Poly ADP-ribose Polymerase Protein Research Articles

A natural product enhances apoptosis via mitochondria/caspase-mediated pathway in HeLa cells.

Cervical cancer is the fourth most lethal human malignancy and the leading cause of death among females around the world. Many antitumor agents have microbial origins. 5'-epi-SPA-6952A is a new 24-membered macrolide isolated from the cultured broth of Streptomyces diastatochromogenes. Therefore, we studied the activity and molecular mechanism of 5'-epi-SPA-6952A in human cervical carcinoma HeLa cell. The results showed that 5'-epi-SPA-6952A significantly inhibited cell proliferation and migration. In addition, 5'-epi-SPA-6952A obviously increased the production of intracellular reactive oxygen species and DNA damage in HeLa cells. Moreover, nuclear shrinkage of cells, decrease in mitochondrial membrane potential, and upregulation of Bax/Bcl-2 ratio resulted in the release of cytochrome c, and activation of caspase-9/3 was observed in HeLa cells treated with 5'-epi-SPA-6952A, which means it enhanced the intrinsic mitochondrial apoptosis. Besides, DNA-damage associated proteins poly (ADP-ribose) polymerase (PARP) and p53 were also studied, and the expressions of cleaved-PARP and p53 were drastically increased in HeLa cells treated with 5'-epi-SPA-6952A. Furthermore, we confirmed that 5'-epi-SPA-6952A affected the survival of HeLa cells by blocking cell cycle progression in the G1 phase. Taken together, the results shows that 5'-epi-SPA-6952A significantly inhibited HeLa cells proliferation via intrinsic mitochondrial apoptosis, cell cycle arrest, and blocking cell migration.

Study on the Metabolic Reprogramming of Lung Cancer Cells Regulated by Docetaxel Based on Metabolomics

背景与目的多西他赛是一种临床常用的抗肿瘤药物，是晚期非小细胞肺癌（non-small cell lung cancer, NSCLC）的一线用药。然而，多西他赛抗NSCLC作用的分子机制尚不明确。研究表明肿瘤细胞的代谢重编程在肿瘤发生发展过程中发挥重要作用。本研究旨在通过结合代谢组学分析及生物学手段来探讨多西他赛所影响的NSCLC细胞代谢通路。方法首先，通过CCK-8实验分析多西他赛对NSCLC细胞活力的影响，筛选合适药物浓度。接下来，通过基于气相色谱质谱联用（gas chromatography-mass spectrometry, GC-MS）的代谢组学技术分析多西他赛处理和未处理的A549和H1299细胞。并通过统计学计算得到处理组和未处理组间的差异代谢物。最后，通过蛋白质免疫印迹分析（Western blot）多西他赛对其所调控的相关代谢途径中关键酶蛋白质表达水平的影响。结果多西他赛可时间依赖和浓度依赖地抑制A549和H1299细胞活力。随着多西他赛处理时间延长，凋亡敏感蛋白质多聚二磷酸腺苷核糖聚合酶[Poly(ADP-)Polymerase, PARP]逐渐被激活裂解形成P89片段。代谢组学分析发现，药物处理后的A549和H1299细胞内，8种代谢物均发生显著变化，主要集中于三羧酸（tricarboxylic acid, TCA）循环代谢通路。同时，药物处理后，TCA循环关键调控酶异柠檬酸脱氢酶蛋白质表达水平显著下降。结论多西他赛诱导NSCLC增殖抑制及凋亡的效应可能与下调异柠檬酸脱氢酶，进而抑制三羧酸循环代谢途径有关。

Downregulation of APE1 potentiates breast cancer cells to olaparib by inhibiting PARP-1 expression.

Targeting DNA repair mechanisms to induce apoptosis may be a promising strategy for breast cancer treatment. Olaparib is proved to have anticancer effect by inhibiting DNA repairing protein poly (ADP-ribose) polymerase (PARP). However, the cytotoxicity of olaparib is very limited to homologous recombination-proficient cells. This study aims to examine the effect and mechanismof olaparib treatment in breast cancer cell lines. We investigated the cytotoxic effect of various doses ofolaparib treatment to MCF-7 and ZR-75-1 cells in vitro. mRNA and proteinlevels of PARP and APE1 were examined by real-time PCR and western blot, respectively. APE1-deficient cell lines were created by RNA interference and used for in vitro cytotoxicity study as well as in vivo study. 2µM or higher concentrations of olapariblead to significant cell death and ROS production. Moreover, olaparib treatment not only inhibits PARP1, but also reduces the expression of APE1 in both mRNA and protein levels. Deficiency of APE1 resulted in increased sensitivity of MCF-7 and ZR-75-1 cells to olaparib treatment. In vivo study showedthat reduction of APE1 significantly reduced the volume and weight of MCF-7 xenografted tumors when treated with olaparib, which suggests the synergistic function of inhibition of APE1 in promoting antitumor effects of olaparib treatment. To acquire better benefits for HR-proficient breast cancer patients, developing chemotherapeutic drugs antagonize APE1 would be an effective strategy to improve the clinical outcome of PARP inhibitors.

Capsaicin induces apoptosis and autophagy in human melanoma cells.

Melanoma is the most lethal type of skin cancer; rapid metastasis and resistance to conventional radio- and chemotherapy make melanoma the most aggressive type of skin cancer. In addition, there is a high recurrence rate within 1 year among patients with melanoma following traditional treatment by chemotherapy or immunotherapy, and these treatment options are only useful in advanced stages. As the efficiency of treatment options for melanoma is not ideal, the present study aimed to confirm that capsaicin has inhibitory effects on the human melanoma A375 and C8161 cell lines in vitro. Capsaicin, the active component of peppers, has been reported to possess substantial anticarcinogenic and antimutagenic activities. Additionally, capsaicin exhibits an inhibitory effect on tumor growth in numerous malignant cell lines. In the present study, flow cytometry, fluorescent puncta detection and western blotting were performed. The experimental results indicated that capsaicin activated apoptosis, and that apoptosis induction was associated with poly(ADP-ribose) polymerase (PARP) cleavage and activation of caspase-3. Additionally, the formation of autophagosomes and accumulation of microtubule-associated proteins 1A/1B light chain 3B-II and beclin 1 suggested that capsaicin induced autophagy in human melanoma cells. Furthermore, inhibiting capsaicin-induced autophagy promoted the activation of cleaved caspase-3 and PARP proteins, which are associated with apoptosis. In addition, inhibition of autophagy using 3-MA enhanced capsaicin-induced cell death, indicating that capsaicin-induced autophagy is a pro-survival process in cells. In conclusion, the results of the present study revealed that capsaicin induced cell apoptosis and autophagy in human melanoma cells and capsaicin may be considered as a novel candidate drug for melanoma treatment.

Poly(ADP-Ribose) Polymerases in Plants and Their Human Counterparts: Parallels and Peculiarities.

Poly(ADP-ribosyl)ation is a rapid and transient post-translational protein modification that was described first in mammalian cells. Activated by the sensing of DNA strand breaks, poly(ADP-ribose)polymerase1 (PARP1) transfers ADP-ribose units onto itself and other target proteins using NAD+ as a substrate. Subsequently, DNA damage responses and other cellular responses are initiated. In plants, poly(ADP-ribose) polymerases (PARPs) have also been implicated in responses to DNA damage. The Arabidopsis genome contains three canonical PARP genes, the nomenclature of which has been uncoordinated in the past. Albeit assumptions concerning the function and roles of PARP proteins in planta have often been inferred from homology and structural conservation between plant PARPs and their mammalian counterparts, plant-specific roles have become apparent. In particular, PARPs have been linked to stress responses of plants. A negative role under abiotic stress has been inferred from studies in which a genetic or, more commonly, pharmacological inhibition of PARP activity improved the performance of stressed plants; in response to pathogen-associated molecular patterns, a positive role has been suggested. However, reports have been inconsistent, and the effects of PARP inhibitors appear to be more robust than the genetic abolition of PARP gene expression, indicating the presence of alternative targets of those drugs. Collectively, recent evidence suggests a conditionality of stress-related phenotypes of parp mutants and calls for a reconsideration of PARP inhibitor studies on plants. This review critically summarizes our current understanding of poly(ADP-ribosylation) and PARP proteins in plants, highlighting similarities and differences to human PARPs, areas of controversy, and requirements for future studies.

Novel PNKP mutations causing defective DNA strand break repair and PARP1 hyperactivity in MCSZ

ObjectiveTo address the relationship between novel mutations in polynucleotide 5'-kinase 3'-phosphatase (PNKP), DNA strand break repair, and neurologic disease.MethodsWe have employed whole-exome sequencing, Sanger sequencing, and molecular/cellular biology.ResultsWe describe here a patient with microcephaly with early onset seizures (MCSZ) from the Indian sub-continent harboring 2 novel mutations in PNKP, including a pathogenic mutation in the fork-head associated domain. In addition, we confirm that MCSZ is associated with hyperactivation of the single-strand break sensor protein protein poly (ADP-ribose) polymerase 1 (PARP1) following the induction of abortive topoisomerase I activity, a source of DNA strand breakage associated previously with neurologic disease.ConclusionsThese data expand the spectrum of PNKP mutations associated with MCSZ and show that PARP1 hyperactivation at unrepaired topoisomerase-induced DNA breaks is a molecular feature of this disease.

Characterisation of Deubiquitylating Enzymes in the Cellular Response to High-LET Ionizing Radiation and Complex DNA Damage

PurposeIonizing radiation, particular high-linear energy transfer (LET) radiation, can induce complex DNA damage (CDD) wherein 2 or more DNA lesions are induced in close proximity, which contributes significantly to the cell killing effects. However, knowledge of the enzymes and mechanisms involved in coordinating the recognition and processing of CDD in cellular DNA are currently lacking.Methods and MaterialsA small interfering RNA screen of deubiquitylation enzymes was conducted in HeLa cells irradiated with high-LET α-particles or protons, versus low-LET protons and x-rays, and cell survival was monitored by clonogenic assays. Candidates whose depletion led to decreased cell survival specifically in response to high-LET radiation were validated in both HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A) cells, and the association with CDD repair was confirmed using an enzyme modified neutral comet assay.ResultsDepletion of USP6 decreased cell survival specifically after high-LET α-particles and protons, but not low-LET protons or x-rays. USP6 depletion caused cell cycle arrest and a deficiency in CDD repair mediated through instability of poly(ADP-ribose) polymerase-1 (PARP-1) protein. Increased radiosensitivity of cells to high-LET protons as a consequence of defective CDD repair was furthermore mimicked using the PARP inhibitor olaparib, and through PARP-1 small interfering RNA.ConclusionsUSP6 controls cell survival in response to high-LET radiation by stabilizing PARP-1 protein levels, which is essential for CDD repair. We also describe synergy between CDD induced by high-LET protons and PARP inhibition, or PARP-1 depletion, in effective cancer cell killing.

Identification and quantification of DNA repair protein poly(ADP ribose) polymerase 1 (PARP1) in human tissues and cultured cells by liquid chromatography/isotope-dilution tandem mass spectrometry

Poly(ADP ribose) polymerase 1 (PARP1) is a multifunctional DNA repair protein of the base excision repair pathway and plays a major role in the repair of DNA strand breaks and in replication and transcriptional regulation among other functions. Mounting evidence points to the predictive and prognostic value of PARP1 expression in human cancers. Thus, PARP1 has become an important target in cancer therapy, leading to the development of inhibitors as anticancer drugs. In the past, PARP1 expression levels in tissue samples have generally been estimated by indirect and semi-quantitative immunohistochemical methods. Accurate measurement of PARP1 in normal tissues and malignant tumors of patients will be essential for evaluating PARP1 as a predictive and prognostic biomarker in cancer and other diseases, and for the development and use of its inhibitors in cancer therapy. In this work, we present an approach involving liquid chromatography–isotope-dilution tandem mass spectrometry to positively identify and accurately quantify PARP1 in human tissues and cultured cells. We identified and quantified PARP1 in human normal ovarian tissues and malignant ovarian tumors, and in three pairs of human cell lines, each pair consisting of a normal cell line and its cancerous counterpart. Significantly greater expression of PARP1 was observed in malignant ovarian tissues than in normal ovarian tissues. In the case of one pair of cell lines, the cancerous cell line also exhibited greater expression of PARP1 than in normal cell line. We also show the simultaneous measurement of PARP1 and apurinic/apyrimidinic endonuclease 1 (APE1) in a given protein extract. The approach presented in this work is expected to contribute to the accurate quantitative assessment of PARP1 levels in basic research and clinical studies.

Resveratrol attenuates oxidative injury in human umbilical vein endothelial cells through regulating mitochondrial fusion via TyrRS-PARP1 pathway

Background/aimsOxidative stress-induced damage in endothelial cells is a crucial initiator of atherosclerosis (AS), which is highly related to excessive reactive oxygen species (ROS) and mitochondrial dynamics. Resveratrol (RSV) exerts beneficial effects against endothelial oxidative injury, while the underlying mechanisms have not been fully elucidated. Thus, we aimed to explore the role of mitochondria dynamics during the anti-oxidative activities of RSV in palmitic acid (PA)-stimulated human umbilical vein endothelial cells (HUVECs) and to verify whether tyrosyl transfer- RNA synthetase (TyrRS) and poly (ADP-ribose) polymerase 1 (PARP1) are targeted during this process.MethodsHUVECs were exposed to 200 μM of PA for 16 h before treated with 10 μM of RSV for 8 h. Cell viability was detected using Cell counting kit-8 (CCK-8) assay. The intracellular ROS level and mitochondria membrane potential (MMP) were measured using microplate reader and flow cytometry. The malondialdehyde and superoxide dismutase were measured using the microplate reader. The mitochondrial morphology and fusion process was observed under transmission electron microscopy and confocal microscopy. TyrRS and PARP1 were knocked down with the specific small interference RNAs (siRNA), and the protein expressions of TyrRS, PARP1, and mitochondrial fusion proteins (MFN1, MFN2, and OPA1) were measured by western blot.ResultsRSV treatment suppressed the PA-induced injuries in HUVECs, including the damage to cell viability, oxidative stress, and loss of MMP. Additionally, RSV improved the protein levels of MFN1, MFN2, and OPA1 as well as inhibited the PA-induced fragmentation of mitochondria. However, the effects of RSV on oxidative stress and mitochondrial fusion were abolished by the pretreatment of siRNAs of TyrRS and PARP1, indicating that these effects of RSV were dependent on the TyrRS-PARP1 pathway.ConclusionsRSV attenuated endothelial oxidative injury by regulating mitochondrial fusion via TyrRS-PARP1 signaling pathway.

Poly(ADP-ribose) polymerase 1 induces cardiac fibrosis by mediating mammalian target of rapamycin activity.

Cardiac fibrosis is involved in nearly all forms of heart diseases and is characterized by excessive deposition of extracellular matrix proteins by cardiac fibroblasts (CFs). We and others have reported the possibility of poly(ADP-ribose) polymerase 1 (PARP1), the founding subtype of the PARPs enzyme family, as a novel therapeutic target of heart diseases. The cardiac fibrotic induction of mammalian target of rapamycin (mTOR) is mainly due to collagen expression, Smad3- and p53/JNK-mediated apoptosis. However, the possible link between PARP1 and mTOR in the progression of cardiac fibrosis remains unclear. In this study, PARP1 protein expression, and the activity of mTOR and its three target substrates (p70 ribosomal S6 Kinase 1, eukaryotic initiation factor 4E--binding protein 1, and UNC-51-like kinase 1) were augmented; meanwhile, the nicotinamide adenine dinucleotide (NAD) content was significantly reduced in the process of cardiac fibrosis in vivo and in vitro. Sprague-Dawley rats were intraperitoneally injected with 3-aminobenzamide (3AB) (20 mg/kg/d; a well-established PARP1 inhibitor) or rapamycin (Rapa; 1 mg/kg/d; used for mTOR inhibition) 7 days after abdominal aortic constriction (AAC) surgery for 6 weeks. Pretreatment of 3AB or Rapa both relieved AAC-caused cardiac fibrosis and heart dysfunction. Overexpression of PARP1 with adenovirus carrying PARP1 gene specifically transduced into the hearts via intramyocardial multipoint injection caused similar myocardial damage. In CFs, preincubation with PARP1 or mTOR inhibitors all blocked TGF-β1 induced cardiac fibrosis. PARP1 overexpression evoked cardiac fibrosis, which could be antagonized by mTOR inhibitors or NAD supplementation in CFs. These results provide novel and compelling evidence that PARP1 exacerbated cardiac fibrosis, which was partially attributed to NAD-dependent activation of mTOR.

Overexpression of PARP is an independent prognostic marker for poor survival in Middle Eastern breast cancer and its inhibition can be enhanced with embelin co-treatment.

Patients with aggressive breast cancer (BC) subtypes usually don’t have favorable prognosis despite the improvement in treatment modalities. These cancers still remain a major cause of morbidity and mortality in females. This has fostered a major effort to discover actionable molecular targets to treat these patients. Poly ADP ribose polymerase (PARP) is one of these molecular targets that are under comprehensive investigation for treatment of such tumors. However, its role in the pathogenesis of BC from Middle Eastern ethnicity has not yet been explored. Therefore, we examined the expression of PARP protein in a large cohort of over 1000 Middle Eastern BC cases by immunohistochemistry. Correlation with clinico-pathological parameters were performed. Nuclear PARP overexpression was observed in 44.7% of all BC cases and was significantly associated with aggressive clinico-pathological markers. Interestingly, nuclear PARP overexpression was an independent predictor of poor prognosis. PARP overexpression was also directly associated with XIAP overexpression, with PARP and XIAP co-expression in 15.8% (159/1008) of our cases. We showed that combined inhibition of PARP by olaparib and XIAP by embelin significantly and synergistically inhibited cell growth and induced apoptosis in BC cell lines. Finally, co-treatment of olaparib and embelin regressed BC xenograft tumor growth in nude mice. Our results revealed the role of PARP in Middle Eastern BC pathogenesis and prognosis. Furthermore, our data support the potential clinical development of combined inhibition of PARP and XIAP, which eventually could extend the utility of olaparib beyond BRCA deficient cancer.

Nuclear Import of Arabidopsis Poly(ADP-Ribose) Polymerase 2 Is Mediated by Importin-α and a Nuclear Localization Sequence Located Between the Predicted SAP Domains.

Proteins of the Poly(ADP-Ribose) Polymerase (PARP) family modify target proteins by covalent attachment of ADP-ribose moieties onto amino acid side chains. In Arabidopsis, PARP proteins contribute to repair of DNA lesions and modulate plant responses to various abiotic and biotic stressors. Arabidopsis PARP1 and PARP2 are nuclear proteins and given that their molecular weights exceed the diffusion limit of nuclear pore complexes, an active import mechanism into the nucleus is likely. Here we use confocal microscopy of fluorescent protein-tagged Arabidopsis PARP2 and PARP2 deletion constructs in combination with site-directed mutagenesis to identify a nuclear localization sequence in PARP2 that is required for nuclear import. We report that in co-immunoprecipitation assays PARP2 interacts with several isoforms of the importin-α group of nuclear transport adapters and that PARP2 binding to IMPORTIN-α2 is mediated by the identified nuclear localization sequence. Our results demonstrate that PARP2 is a cargo protein of the canonical importin-α/β nuclear import pathway.

Effects of mdig on proliferation and apoptosis of lung cancer cells.

Expression of mineral dust-induced gene (mdig) in lung cancer NCI-H1650 cells was detected to investigate the effects of mdig on proliferation and apoptosis of NCI-H1650 cells. NCI-H1650 lung cancer cells were cultured in vitro. The expression of mdig in NCI-H1650 cells was lowered using ribonucleic acid interference (RNAi) technique. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were used to detect the effects of mdig small interfering RNA (siRNA) on messenger RNA (mRNA) and the protein expression of mdig in lung cancer NCI-H1650 cells, respectively. The effect of mdig on the proliferation of NCI-H1650 cells was observed through 3-(4,5)-dimethylthiazol (-z-y1)-3,5-di-phenyl tetrazolium bromide (MTT) assay, and flow cytometry was used to detect the impact of mdig on cell cycle and apoptosis of NCI-H1650 cells. The influence of mdig on caspase-3 and poly (ADP-ribose) polymerase 1 (PARP1) proteins in NCI-H1650 cells were investigated via western blot analysis. The results of RT-qPCR and western blot analysis showed that mdig siRNA obviously inhibited the expression of mRNA and protein of mdig in NCI-H1650 cells. Results of the MTT assay showed mdig siRNA could significantly reduce the proliferation ability of NCI-H1650 cells. In addition cell cycle detection showed that mdig siRNA caused NCI-H1650 cell arrest at G1 phase. Apoptosis detection results indicated that mdig siRNA promoted apoptosis of NCI-H1650 cells. Western-blot analysis revealed that mdig siRNA upregulated the expression of cleaved caspase-3 and cleaved PARP1 proteins in NCI-H1650 cells. Mdig is highly expressed in lung cancer NCI-H1650 cells while mdig siRNA can inhibit proliferation of NCI-H1650 cells and accelerate apoptosis. The underlying mechanism may be related to inhibited cell cycle progression and upregulated expression of cleavage proteins (cleaved caspase-3 and cleaved PARP1).

Identification of amentoflavone as a potent highly selective PARP-1 inhibitor and its potentiation on carboplatin in human non-small cell lung cancer

Nuclear protein poly (ADP-ribose) polymerase-1 (PARP-1) is a key enzyme in the repair of DNA and is a promising target in the development of chemosensitizers. This study first investigated the inhibitory effects of amentoflavone (AMF) and its derivatives on PARP-1 and the potentiation of AMF on carboplatin (CBP) in non-small cell lung cancer (NSCLC). This study aims to evaluate the inhibitory effect of AMF against PARP-1 and its potentiation on CBP in lung cancer both in vitro and in vivo. The inhibitory effect of AMF on PARP-1 was investigated using molecular docking and cell-free model of PARP-1 assay. Its potentiation on CBP in lung cancer was also evaluated. Fluorescence resonance energy transfer assay was used to detect the inhibitory effects of AMF and its analogues on PARP-1. Molecular docking was employed to predict the binding mode of AMF and PARP-1. MTT assay, isobologram analysis, Hoechst staining, and Annexin V-PI double staining were used to confirm the potentiation of AMF on CBP in vitro. siRNA (PARP-1)-A549 cells were used to reveal the action target of AMF. Western blot analysis, immunohistochemistry, and Tunnel assay were employed to evaluate the potentiation of AMF on CBP in A549 xenograft mice. AMF and its analogues exerted excellent inhibitory effects on PARP-1 with IC50 values ranging from 0.198 μM to 0.409 μM. Docking experiment showed that AMF can stably bind to PARP-1 with a comparable binding energy to olaparib. AMF can decrease the expression of PAR induced by H2O2in vitro. AMF synergistically increased the CBP anti-proliferative effect in A549. However, its potentiation nearly disappeared when the cells were transfected with siRNAs against PARP-1. Oral administration of AMF (100 mg/kg), combined with CBP, remarkably inhibited A549 tumor growth and ki67 expression, and increased apoptosis compared with CBP-alone group. All results suggest that AMF can be a potential PARP-1 inhibitor and a candidate adjuvant agent to boost the anticancer effect of CBP in NSCLC.

Endonuclease G takes part in AIF-mediated caspase-independent apoptosis in Mycobacterium bovis-infected bovine macrophages

Mycobacterium bovis, the causative agent of bovine tuberculosis encodes different virulence mechanisms to survive inside of host cells. One of the possible outcomes in this host–pathogen interaction is cell death. Previous results from our group showed that M. bovis induces a caspase-independent apoptosis in bovine macrophages with the possible participation of apoptosis inducing factor mitochondria associated 1 (AIFM1/AIF), a flavoprotein that functions as a cell-death regulator. However, contribution of other caspase-independent cell death mediators in M. bovis-infected macrophages is not known. In this study, we aimed to further characterize M. bovis-induced apoptosis, addressing Endonuclease G (Endo G) and Poly (ADP-ribose) polymerase 1 (PARP-1). In order to accomplish our objective, we infected bovine macrophages with M. bovis AN5 (MOI 10:1). Analysis of M. bovis-infected nuclear protein extracts by immunoblot, identified a 15- and 43-fold increase in concentration of mitochondrial proteins AIF and Endo G respectively. Interestingly, pretreatment of M. bovis-infected macrophages with cyclosporine A, a mitochondrial permeability transition pore inhibitor, abolished AIF and Endo G nuclear translocation. In addition, it also decreased macrophage DNA fragmentation to baseline and caused a 26.2% increase in bacterial viability. We also demonstrated that PARP-1 protein expression in macrophages did not change during M. bovis infection. Furthermore, pretreatment of M. bovis-infected bovine macrophages with 3-aminobenzamide, a PARP-1 inhibitor, did not change the proportion of macrophage DNA fragmentation. Our results suggest participation of Endo G, but not PARP-1, in M. bovis-induced macrophage apoptosis. To the best of our knowledge this is the first report associating Endo G with caspase-independent apoptosis induced by a member of the Mycobacterium tuberculosis complex.

Abstract 4844: Evidence of PARP-1 dependent cytotoxic agents with low target affinity

Abstract Introduction: Inhibition of nuclear protein poly (ADP-ribose) polymerase-1 (PARP1) is synthetically lethal in BRCA mutated cancers. However, the mechanism by which PARPi exert cytotoxic effects is not fully understood. In this study, we compare 16 olaparib analogs and measured their potency in a radioligand binding assay to measure their affinity for PARP1, and in a cytotoxicity assay to measure their ability to serve as a chemotherapeutic. Methods: Compounds were prepared by replacing the piperazine core on olaparib with a series of diazaspiro ring systems. PARP1 affinity was assessed using radioligand binding competition assays. In vitro cytotoxicity was performed on BRCA1 methylated and PARP1-/- ovarian cancer cell lines to evaluate the relative potency of the analog compounds. Results: The 16 analogs displayed lower affinity to PARP1 but comparable or higher potency in cell kill assays when compared to olaparib. Of the 16 compounds, those that contained the diazaspiro core with an N-Boc-functional group displayed the highest degree of potency. Cytotoxicity assays were performed for each compound using PARP1 -/- and PARP2 -/- cells to assess off-target effects. Dose response curves for PARP1-/- cells resulted in a rightward shift; no changes in the dose-response curves were detected for PARP2 -/- cells. Conclusion: Radioligand binding assays demonstrated that these compounds have low PARP1 affinity. Evaluation of the relative potency of these compounds revealed that compounds having a lower PARP1 affinity relative to olaparib still retained a high potency in cell kill assays. In vitro cytotoxicity assays on PARP1 -/- and PARP2 -/- cells confirmed that these compounds are PARP1 but not PARP2 dependent. Future studies will focus on assessing catalytic inhibition of PARP1 and PARP1 trapping in order to better elucidate the antiproliferative effects of these compounds. Citation Format: Laura Puentes, Sean Reilly. Evidence of PARP-1 dependent cytotoxic agents with low target affinity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4844.

Abstract 1851: Combined inhibition of PARP and the unfolded protein response synergistically reduces viability in BRCA1-mutant breast cancer cells

Abstract As single agents, small molecule inhibitors of poly-ADP-ribose polymerase (PARP) have shown remarkable efficacy in BRCA1/2-mutant tumors due to their synthetic lethality in DNA damage response-deficient cells. While PARP inhibitors, such as olaparib, have gained FDA-approval for gynecological malignancies, recurrent disease remains a challenge. To identify drug combinations with the potential to improve the initial response to PARP inhibition, we carried out a high-throughput drug screen in which we examined the ability of olaparib to synergize with more than 500 small molecules in BRCA1-mutant breast cancer cells. The compounds investigated included not only conventional “anti-cancer” agents but also lesser-studied metabolic and stress-inducing drugs. All compounds were tested across 10-point dose curves using an image-based readout to avoid false positives/negatives associated with more traditional metabolic readouts of cell viability (i.e. Cell Titer Glo). This screening approach revealed that inhibition of the unfolded protein response (UPR) sensitizes breast cancer cells to PARP inhibition. We have further characterized interaction of one class of inhibitors that target IRE1 (inositol-requiring enzyme 1), which initiates adaptive responses to ER stress through unconventional mRNA splicing of the transcription factor X-box binding protein 1 (XBP1). Combining olaparib with an inhibitor of IRE1-mediated XBP1 splicing (STF-0831010) synergistically reduced cancer cell viability in BRCA1-mutant SUM149 breast cancer cells. This combination led to increased DNA damage, indicated by increased γ-H2AX staining. This study provides evidence that UPR inhibition sensitizes cells to PARP inhibition, suggesting a previously un-recognized connection between PARP inhibition and protein homeostasis. Furthermore, it identifies a potential new drug combination to increase the efficacy of PARP inhibitors in the clinic. Citation Format: Jennifer E. Endress, Isaac S. Harris, Jacob Stewart-Ornstein, Galit Lahav, Joan S. Brugge. Combined inhibition of PARP and the unfolded protein response synergistically reduces viability in BRCA1-mutant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1851.

PO-091 Induction of anti-Fas regulates PUMA and MYC: P53-dependent apoptosis pathway in hepatocellular carcinoma cells

IntroductionIncreased oxidative stress is a hallmark of cancer cells, which makes them more vulnerable to induction of reactive oxygen species (ROS). P53 plays a crucial role in sensing and removing oxidative damage to DNA, and inactivating mutations in the TP53 gene attenuate this function. In addition, it was shown that mutant p53 is able to suppress the function of major antioxidant factors. Therefore, mutant p53 renders cancer cells even more susceptible to the induction of oxidative stress. Besides p53, the poly (ADP-ribose) polymerase 1 (PARP-1) protein plays an important role in the repair of ROS-induced DNA-damage. This led us to explore the potential of combining oxidative stress induction with the targeted inhibition of the PARP-1 protein to selectively target mutant p53 NSCLC cancer cells.Material and methodsAPR-246 and Auranofin (inhibition glutathione (GSH) and/or thioredoxin reductase 1 (TrxR1)) and Olaparib (PARP-1 inhibitor) were used. The cytotoxicity (SRB-assay) of these compounds was determined in a panel of NSCLC cell lines with different p53 status, including isogenic cell lines (p53 shRNA-knockdown, p53 knock-in). Total GSH content (GSH/GSSG-GloTM) and ROS content (CellROX) were determined. N-acetyl-l-cysteine (NAC) was used as a potent ROS-scavenger. Induction of apoptosis/cell death was determined by the AnnV/PI assay (FC) or the Cytotox Reagent (IncuCyte). DNA-damage was assessed by g-H2AX foci (IF) and the Comet Assay. Synergism was determined using the Additive model.Results and discussionsP53Mut knock-down reduces the cytotoxic effect of APR-246, Auranofin and Olaparib, while p53Mut knock-in sensitised cells for all three compounds. APR-246/Olaparib treatment reduced GSH levels and increased ROS content, resulting in a strong accumulation of DNA-damage and synergistic induction of cell death. Co-treatment with NAC or p53-knockdown significantly reduced this cytotoxic response. Similar synergistic effects were observed for Auranofin/Olaparib treatment in several cell lines with clinically relevant p53 mutations.ConclusionMutant p53 protein expression renders NSCLC cells more susceptible to APR-246, Auranofin and Olaparib treatment. In addition, the combination of oxidative stress induction (APR-246, Auranofin) with PARP-1 inhibition (Olaparib) results in remarkable synergistic effects in the presence of mutant p53. Therefore, this combination strategy could be a promising and selective treatment option for mutant p53 NSCLC patients in which resistance to standard therapies often occurs.

Abstract 2773: A DNA-damaging lupus autoantibody synergizes with PARP inhibitors against DNA repair-deficient tumor cells

Abstract The lupus anti-DNA autoantibody 3E10 is a compelling candidate for development as a targeted therapy for DNA repair-deficient malignancies. 3E10 has previously been shown to localize to tumors due to its attraction to DNA released by dying cancer cells, penetrate into cell nuclei, inhibit DNA repair, and kill cancer cells with defects in homology-directed repair (HDR) of DNA double-strand breaks. A more potent derivative of 3E10 with increased affinity for DNA has been developed (referred to here as 3E10EN), and identification of optimal combination therapies with 3E10EN is needed to facilitate planning for upcoming clinical trials. In the present study, we found that 3E10EN increases the activity of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP) in HDR-deficient cells and hypothesized that combination treatment with 3E10EN and PARP inhibitors (PARPi) would yield synergistic effects on HDR-deficient cancer cell survival. PARP content and activity in HDR-deficient and proficient cells prior to and following treatment with 3E10EN was evaluated. 3E10EN did not impact PARP protein content but yielded a significant increase in pADPr signal in HDR-deficient cells, which suggests a compensatory increase in PARP activity in response to DNA damage accumulation in HDR-deficient cells. Combinations of 3E10EN and the PARPi olaparib were tested on a panel of HDR-deficient cells, and a matched pair of BRCA2-deficient and proficient DLD1 cells. Olaparib inhibited the increase in pADPr caused by 3E10EN, and colony formation assays analyzed by the Chou-Talalay method confirmed that 3E10EN and olaparib synergized against HDR-deficient cancer cells. Conversely, HDR-proficient cells were resistant to 3E10EN and olaparib combination treatment. The original 3E10 is a murine antibody isolated from a lupus mouse model, and in preparation for its further development as a new drug we have recently designed Deoxymab 1 (DX1), a humanized version of 3E10EN. DX1 exhibits improved activity relative to the 3E10EN prototype, and when tested on a panel of HDR-deficient and proficient cells, DX1 and olaparib exhibited synergistic effects similar to that observed with the 3E10EN prototype. In conclusion, we have found that both the prototype 3E10EN and humanized DX1 synergize with PARPi against HDR-deficient tumor cells. These findings provide the rationale for further studies to determine the potential for this approach to be translated into a clinically relevant therapeutic strategy. Citation Format: Zahra Rattray, Jaymin M. Patel, Philip W. Noble, Valentina Dubljevic, Deanne L. Greenwood, James A. Campbell, James E. Hansen. A DNA-damaging lupus autoantibody synergizes with PARP inhibitors against DNA repair-deficient tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2773.

IDENTIFICATION OF PARP1 AS A POTENTIAL THERAPEUTIC TARGET IN TRIPLE NEGATIVE AND BASAL-LIKE BREAST CANCER MOLECULAR SUBTYPES AMONG WEST ALGERIAN PATIENTS

Purpose: Breast cancer is partly responsible for the therapeutic failures of tumors with the most worsening prognoses. Increasing understanding of the cellular aberrations inherent to cancer cells was an important step towards therapy individualization. Poly (ADP-ribose) polymerase-1 (PARP1) as a promising novel target in breast cancer was an example of this strategy. However, the biological and clinical significance of PARP1 expression and its associations with outcome remain to be defined. We aimed in present study to explore PARP1 expression in breast cancers samples among west Algerian patients. Materials and Methods: We classified 78 primary breast cancers collected and diagnosed at the Regional Military Hospital of Oran (Algeria) using immunohistochemical biomarkers then; we examined immunohistochemical PARP1 expression and assessed its correlation with clinicopathological parameters and breast cancer molecular subtypes. Results: PARP1 nuclear overexpression was found in (44, 8%) of all infiltrating breast carcinomas. PARP-1 protein overexpression was associated to higher tumor grade (P=0.036), hormone-negative tumors (P=0.005) and estrogen-negative tumors (P=0.003). Moreover, PARP1 was significantly expressed in Triple-negative (P=0.015) and basal-like breast cancers, but was also present in HER2 positive breast cancers (P=0.014). Conclusions: Our data highlights the interest of immunohistochemical biomarkers in breast cancer molecular classification. We found that PARP1 overexpression is associated with triple-negative and basal-like tumors and could be a potential therapeutic in these two pejorative breast cancer subtypes. Furthermore, PARP1 overexpression could be a predictive biomarker of therapeutic resistance in HER2 positive tumors.The assessment of PARP1 protein expression may improve the selection of patients for PARP1 inhibitor therapy.