Significance of salivary poly (ADP-ribose)-polymerase in the assessment of age-dependent pathological processes in the oral cavity.

Age-related changes in the oral cavity are accompanied by the development of age-related pathology, such as chronic periodontitis (CP). Although apoptosis plays a certain role in its pathogenesis, this fact, however, has not been evaluated clinically, and the diagnostic information content of biomarkers of apoptosis and aging has not been determined. The aim of the study was to evaluate the content of cleaved poly-(ADP-ribose)-polymerase (cPARP) and caspase-3 (Casp3) in mixed saliva of elderly patients with age-related dental diseases and in mature patients with mild to moderate CP. The study included 69 people. The control group included 22 healthy young volunteers aged 18 to 44 years. The main group included 22 elderly patients aged 60 to 74 years. They were divided into subgroups according to clinical manifestations: occlusion (comparison group), periodontal, and dystrophic syndromes. Additionally, a group of 25 patients of mature age from 45 to 59 years old with mild to moderate CP was analyzed. The content of salivary Casp3 in patients with occlusion syndrome was lower than in healthy young people (p=0.014). In patients with the periodontal syndrome, the content of cPARP was higher than in the comparison group (p=0.031). The group with dystrophic syndrome had the highest level of Casp3 in comparison with the control group and the comparison group (p=0.012, p=0.004, respectively). There were no statistically significant differences between patients of different age groups with mild to moderate CP. Evaluation of the correlation between cPARP and Casp3 levels revealed a direct relationship in the group of elderly patients and in patients with mild CP (r=0.69, r=0.81, respectively). We assessed the effect of Casp3 levels on changes in the cPARP levels by means of a simple linear regression analysis. The cPARP level correlated with the content of Casp3 (r²=0.555). According to the results of the ROC analysis, it was found that using the cPARP indicator it would be possible to distinguish between groups of elderly patients with periodontal and occlusion syndromes (AUC=0.71), while using Casp3 it would be possible to distinguish patients with the occlusion syndrome and the control group (AUC=0.78). Since the level of Casp3 in young people is significantly higher than in the elderly patients, its decrease can be considered as a potential salivary biomarker of aging. The level of studied cPARP in the elderly has clinical value in periodontal syndrome and low age dependence.

DoE Optimization Empowers the Automated Preparation of Enantiomerically Pure [18F]Talazoparib and its In Vivo Evaluation as a PARP Radiotracer.

Given the clinical potential of poly(ADP-ribose) polymerases (PARP) imaging for the detection and stratification of various cancers, the development of novel PARP imaging probes with improved pharmacological profiles over established PARP imaging agents is warranted. Here, we present a novel 18F-labeled PARP radiotracer based on the clinically superior PARP inhibitor talazoparib. An automated radiosynthesis of [18F]talazoparib (RCY: 13 ± 3.4%; n = 4) was achieved using a "design of experiments" (DoE) optimized copper-mediated radiofluorination reaction. The chiral product was isolated from the reaction mixture using 2D reversed-phase/chiral radio-HPLC (>99% ee). (8S,9R)-[18F]Talazoparib demonstrated PARP binding in HCC1937 cells in vitro and showed an excellent tumor-to-blood ratio in xenograft-bearing mice (10.2 ± 1.5). Additionally, a favorable pharmacological profile in terms of excretion, metabolism, and target engagement was observed. This synthesis of [18F]talazoparib exemplifies how DoE can enable the radiosyntheses of synthetically challenging radiolabeled compounds of high interest to the imaging community.

Identification of poly(ADP-ribose)polymerase 1 and 2 (PARP1/2) as targets of andrographolide using an integrated chemical biology approach.

Here, we describe the identification of PARP1/2 as direct binding proteins of andrographolide (Andro) using protein microarray, surface plasmon resonance (SPR), and enzyme activity assays. We then evaluated the proliferation inhibition, apoptosis, and cell migration effects of Andro on the MDA-MB-436 cell line in vitro. The final biological evaluation confirmed that Andro was a highly effective single agent in the MDA-MB-436 xenograft model and had a low hERG-mediated cardiac toxicity. Therefore, Andro represents the first natural product, non-amide member of a novel nanomolar-potency PARP1/2 inhibitor family.

Fluorescent detection of PARP activity in unfixed tissue.

Poly-ADP-ribose-polymerase (PARP) relates to a family of enzymes that can detect DNA breaks and initiate DNA repair. While this activity is generally seen as promoting cell survival, PARP enzymes are also known to be involved in cell death in numerous pathologies, including in inherited retinal degeneration. This ambiguous role of PARP makes it attractive to have a simple and fast enzyme activity assay, that allows resolving its enzymatic activity in situ, in individual cells, within complex tissues. A previously published two-step PARP activity assay uses biotinylated NAD+ and streptavidin labelling for this purpose. Here, we used the fluorescent NAD+ analogues ε-NAD+ and 6-Fluo-10-NAD+ to assess PARP activity directly on unfixed tissue sections obtained from wild-type and retinal degeneration-1 (rd1) mutant retina. In standard UV microscopy ε-NAD+ incubation did not reveal PARP specific signal. In contrast, 6-Fluo-10-NAD+ resulted in reliable detection of in situ PARP activity in rd1 retina, especially in the degenerating photoreceptor cells. When the 6-Fluo-10-NAD+ based PARP activity assay was performed in the presence of the PARP specific inhibitor olaparib, the activity signal was completely abolished, attesting to the specificity of the assay. The incubation of live organotypic retinal explant cultures with 6-Fluo-10-NAD+, did not produce PARP specific signal, indicating that the fluorescent marker may not be sufficiently membrane-permeable to label living cells. In summary, we present a new, rapid, and simple to use fluorescence assay for the cellular resolution of PARP activity on unfixed tissue, for instance in complex neuronal tissues such as the retina.

Analysis of Redox Relationships in the Plant Cell Cycle: Determination of Ascorbate, Glutathione, and Poly(ADPribose)polymerase (PARP) in Plant Cell Cultures.

Reactive oxygen species (ROS) and low molecular weight antioxidants, such as glutathione and ascorbate, are powerful signalling molecules that participate in the control of plant growth and development, and modulate progression through the mitotic cell cycle. Enhanced ROS accumulation or low levels of ascorbate or glutathione cause the cell cycle to arrest and halt progression especially through the G1 checkpoint. Plant cell suspension cultures have proved to be particularly useful tools for the study of cell cycle regulation. Here we provide effective and accurate methods for the measurement of changes in the cellular ascorbate and glutathione pools and the activities of related enzymes such poly(ADP-ribose)polymerase (PARP) during mitosis and cell expansion, particularly in cell suspension cultures. These methods can be used in studies seeking to improve current understanding of the roles of redox controls on cell division and cell expansion.

Initial evaluation of Cu-64 labeled PARPi-DOTA PET imaging in mice with mesothelioma.

Poly(ADP-ribose) polymerase (PARP) has emerged as an important molecular target for the treatment of several oncological diseases. A couple of molecular probes based on Olaparib scaffold have been developed by incorporation of F-18 or fluorophore for positron emission tomography (PET) or optical imaging in several types of tumor. PARP has been reported overexpressed in mesothelioma. We hereby synthesized an analogue of Olaparib containing DOTA moiety and radiolabeled it with Cu-64 to evaluate its utility of PET tracer for mesothelioma. The Cu-64 labeling was conveniently achieved at 90% yield with final compound at >99% radiochemistry purity. The biodistribution and PET imaging were performed at 0.5, 1, 2 and 18h to confirm the in vivo tumor targeting. The tumor uptake in study group was significant higher than that in control group (3.45±0.47% ID/g vs 2.26±0.30% ID/g) and tumor were clearly detected by PET imaging. These results suggest the feasibility to develop an Olaparib-based theranostic agent for mesothelioma.

The Prognostic Value of BRCA1 and PARP Expression in Epithelial Ovarian Carcinoma: Immunohistochemical Detection.

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 pathway (BRCAness phenomenon), which is important when treatment with poly (adenosine-diphosphate-ribose) polymerase (PARP) inhibitors is considered. The aim of this study was to investigate immunohistochemical detection of BRCA1 and PARP expression in EOC and their possible prognostic relevance. Tumor 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 and clinicopathologic characteristics and overall survival. BRCA1 expression was reduced in 21.2 % of the tumors and 36.5% showed high PARP expression. No correlation between the 2 parameters or between PARP and clinicopathologic features was found. Overall survival was significantly increased in the BRCA1-reduced and equivocal groups [median survival 2.4 y (95% CI, 1.6-6.6) and 4.9 y (95 % CI, 2.3-6.7) vs. 1.5 y (95% CI, 1.3-1.9); P=0.0002]. Multivariate analysis confirmed these findings; hazard ratio=0.53 (95% CI, 0.34-0.81; P=0.0037; loss of BRCA1 expression). In conclusion, immunohistochemical BRCA1 expression in EOC holds considerable prognostic information, whereas PARP expression did not influence the outcome. The results call for validation in prospective trials.

Combination of temsirolimus and adriamycin exhibits an enhanced antitumor effect in hepatocellular carcinoma.

OBJECTIVE: The oncogenic PI3K/Akt/mTOR pathway is frequently activated in hepatocellular carcinoma (HCC). The aim of this study is to investigate the anti-HCC effect of combination of temsirolimus, an mTOR inhibitor, and adriamycin, a routinely used drug for treating HCC. METHODS AND MATERIALS: Proliferation of HCC cells exposure to temsirolimus, adriamycin, and their combination was determined using MTT assay in vitro as well as in a nude mice model in vivo. Cell apoptosis was examined using flow cytometry. Expressions of apoptosis-related proteins including caspase-9, -3, PARP, Bax, and Bcl-2 were determined using Western blotting. RESULTS: Temsirolimus plus adriamycin showed an enhanced inhibitory effect on cell proliferation compared to temsirolimus or adriamycin in HCC cells PLC/PRF/5, BEL7402, and HuH7 in vitro. The drug combination solicited a higher percentage of apoptosis cells and induced higher levels of cleaved caspase-9, -3, and PARP than temsirolimus or adriamycin used alone. The ratio of Bax/Bcl-2 was increased in cells exposed to the combination treatment. The enhanced anti-tumor effect of this drug combination was verified in a nude mice model. We also observed that half doses of temsirolimus and adriamycin used in combination achieved a comparable tumor growth inhibitor rate with full dose of temsirolimus or adriamycin used alone. CONCLUSION: Temsirolimus plus adriamycin exhibited an enhanced antitumor effect in HCC and this drug combination might have a potential value in treatment of HCC. Studies are warranted to comprehensively evaluate the efficacy and safety of this regimen in the future.

Pharmacodynamic analyses in a multi-laboratory network: lessons from the poly(ADP-ribose) assay.

Clinical pharmacodynamic assays need to meet higher criteria for sensitivity, precision, robustness, and reproducibility than those expected for research-grade assays because of the long duration of clinical trials and the potentially unpredictable number of laboratories running the assays. This report describes the process of making an immunoassay based on commercially available reagents "clinically ready". The assay was developed to quantify poly(ADP-ribose) (PAR) levels as a marker of PAR polymerase inhibitor activity for a proof-of-concept phase 0 clinical trial at the National Cancer Institute (NCI) and subsequent clinical trials. In this publication, we retrospectively examine the measures taken to validate the published PAR immunoassay and outline key lessons learned during the development and implementation of these procedures at both internal and external clinical trial sites; these measures included optimizing PAR measurements in tumor biopsies and peripheral blood mononuclear cells (PBMCs), reagent qualification, analytical validation and assay quality control, instrument qualification and method quality control, and support for external laboratories.

The PARP family: insights into functional aspects of poly (ADP-ribose) polymerase-1 in cell growth and survival.

PARP family members can be found spread across all domains and continue to be essential molecules from lower to higher eukaryotes. Poly (ADP-ribose) polymerase 1 (PARP-1), newly termed ADP-ribosyltransferase D-type 1 (ARTD1), is a ubiquitously expressed ADP-ribosyltransferase (ART) enzyme involved in key cellular processes such as DNA repair and cell death. This review assesses current developments in PARP-1 biology and activation signals for PARP-1, other than conventional DNA damage activation. Moreover, many essential functions of PARP-1 still remain elusive. PARP-1 is found to be involved in a myriad of cellular events via conservation of genomic integrity, chromatin dynamics and transcriptional regulation. This article briefly focuses on its other equally important overlooked functions during growth, metabolic regulation, spermatogenesis, embryogenesis, epigenetics and differentiation. Understanding the role of PARP-1, its multidimensional regulatory mechanisms in the cell and its dysregulation resulting in diseased states, will help in harnessing its true therapeutic potential.

A Radiotracer Strategy to Quantify PARP-1 Expression In Vivo Provides a Biomarker That Can Enable Patient Selection for PARP Inhibitor Therapy.

Despite the availability of PARP inhibitors for cancer therapy, a biomarker to clearly stratify patients for selection of this treatment remains lacking. Here we describe a radiotracer-based method that addresses this issue, using the novel compound [(125)I] KX1: as a PARP-1-selective radiotracer that can accurately measure PARP-1 expression in vitro and in vivo The pharmacologic properties of the PARP radiotracer [(125)I] KX1: was characterized in multiple cell lines where single-agent sensitivity was correlated with [(125)I] KX1: binding to PARP-1. In vivo evaluation of [(125)I] KX1: verified in vitro results, validating PARP radiotracers to define PARP-1 enzyme expression as an in vivo biomarker. Notably, PARP-1 expression as quantified by [(125)I] KX1: correlated positively with the cytotoxic sensitivity of cell lines evaluated with PARP inhibitors. Overall, our results defined a novel technology with the potential to serve as a companion diagnostic to identify patients most likely to respond therapeutically to a PARP inhibitor. Cancer Res; 76(15); 4516-24. ©2016 AACR.

Human colon cancer HT-29 cell death responses to doxorubicin and Morus Alba leaves flavonoid extract.

The mechanistic basis for the biological properties of Morus alba flavonoid extract (MFE) and chemotherapy drug of doxorubicin on human colon cancer HT-29 cell line death are unknown. The effect of doxorubicin and flavonoid extract on colon cancer HT-29 cell line death and identification of APC gene expression and PARP concentration of HT-29 cell line were investigated. The results showed that flavonoid extract and doxorubicin induce a dose dependent cell death in HT-29 cell line. MFE and doxorubicin exert a cytotoxic effect on human colon cancer HT-29 cell line by probably promoting or induction of apoptosis.

Altered expression profile of apoptosis-related molecules correlated with clinicopathological factors in non-small-cell lung cancer.

Apoptosis-related molecules can be abnormally expressed in cancers and underscore the hallmark of resisting cell death in cancer cells. This study was aimed to observe the expression patterns of apoptosis-related molecules in lung cancer and paired non-cancerous tissues, and to observe if there is a correlation between the expression of these apoptotic molecules and clinicopathologic parameters. Immunohistochemistry (IHC) was performed to analyze the expression level of CASP3, CASP8, CASP9, PARP1, Cleaved CASP3 (C-CASP3), Cleaved PARP1 (C-PARP1), XIAP, BIRC5 (Survivin) and BCL2 in lung cancer and paired non-cancerous tissues. We found that apoptosis-related molecules CASP3, CASP9, BCL2, BIRC5 and PARP1 are abnormally expressed in lung cancer cells and their expression were correlated with histology. BCL2, BIRC5 and PARP1 are expressed at higher levels in SCC than in non-SCC. C-PARP1 expression might be an independent prognostic factor for NSCLC.

Synthesis and Evaluation of a Radioiodinated Tracer with Specificity for Poly(ADP-ribose) Polymerase-1 (PARP-1) in Vivo.

Interest in nuclear imaging of poly(ADP-ribose) polymerase-1 (PARP-1) has grown in recent years due to the ability of PARP-1 to act as a biomarker for glioblastoma and increased clinical use of PARP-1 inhibitors. This study reports the identification of a lead iodinated analog 5 of the clinical PARP-1 inhibitor olaparib as a potential single-photon emission computed tomography (SPECT) imaging agent. Compound 5 was shown to be a potent PARP-1 inhibitor in cell-free and cellular assays, and it exhibited mouse plasma stability but approximately 3-fold greater intrinsic clearance when compared to olaparib. An (123)I-labeled version of 5 was generated using solid state halogen exchange methodology. Ex vivo biodistribution studies of [(123)I]5 in mice bearing subcutaneous glioblastoma xenografts revealed that the tracer had the ability to be retained in tumor tissue and bind to PARP-1 with specificity. These findings support further investigations of [(123)I]5 as a noninvasive PARP-1 SPECT imaging agent.

Chemotherapy reduces PARP1 in cancers of the ovary: implications for future clinical trials involving PARP inhibitors.

BACKGROUND: PARP inhibitors have shown promising clinical results in cancer patients carrying BRCA1/2 mutations. Their clinical efficacy could logically be influenced by PARP1 protein levels in patient tumors. METHODS: We screened three cohorts of patients with ovarian cancer, totaling 313 samples, and evaluated PARP1 protein expression by immunohistochemistry with further validation by western blotting. RESULTS: We observed that up to 60 % of tumors showed little PARP1 protein expression. In serous ovarian tumors, comparing intratumoral PARP1 expression between chemo-naïve and post-chemotherapy patients revealed a decrease in intratumoral PARP1 following chemotherapy in all three cohorts (immunohistochemistry: p < 0.001, n = 239; western blot: p = 0.012, n = 74). The findings were further confirmed in a selection of matched samples from the same patients before and after chemotherapy. CONCLUSION: Our data suggest that patients should be screened for PARP1 expression prior to therapy with PARP inhibitors. Further, the observed reduction of intratumoral PARP1 post-chemotherapy suggests that treating chemo-naïve patients with PARP inhibitors prior to the administration of chemotherapy, or concurrently, might increase the responsiveness to PARP1 inhibition. Thus, a change in the timing of PARP inhibitor administration may be warranted for future clinical trials.

Polymorphisms in poly (ADP-ribose) polymerase-1 (PARP1) promoter and 3' untranslated region and their association with PARP1 expression in breast cancer patients.

Within the past several years, inhibition of the PARP1 activity has been emerged as one of the most exciting and promising strategies for triple-negative breast cancer (TNBC) therapy. The purpose of this study is to assess PARP1 expression in TNBCs and to evaluate the association between polymorphisms in PARP1 promoter or 3' untranslated region (3'UTR) and PARP1 expression. It was found that PARP1 was overexpressed in nuclear (nPARP1), cytoplasm (cPARP1) and nuclear-cytoplasmic coexisting (coPARP1) of 187 TNBCs in comparison to that of 115 non-TNBCs (nPARP1, p<0.001; cPARP1, p<0.001; coPARP1, p<0.001). High expression of nPARP1 and cPARP1 in breast cancer was related to worse progression-free survival (nPARP1, p=0.007, cPARP1, p=0.003). Additionally, we identified seven published polymorphism sites in the promoter region and in 3'UTR of PARP1 by sequencing. rs7527192 and rs2077197 genotypes were found to be significantly associated with the cPARP1 expression in TNBC patients (rs7527192 AA+GA versus GG, p=0.014; rs2077197 AA+GA versus GG, p=0.041). These findings were confirmed in an independent validation set of 88 TNBCs (rs7527192 GG versus GA+AA, p=0.030; rs2077197 GG versus GA+AA, p=0.030). The PARP1 over-expression including nuclear, cytoplasm and nuclear-cytoplasmic coexisting is a feature of TNBCs and the assessment of its expression may help to predict the efficacy of chemotherapy with PARP1 inhibitor.

A poly(ADP-ribose) polymerase-1 activity assay based on the FRET between a cationic conjugated polymer and supercharged green fluorescent protein.

A label-free and convenient strategy for PARP-1 activity assay and inhibitors assessment has been developed based on the fluorescence resonance energy transfer (FRET) between a cationic conjugated polymer (CCP) and supercharged green fluorescent protein (scGFP).

Cleaved PARP-1, an Apoptotic Marker, can be Detected in Ram Spermatozoa.

The presence of apoptotic features in spermatozoa has been related to lower quality and functional impairment. Members of the poly-ADP-ribose polymerases (PARP) familyare involved in both DNA repair and apoptosis, playing important roles in spermatogenesis. Poly-ADP-ribose polymerase can be cleaved by caspases, and the presence of its cleavage product (cPARP) in spermatozoa has been related to chromatin remodelling during spermatogenesis and to the activation of apoptotic pathways. There are no reports on immunodetection of cPARP in ram spermatozoa; thus, we have tested a commercially available antibody for this purpose. cPARP was microscopically detected in the acrosomal ridge of some spermatozoa (indirect immunofluorescence). A preliminary study was carried out by flow cytometry (direct immunofluorescence, FITC). Ram semen was extended in TALP and incubated for 4 h with apoptosis inducers staurosporine (10 µm) or betulinic acid (200 µm). Both inducers and incubation caused a significant increase in cPARP spermatozoa (0 h, control: 21.4±3.3%, inducers: 44.3±1.4%; 4 h, control: 44.3±2.4%, inducers: 53.3±1.4%). In a second experiment, we compared the sperm fractions after density gradient separation (pellet and interface). The pellet yielded a slightly lower proportion of cPARP spermatozoa (28.5±1.2% vs 36.2±2.0% in the interface; p < 0.001), and a 12-h incubation increased cPARP similarly in both fractions (p < 0.001). cPARP seems to be an early marker of apoptosis in ram semen, although its presence in untreated samples was weakly related to worse quality (pellet/interface). We suggest to study the relationship of PARP and cPARP levels with between-male differences on sperm fertility.

Emerging concepts on drug resistance in bladder cancer: Implications for future strategies.

The combination chemotherapies with methotrexate plus vinblastine, doxorubicin and cisplatin (MVAC or CMV regimens) or gemcitabine plus cisplatin represent the standard as first-line therapy for patients with metastatic urothelial cancer. In Europe, vinflunine is an option for second-line therapy for patients progressed during first-line or perioperative platinum-containing regimen. Alternative regimens containing taxanes and/or gemcitabine may be valuated case by case. Furthermore, carboplatin should be considered in patients unfit for cisplatin both in the first and second-line setting. Based on these findings, a better comprehension of the mechanisms underlying the development of drug resistance in patients with bladder cancer will represent a major step forward in optimizing patients' outcome. This article reviews the current knowledge of the mechanisms and emerging strategies to overcome resistance in patients with advanced urothelial cancer.

A Clickable Aminooxy Probe for Monitoring Cellular ADP-Ribosylation.

ADP-ribosylation is essential for cell function, yet there is a dearth of methods for detecting this post-translational modification in cells. Here, we describe a clickable aminooxy alkyne (AO-alkyne) probe that can detect cellular ADP-ribosylation on acidic amino acids following Cu-catalyzed conjugation to an azide-containing reporter. Using AO-alkyne, we show that PARP10 and PARP11 are auto-ADP-ribosylated in cells. We also demonstrate that AO-alkyne can be used to monitor stimulus-induced ADP-ribosylation in cells. Functional studies using AO-alkyne support a previously unknown mechanism for ADP-ribosylation on acidic amino acids, wherein a glutamate or aspartate at the initial C1'-position of ADP-ribose transfers to the C2' position. This new mechanism for ADP-ribosylation has important implications for how glutamyl/aspartyl-ADP-ribose is recognized by proteins in cells.