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.

PD-L1, PARP1, and MMRs as potential therapeutic biomarkers for neuroendocrine cervical cancer.

OBJECTIVE: Neuroendocrine cervical cancer (NECC) is a rare cervical cancer with high aggressivity that causes poor prognosis even in the early stage. Given other neuroendocrine carcinomas and other types of cervical cancer have been proved to have expression of programmed cell death protein 1 ligand 1(PD-L1) and poly ADP-ribose polymerase-1(PARP1), we would measure and analyze these proteins in this invasive cancer. The purpose of this study is to investigate the application value of PD-1/PD-L1 and PARP1 inhibitors in NECC. METHODS: The NECC cases in our center with formalin-fixed paraffin-embedded tissue blocks were collected, and immunohistochemical (IHC) staining of PD-L1, PARP1, Mismatch repair proteins (MMRs), and P53 was performed. Chi-square test was used to analyze associations between various protein expressions. We analyzed the efficacy of immunotherapy in a recent patient with secondary recurrence after two courses of chemotherapy. RESULTS: After rigorous screening, 20 cases were finally included. Three cases did not undergo surgical treatment because of their advanced stage. Twelve (60%) developed distant metastases or relapsed within five years, and most of them within two years. The positive rate of PD-L1 and PARP1 were 70% and 75% respectively. Among all the cases, microsatellite instability (MSI) was seen in six cases (30%) and abnormal p53 expression was in 15 patients (75%). PD-L1 was associated with PARP1 expression in the MSI subgroup. The patient treated with chemotherapy + VEGF inhibitor (VEGFi) + programmed cell death protein 1(PD-1) inhibitor had an excellent improvement in clinical symptoms, tumor markers, and mass size. CONCLUSION: The IHC results of PD-L1, PARP1, and MMRs suggested that NECC was the target of immunotargeted therapy. Our case confirmed that immune checkpoint therapy was effective in patients with PD-L1 positive and MMRs loss. Considering the clinical practicability, more cases should be collected, and effective biomarkers still need to be further searched.

Role of chromatin assembly factor-1/p60 and poly [ADP-ribose] polymerase 1 in mycosis fungoides.

Mycosis fungoides (MF) represents the most common type of cutaneous lymphoma. In the majority of patients, the disease has a slow evolution and a protracted course; however, a subset of patients shows poor oncologic outcomes. Unfortunately, there are no reliable prognostic markers for MF, and the currently available treatments are only effective in a minority of patients. This study aimed to evaluate the expression and clinical significance of PARP-1 and CAF-1/p60 in MF. Sixty-four MF representatives of the different stages of disease were assessed by immunohistochemistry for PARP-1 and CAF-1/p60. The association of PARP-1 and CAF-1/p60 with the MF stage and outcome was assessed by using Fisher's exact test and Kaplan-Meier survival analysis with the Log-rank test; a p value < 0.05 was considered significant. PARP-1 was overexpressed in 57.9% of MF and was significantly associated with a MF stage > II (p = 0.034) but not with the risk of death (p = 0.237). CAF-1/p60 was overexpressed in 26.8% of MF and was significantly associated with decreased overall survival (p < 0.001) but not with the MF stage (p = 1). A significant association was found between PARP-1 overexpression and CAF-1/p60 overexpression (p = 0.0025). Simultaneous overexpression of PARP-1 and CAF-1/p60 was significantly associated with decreased overall survival (p < 0.001), although less strongly than CAF-1/p60 alone (χ2 = 14.916 vs 21.729, respectively). In MF, PARP-1 is overexpressed in advanced stages, while CAF-1/p60 is overexpressed in the cases with shorter overall survival, appearing as a significant prognostic marker. A role for PARP-1 inhibitors and anti-CAF-1/p60 targeted therapy may be reasonably hypothesized in MF.

Olaparib-Based Photoaffinity Probes for PARP-1 Detection in Living Cells.

The poly-ADP-ribose polymerase (PARP) is a protein from the family of ADP-ribosyltransferases that catalyzes polyadenosine diphosphate ribose (ADPR) formation in order to attract the DNA repair machinery to sites of DNA damage. The inhibition of PARP activity by olaparib can cause cell death, which is of clinical relevance in some tumor types. This demonstrates that quantification of PARP activity in the context of living cells is of great importance. In this work, we present the design, synthesis and biological evaluation of photo-activatable affinity probes inspired by the olaparib molecule that are equipped with a diazirine for covalent attachment upon activation by UV light and a ligation handle for the addition of a reporter group of choice. SDS-PAGE, western blotting and label-free LC-MS/MS quantification analysis show that the probes target the PARP-1 protein and are selectively outcompeted by olaparib; this suggests that they bind in the same enzymatic pocket. Proteomics data are available via ProteomeXchange with identifier PXD018661.

Renewable electrochemical sensor for PARP-1 activity detection based on host-guest recognition.

Poly (ADP-ribose) polymerase-1 (PARP-1) was defined as a new biomarker, which has achieved wide attention in recent years. In this work, we designed a renewable electrochemical sensor based on host-guest recognition for the detection of PARP-1 activity. Mono-(6-Mercapto-6-deoxy)-beta-Cyclodextrin (SH-ß-CD) was modified on the electrode surface to recognize the trans-azobenzene labeled dsDNA (Abz-dsDNA). In the presence of PARP-1, PAR with abundant PO43- was generated and reacted with MoO42- to form PMo12O403-, producing strong current. The proposed method avoided the unspecific adsorption effectively and improved the detection accuracy. Under UV irradiation, Abz-dsDNA was removed from the electrode surface because of the configuration change of azobenzene from trans to cis structure, allowing the electrode to be recycled. The sensor realized the linear detection of PARP-1, ranging from 0.01 U to 1.0 U with a detection limit of 0.008 U, which is comparable to results from reported methods. It is expected to be a potential tool for clinical detection because of its high sensitivity and selectivity.

Quartz Crystal Microbalance Detection of Poly(ADP-ribose) Polymerase-1 Based on Gold Nanorods Signal Amplification.

Recent findings have thrust poly(ADP-ribose) polymerase-1 (PARP-1) into the limelight as a potential biomarker and chemotherapeutic target for cancer. Thus, a sensitive method for detection of PARP-1 is necessary for early diagnosis of cancer and drug development. However, the poor electrochemical and optical activity of PARP-1 and its product poly(ADP-ribose) (PAR) prompted researchers to develop more methods. Here, we developed an efficient method for the determination of PARP-1 by using quartz crystal microbalance (QCM) because it is mass-sensitive. Once activated by the specific DNA, PARP-1 cleaves nicotinamideadenine dinucleotide (NAD+) into nicotinamide and ADP-ribose to synthesize a hyperbranched poly(ADP-ribose) polymer. Although QCM is mass-sensitive, it is not sensitive enough to discern PAR effectively. So, positively charged cetyltrimethylammonium bromide (CTAB)-coated gold nanorods (GNRs) were introduced to increase the frequency change significantly because of the strong electrostatic interaction between them with negatively charged PAR. PARP-1 ranging from 0.06 to 3 nM can be facilely detected with a low detection limit of 0.04 nM. The strategy has been used to evaluate PARP-1 inhibitors and to detect PARP-1 activity in real cancer cells lysate with satisfactory results, indicating that it was a promising candidate for clinical diagnosis and drug screening in the future.

Simultaneous dual-channel imaging to quantify interdependent protein recruitment to laser-induced DNA damage sites.

Fluorescence microscopy in combination with the induction of localized DNA damage using focused light beams has played a major role in the study of protein recruitment kinetics to DNA damage sites in recent years. Currently published methods are dedicated to the study of single fluorophore/single protein kinetics. However, these methods may be limited when studying the relative recruitment dynamics between two or more proteins due to cell-to-cell variability in gene expression and recruitment kinetics, and are not suitable for comparative analysis of fast-recruiting proteins. To tackle these limitations, we have established a time-lapse fluorescence microscopy method based on simultaneous dual-channel acquisition following UV-A-induced local DNA damage coupled with a standardized image and recruitment analysis workflow. Simultaneous acquisition is achieved by spectrally splitting the emitted light into two light paths, which are simultaneously imaged on two halves of the same camera chip. To validate this method, we studied the recruitment of poly(ADP-ribose) polymerase 1 (PARP1), poly (ADP-ribose) glycohydrolase (PARG), proliferating cell nuclear antigen (PCNA) and the chromatin remodeler ALC1. In accordance with the published data based on single fluorophore imaging, simultaneous dual-channel imaging revealed that PARP1 regulates fast recruitment and dissociation of PARG and that in PARP1-depleted cells PARG and PCNA are recruited with comparable kinetics. This approach is particularly advantageous for analyzing the recruitment sequence of fast-recruiting proteins such as PARP1 and ALC1, and revealed that PARP1 is recruited faster than ALC1. Split-view imaging can be incorporated into any laser microirradiation-adapted microscopy setup together with a recruitment-dedicated image analysis package.

Detection of PARP-1 activity based on hyperbranched-poly (ADP-ribose) polymers responsive current in artificial nanochannels.

The cellular enzyme poly ADP (ADP: adenosine diphosphate)-ribose polymerase-1 (PARP-1) plays key roles in DNA repair. Its activity is closely related to various cancer developments. Detection of PARP-1 activity is significant, however, it is relatively difficult since it lacks superiority property that can be used to detect conveniently. PARP-1 lead to the synthesis of hyperbranched poly (ADP-ribose) polymers (PAR) using nicotinamide adenine dinucleotide (NAD+) as substrate during DNA damage repairing. In this paper, we found that hyper-branched PAR increased the steric hindrance and reduced the flux of probe ions effectively in anodic aluminum oxide (AAO) nanochannels. To the best of our knowledge, few papers have been reported that hyper-branched polymer has the similar effects in nanochannels as G-quadruplex DNA. Thus, a novel and simple strategy for PARP-1 detection has been proposed due to its great impacts on the diffusion flux of ferricyanide in AAO. It is also proved that electrostatic repulsion is another important factor to influence the current. The method is label-free, simple and sensitive. Quantitative detection of PARP-1 activity was achieved with the detection limit of 0.006 U, which is lower or comparable to the most reported methods. The method has good accuracy and reproducibility. The strategy has been used to detect PARP-1 activity in real breast cancer cells and to evaluate PARP-1 inhibitors with satisfactory results, indicating that it is a potential powerful tool for clinical diagnosis and drug development in the future.

A rapid fluorescent method for the real-time measurement of poly(ADP-ribose) polymerase 1 activity.

Poly(ADP-ribose) polymerase 1 (PARP1) is a key enzyme that regulates important cellular processes, including DNA repair. PARP1 binds to a DNA damage site and synthesizes poly(ADP-ribose) chains (PARs), which serve as a signal of DNA damage for other DNA repair enzymes. PARP1 is a recognized target for the development of anti-cancer drugs. In this work, a method is developed that makes it possible to investigate the complex formation of PARP1 with DNA as well as its dissociation by detecting the fluorescence anisotropy of this complex during the poly(ADP-ribose) synthesis. The method allows investigation of the inhibition of PARP1 activity in the presence of its inhibitors. In this work, we demonstrated that PARP1 is activated by DNA duplexes containing a damage and a fluorophore at the 3'-end of one of the DNA duplex chains. The effects of the clinical inhibitor olaparib on the activity of PARP1 was studied. It was shown that olaparib has no influence on the binding of PARP1 to the model DNA structures used, but it significantly inhibits the poly(ADP-ribosyl)ation of PARP1. The proposed convenient method for the real-time determination of the PARP1 activity can be used to screen PARP1 inhibitors with the calculation of quantitative inhibition parameters.

Ultrasensitive electrochemical detection of poly (ADP-ribose) polymerase-1 via polyaniline deposition.

Recent findings have thrust poly ADP (ADP: adenosine diphosphate)-ribose polymerase-1 (PARP-1) into the limelight as potential chemotherapeutic target because it is closely related to the development of tumor. So, studies on its detection and inhibitors evaluation have attracted more attention. It is interesting that poly (ADP-ribose) (PAR), the catalytic product of PARP-1 in the existence of nicotinamide adenine dinucleotide (NAD+), possess twice charge density of DNA strands. PAR contain 200 units, i.e., about 400bp bases, and multiple branched strands. So, plentiful negative charges on PAR supplied exquisite environment for PANI deposition, which was triggered by horseradish peroxidase (HRP). Because of the unique electrochemical property of PANI, ultrasensitive electrochemical detection of PARP-1 was proposed. Under optimum conditions, DPV intensity linearly increased with the increment of PARP-1 in the range of 0.005-1.0 U. The detection limit was 0.002 U, which was comparable or more sensitive than that obtained from previously reported strategies.

Expression and Single Nucleotide Polymorphism of Poly (ADPRibose) Polymerase-1 in Gastrointestinal Tumours: Clinical Involvement.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that plays a critical role in diverse cellular functions, such as DNA damage detection and repair, transcriptional regulation and cell death. Furthermore, PARP-1 has emerged as a key player in the pathogenesis of multiple inflammatory diseases and has become a promising target for the treatment of cardiovascular disorders, neurodegenerative diseases and cancer. An increasing body of evidence has linked alterations in the expression levels of PARP-1, enzymatic activity and presence of polymorphism to gastrointestinal malignancies, including oesophageal, gastric, pancreas, liver and colorectal cancers. PARP inhibition has been proposed as a valuable strategy for treating these gastrointestinal disorders. This paper summarises the most significant current literature on the involvement of PARP-1 in gastrointestinal cancer, focusing in particular on its role in the development and occurrence of tumours, providing information about clinical trials and exploring therapeutic possibilities.

Biomarker-Based PET Imaging of Diffuse Intrinsic Pontine Glioma in Mouse Models.

Diffuse intrinsic pontine glioma (DIPG) is a childhood brainstem tumor with a universally poor prognosis. Here, we characterize a positron emission tomography (PET) probe for imaging DIPG in vivo In human histological tissues, the probes target, PARP1, was highly expressed in DIPG compared to normal brain. PET imaging allowed for the sensitive detection of DIPG in a genetically engineered mouse model, and probe uptake correlated to histologically determined tumor infiltration. Imaging with the sister fluorescence agent revealed that uptake was confined to proliferating, PARP1-expressing cells. Comparison with other imaging technologies revealed remarkable accuracy of our biomarker approach. We subsequently demonstrated that serial imaging of DIPG in mouse models enables monitoring of tumor growth, as shown in modeling of tumor progression. Overall, this validated method for quantifying DIPG burden would serve useful in monitoring treatment response in early phase clinical trials. Cancer Res; 77(8); 2112-23. ©2017 AACR.

A Phase 1 Study of the PARP Inhibitor Veliparib in Combination with Temozolomide in Acute Myeloid Leukemia.

PURPOSE: In preclinical studies, the PARP inhibitor veliparib enhanced the antileukemic action of temozolomide through potentiation of DNA damage. Accordingly, we conducted a phase 1 study of temozolomide with escalating doses of veliparib in patients with relapsed, refractory acute myeloid leukemia (AML) or AML arising from aggressive myeloid malignancies. EXPERIMENTAL DESIGN: Patients received veliparib [20-200 mg once a day on day 1 and twice daily on days 4-12 in cycle 1 (days 1-8 in cycle ≥2)] and temozolomide [150-200 mg/m2 daily on days 3-9 in cycle 1 (days 1-5 in cycle ≥2)] every 28 to 56 days. Veliparib pharmacokinetics and pharmacodynamics [ability to inhibit poly(ADP-ribose) polymer (PAR) formation and induce H2AX phosphorylation] were assessed. Pretreatment levels of MGMT and PARP1 protein, methylation of the MGMT promoter, and integrity of the Fanconi anemia pathway were also examined. RESULTS: Forty-eight patients were treated at seven dose levels. Dose-limiting toxicities were oral mucositis and esophagitis lasting >7 days. The MTD was veliparib 150 mg twice daily with temozolomide 200 mg/m2 daily. The complete response (CR) rate was 17% (8/48 patients). Veliparib exposure as well as inhibition of PAR polymer formation increased dose proportionately. A veliparib-induced increase in H2AX phosphorylation in CD34+ cells was observed in responders. Three of 4 patients with MGMT promoter methylation achieved CR. CONCLUSIONS: Veliparib plus temozolomide is well tolerated, with activity in advanced AML. Further evaluation of this regimen and of treatment-induced phosphorylation of H2AX and MGMT methylation as potential response predictors appears warranted. Clin Cancer Res; 23(3); 697-706. ©2016 AACR.

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.

Assessment of PARP protein expression in epithelial ovarian cancer by ELISA pharmacodynamic assay and immunohistochemistry.

Targeting Poly (ADP-ribose) polymerase 1 (PARP-1) involved in base excision repair (BER) has been shown to be a clinically effective treatment strategy in epithelial ovarian cancer (EOC) defective in homologous recombination (HR). The aim of this study was to evaluate fresh EOC tumor tissue in regard to PAR (Poly (ADP-ribose)) concentration as a surrogate marker for PARP activity and PARP protein expression in archival samples by immunohistochemistry (IHC). The prospective study cohort consisted of 57 fresh tumor samples derived from patients undergoing primary (n = 38) or interval debulking surgery (n = 19) for EOC and parallel archival paraffin-embedded tumor samples. PARP activity in fresh frozen tumor tissue was assessed by an enzymatic chemiluminescence assay and PARP protein expression in paraffin-embedded tumor tissue by IHC. No correlation was detected between PARP enzyme activity and PARP staining by IHC (p = 0.82). High PARP activity was associated with platinum sensitivity both in the entire study cohort (p = 0.022) and in the high-grade subgroup (p = 0.017). High PARP activity was also associated with improved progression-free survival (PFS) (32 vs 14 months, log-rank p = 0.009). However, PARP immunostaining pattern was not predictive of patient survival. In conclusion, we present a novel finding of high PARP activity associated with platinum sensitivity and improved PFS in EOC. There was no association between PARP IHC and pharmacodynamic assay, and the correlation of PARP IHC with clinico-pathological characteristics and patient survival was poor. Pharmacodynamic assay rather than IHC seems to reflect better biologically significant PARP.

Assessment of calpain and caspase systems activities during ageing of two bovine muscles by degradation patterns of αII spectrin and PARP-1.

The activities of calpain and caspase systems during ageing in Longissimus lumborum (LL) and Infraspinatus (IS) muscles of Italian Simmental young bulls (Bos taurus) were assessed. Samples from 10 animals were collected within 20 min of exsanguination (T0), after 48 h (T1) and 7 days (T2) post mortem. Calpain and caspase activity were evaluated based on the formation of αII spectrin cleavage products of 145 kDa (SBDP145) and 120 kDa (SBDP120), respectively. Caspase activity was also assessed by the presence of poly (adenosine diphosphate-ribose) polymerase-1 (PARP-1) cleavage product. At T0, LL showed higher levels of SBDP145 than IS (P < 0.01), while SBDP120 and PARP-1 degradation products were similar between muscles. At T1, no difference was found in the level of SBDP145 between muscles, while SBDP120 and PARP-1 cleavage products were not detected. At T2 neither αII spectrin nor PARP-1 cleavage products were found. LL and IS showed different proteolysis after slaughter that was influenced more by calpain than caspase activity, which was detectable only in the early post mortem period.

Enhanced efficacy of combined HDAC and PARP targeting in glioblastoma.

Recent clinical trials have demonstrated that targeting chromatin remodeling factors is as a promising strategy for the treatment of glioblastoma (GBM). We and others have shown constitutive activation of DNA damage response (DDR) pathways in gliomas and suggested that targeting the DDR may improve the currently grim prognosis for patients. Based on our previous findings that inhibition of poly(ADP-ribose) polymerase (PARP) increases radio-sensitivity of the notoriously radio-resistant GBM cells, we hypothesized that epigenetic down-regulation of the DDR responses and induction of oxidative stress via HDAC inhibition would contribute to more efficient targeting of this deadly disease. Our data show that SAHA, an HDAC class I + II inhibitor, in combination with olaparib (PARP inhibitor): i) enhanced inhibition of GBM cell survival, ii) induced apoptosis, and iii) impaired cell cycle progression. These results provide a pre-clinical rationale for combined administration of SAHA and olaparib, which are already individually in clinical trials.