as Corrosion Inhibitor for Dissolution of Zinc in 1.0 M HCl

    Directory of Open Access Journals (Sweden)

    M. Sobhi

    2013-01-01

    Full Text Available In this study, water marjoram (Origanum marjorana L. extract was evaluated as corrosion inhibitor for zinc in 1.0 M HCl solution. The polarization measurements showed that this inhibitor is acting as mixed inhibitors for both anodic and cathodic reactions. The results showed that the inhibition efficiency was increased by increasing the inhibitor doses and reached the maximum at 500 ppm. The adsorption of marjoram extract on zinc surface was found to obey Langmuir type isotherm. The efficiency obtained from the impedance measurements was in good agreement with those obtained from the gravimetrical, thermometric, and polarization techniques which prove the validity of these tolls in the measurements of the tested inhibitor.

  12. Sensitization of multidrug-resistant human cancer cells to Hsp90 inhibitors by down-regulation of SIRT1

    Science.gov (United States)

    Kim, Hak-Bong; Lee, Su-Hoon; Um, Jee-Hyun; Oh, Won Keun; Kim, Dong-Wan; Kang, Chi-Dug; Kim, Sun-Hee

    2015-01-01

    The effectiveness of Hsp90 inhibitors as anticancer agents was limited in multidrug-resistant (MDR) human cancer cells due to induction of heat shock proteins (Hsps) such as Hsp70/Hsp27 and P-glycoprotein (P-gp)-mediated efflux. In the present study, we showed that resistance to Hsp90 inhibitors of MDR human cancer cells could be overcome with SIRT1 inhibition. SIRT1 knock-down or SIRT1 inhibitors (amurensin G and EX527) effectively suppressed the resistance to Hsp90 inhibitors (17-AAG and AUY922) in several MDR variants of human lymphoblastic leukemia and human breast cancer cell lines. SIRT1 inhibition down-regulated the expression of heat shock factor 1 (HSF1) and subsequently Hsps and facilitated Hsp90 multichaperone complex disruption via hyperacetylation of Hsp90/Hsp70. These findings were followed by acceleration of ubiquitin ligase CHIP-mediated mutant p53 (mut p53) degradation and subsequent down-regulation of P-gp in 17-AAG-treated MDR cancer cells expressing P-gp and mut p53 after inhibition of SIRT1. Therefore, combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be a more effective therapeutic approach for Hsp90 inhibitor-resistant MDR cells via down-regulation of HSF1/Hsps, mut p53 and P-gp. PMID:26416354

  13. Perinatal asphyxia: CNS development and deficits with delayed onset

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    Mario eHerrera-Marschitz

    2014-03-01

    Full Text Available Perinatal asphyxia constitutes a prototype of obstetric complications occurring when pulmonary oxygenation is delayed or interrupted. The primary insult relates to the duration of the period lacking oxygenation, leading to death if not re-established. Re-oxygenation leads to a secondary insult, related to a cascade of biochemical events required for restoring proper function. Perinatal asphyxia interferes with neonatal development, resulting in long-term deficits associated to mental and neurological diseases with delayed clinical onset, by mechanisms not yet clarified.In the experimental scenario, the effects observed long after perinatal asphyxia have been explained by over expression of sentinel proteins, such as poly(ADP-ribose polymerase-1 (PARP-1, competing for NAD+ during re-oxygenation, leading to the idea that sentinel protein inhibition constitutes a suitable therapeutic strategy. Asphyxia induces transcriptional activation of pro-inflammatory factors, in tandem with PARP-1 overactivation, and pharmacologically induced PARP-1 inhibition also down-regulates the expression of proinflammatory cytokines. Nicotinamide has been proposed as a suitable PARP-1 inhibitor. Its effect has been studied in an experimental model of global hypoxia in rats. In that model, the insult is induced by immersing rat foetuses into a water bath for various periods of time. Following asphyxia, the pups are delivered, treated, and nursed by surrogate dams, pending further experiments. Nicotinamide rapidly distributes into the brain following systemic administration, reaching steady state concentrations sufficient to inhibit PARP-1 activity for several hours, preventing several of the long-term consequences of perinatal asphyxia, supporting the idea that it constitutes a lead for exploring compounds with similar or better pharmacological profiles.

  14. Case Reports That Illustrate the Efficacy of SGLT2 Inhibitors in the Type 1 Diabetic Patient

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    David S. H. Bell

    2015-01-01

    Full Text Available SGLT2 inhibitors are only approved for use in adults with type 2 diabetes. However, because SGLT2 inhibitors have a mechanism of action that does not require the presence of endogenous insulin, these drugs should also be efficacious in type 1 diabetes where endogenous insulin production is greatly reduced or absent. Herein, I present five cases which illustrate the benefits of utilizing an SGLT2 inhibitor with type 1 diabetes. In these cases the use of SGLT2 inhibitors resulted not only in better glycemic control in most patients but also in some patients’ less hypoglycemia, weight loss, and decreased doses of insulin. In type 1 diabetes Candida albicans vaginitis and balanitis may occur more frequently than in type 2 diabetes. These cases show that a large randomized clinical trial of SGLT2 inhibitors in type 1 diabetes needs to be performed.

  15. Inhibition of SIRT1 by a small molecule induces apoptosis in breast cancer cells.

    Science.gov (United States)

    Kalle, Arunasree M; Mallika, A; Badiger, Jayasree; Alinakhi; Talukdar, Pinaki; Sachchidanand

    2010-10-08

    Overexpression of SIRT1, a NAD+-dependent class III histone deacetylases (HDACs), is implicated in many cancers and therefore could become a promising antitumor target. Here we demonstrate a small molecule SIRT1 inhibitor, ILS-JGB-1741(JGB1741) with potent inhibitory effects on the proliferation of human metastatic breast cancer cells, MDA-MB 231. The molecule has been designed using medicinal chemistry approach based on known SIRT1 inhibitor, sirtinol. The molecule showed a significant inhibition of SIRT1 activity compared to sirtinol. Studies on the antitumor effects of JGB on three different cancer cell lines, K562, HepG2 and MDA-MB 231 showed an IC₅₀ of 1, 10 and 0.5 μM, respectively. Further studies on MDA-MB 231 cells showed a dose-dependent increase in K9 and K382 acetylation of H3 and p53, respectively. Results also demonstrated that JGB1741-induced apoptosis is associated with increase in cytochrome c release, modulation in Bax/Bcl2 ratio and cleavage of PARP. Flowcytometric analysis showed increased percentage of apoptotic cells, decrease in mitochondrial membrane potential and increase in multicaspase activation. In conclusion, the present study indicates the potent apoptotic effects of JGB1741 in MDA-MB 231 cells. Copyright © 2010 Elsevier Inc. All rights reserved.

  16. Prognosis of hospitalized patients with 2009 H1N1 influenza in Spain: influence of neuraminidase inhibitors

    Science.gov (United States)

    Delgado-Rodríguez, Miguel; Castilla, Jesús; Godoy, Pere; Martín, Vicente; Soldevila, Nuria; Alonso, Jordi; Astray, Jenaro; Baricot, Maretva; Cantón, Rafael; Castro, Ady; Gónzález-Candelas, Fernando; Mayoral, José María; Quintana, José María; Pumarola, Tomás; Tamames, Sonia; Sáez, Marc; Domínguez, Angela

    2012-01-01

    Background The H1N1 influenza pandemic strain has been associated with a poor prognosis in hospitalized patients. The present report evaluates the factors influencing prognosis. Methods A total of 813 patients hospitalized with H1N1 influenza in 36 hospitals (nationwide) in Spain were analysed. Detailed histories of variables preceding hospital admission were obtained by interview, validating data on medications and vaccine with their attending physicians. Data on treatment and complications during hospital stay were recorded. As definition of poor outcome, the endpoints of death and admission to intensive care were combined; and as a further outcome, length of stay was used. Results The mean age was 38.5 years (SD 22.8 years). There were 10 deaths and 79 admissions to intensive care (combined, 88). The use of neuraminidase inhibitors was reported by 495 patients (60.9%). The variables significantly associated with a poor outcome were diabetes (OR = 2.21, 95% CI = 1.21–4.02), corticosteroid therapy (OR = 3.37, 95% CI = 1.39–8.20) and use of histamine-2 receptor antagonists (OR = 2.68, 95% CI = 1.14–6.36), while the use of neuraminidase inhibitors (OR = 0.57, 95% CI = 0.34–0.94) was protective. Neuraminidase inhibitors within the first 2 days after the influenza onset reduced hospital stay by a mean of 1.9 days (95% CI = 4.7–6.6). Conclusions The use of neuraminidase inhibitors decreases the length of hospital stay and admission to intensive care and/or death. PMID:22467633

  17. The Nonglycemic Actions of Dipeptidyl Peptidase-4 Inhibitors

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    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.

  18. Synthesis and characterization of a novel organic corrosion inhibitor for mild steel in 1 M hydrochloric acid

    Science.gov (United States)

    Ahmed, Mohammed H. Othman; Al-Amiery, Ahmed A.; Al-Majedy, Yasmin K.; Kadhum, Abdul Amir H.; Mohamad, Abu Bakar; Gaaz, Tayser Sumer

    2018-03-01

    The synthesis and characterization of a novel organic corrosion inhibitor (4-(3-mercapto-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)phenol), for mild steel in 1 M hydrochloric acid (HCl) has been successfully reported for the first time. The inhibitor evaluated as corrosion inhibitor for mild steel in 1 M of Hydrochloric acid solution using electrochemical impedance spectroscopy (EIS), and electrochemical frequency modulation (EFM) measurement techniques. Changes in the impedance parameters suggested an adsorption of the inhibitor onto the mild steel surface, leading to the formation of protective films. The results show that the inhibition efficiencies increased with increasing the concentrations of the inhibitors and decreased with increasing temperature. The maximum inhibition efficiency up to 67% at the maximum concentration 0.5 mM. This shows that those inhibitors are effective in helping to reduce and slowing down the corrosion process that occurs to mild steel with a hydrochloric acid solution by providing an organic inhibitor for the mild steel that can be weakened by increasing the temperature. The adsorption process of the synthesized organic inhibitor depends on its electronic characteristics in addition to steric effects and the nature of metal surface, temperature degree and the varying degrees of surface-site activity. The synthesized inhibitor molecules were absorbed by metal surface and follow Langmuir isotherms.

  19. The small molecule Mek1/2 inhibitor U0126 disrupts the chordamesoderm to notochord transition in zebrafish

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    Szabó Gábor

    2008-04-01

    Full Text Available Abstract Background Key molecules involved in notochord differentiation and function have been identified through genetic analysis in zebrafish and mice, but MEK1 and 2 have so far not been implicated in this process due to early lethality (Mek1-/- and functional redundancy (Mek2-/- in the knockout animals. Results Here, we reveal a potential role for Mek1/2 during notochord development by using the small molecule Mek1/2 inhibitor U0126 which blocks phosphorylation of the Mek1/2 target gene Erk1/2 in vivo. Applying the inhibitor from early gastrulation until the 18-somite stage produces a specific and consistent phenotype with lack of dark pigmentation, shorter tail and an abnormal, undulated notochord. Using morphological analysis, in situ hybridization, immunhistochemistry, TUNEL staining and electron microscopy, we demonstrate that in treated embryos the chordamesoderm to notochord transition is disrupted and identify disorganization in the medial layer of the perinotochordal basement mebrane as the probable cause of the undulations and bulges in the notochord. We also examined and excluded FGF as the upstream signal during this process. Conclusion Using the small chemical U0126, we have established a novel link between MAPK-signaling and notochord differentiation. Our phenotypic analysis suggests a potential connection between the MAPK-pathway, the COPI-mediated intracellular transport and/or the copper-dependent posttranslational regulatory processes during notochord differentiation.

  20. Inhibition of CUG-binding protein 1 and activation of caspases are critically involved in piperazine derivative BK10007S induced apoptosis in hepatocellular carcinoma cells.

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    Ju-Ha Kim

    Full Text Available Though piperazine derivative BK10007S was known to induce apoptosis in pancreatic cancer xenograft model as a T-type CaV3.1 a1G isoform calcium channel blocker, its underlying antitumor mechanism still remains unclear so far. Thus, in the present study, the antitumor mechanism of BK10007S was elucidated in hepatocellular carcinoma cells (HCCs. Herein, BK10007S showed significant cytotoxicity by 3-[4,5-2-yl]-2,5-diphenyltetra-zolium bromide (MTT assay and anti-proliferative effects by colony formation assay in HepG2 and SK-Hep1 cells. Also, apoptotic bodies and terminal deoxynucleotidyl transferase (TdT dUTP Nick End Labeling (TUNEL positive cells were observed in BK10007S treated HepG2 and SK-Hep1 cells by 4',6-diamidino-2-phenylinodole (DAPI staining and TUNEL assay, respectively. Consistently, BK10007S increased sub G1 population in HepG2 and SK-Hep1 cells by cell cycle analysis. Furthermore, Western blotting revealed that BK10007S activated the caspase cascades (caspase 8, 9 and 3, cleaved poly (ADP-ribose polymerase (PARP, and downregulated the expression of cyclin D1, survivin and for CUG-binding protein 1 (CUGBP1 or CELF1 in HepG2 and SK-Hep1 cells. Conversely, overexpression of CUGBP1 reduced cleavages of PARP and caspase 3, cytotoxicity and subG1 population in BK10007S treated HepG2 cells. Overall, these findings provide scientific evidences that BK10007S induces apoptosis via inhibition of CUGBP1 and activation of caspases in hepatocellular carcinomas as a potent anticancer candidate.