Expression Of Poly ADP-ribose Polymerase Research Articles

Inhibition of PARP1 improves cardiac function after myocardial infarction via up-regulated NLRC5

The incidence and mortality rate of myocardial infarction are increasing per year in China. The polarization of macrophages towards the classically activated macrophages (M1) phenotype is of utmost importance in the progression of inflammatory stress subsequent to myocardial infarction. Poly(ADP-ribose) polymerase 1(PARP1) is the ubiquitous and best characterized member of the PARP family, which has been reported to support macrophage polarization towards the pro-inflammatory phenotype. Yet, the role of PARP1 in myocardial ischemic injury remains to be elucidated. Here, we demonstrated that a myocardial infarction mouse model induced cardiac damage characterized by cardiac dysfunction and increased PARP1 expression in cardiac macrophages. Inhibition of PARP1 by the PJ34 inhibitors could effectively alleviate M1 macrophage polarization, reduce infarction size, decrease inflammation and rescue the cardiac function post-MI in mice. Mechanistically, the suppression of PARP1 increase NLRC5 gene expression, and thus inhibits the NF-κB pathway, thereby decreasing the production of inflammatory cytokines such as IL-1β and TNF-α. Inhibition of NLRC5 promote infection by effectively abolishing the influence of this mechanism discussed above. Interestingly, inhibition of NLRC5 promotes cardiac macrophage polarization toward an M1 phenotype but without having major effects on M2 macrophages. Our results demonstrate that inhibition of PARP1 increased NLRC5 gene expression, thereby suppressing M1 polarization, improving cardiac function, decreasing infarct area and attenuating inflammatory injury. The aforementioned findings provide new insights into the proinflammatory mechanisms that drive macrophage polarization following myocardial infarction, thereby introducing novel potential targets for future therapeutic interventions in individuals affected by myocardial infarction.

Design, synthesis, and biological evaluation of novel chrysin derivatives as poly(ADP-ribose) polymerase 1 (PARP1) inhibitors for the treatment of breast cancer

In this study, we reported the discovery and structure-activity relationship analysis of chrysin derivatives as a new class of inhibitors targeting poly (ADP-ribose) polymerase 1 (PARP1). Among these derivatives, compound 5d emerged as the most effective chrysin-based inhibitor of PARP1, with an IC50 value of 108 nmol·L−1. This compound significantly inhibited the proliferation and migration of breast cancer cell lines HCC-1937 and MDA-MB-436 by inducing DNA damage. Furthermore, 5d induced apoptosis and caused an extended G1/S-phase in these cell lines. Molecular docking studies revealed that 5d possesses a strong binding affinity toward PARP1. In vivo, in a xenograft model, 5d effectively reduced tumor growth by downregulating PARP1 expression. Overall, compound 5d shows promise as a potential therapeutic agent for the treatment of BRCA wild-type breast cancer.

Poly (ADP-ribose) polymerase-1 (PARP-1) is a good prognostic marker for pancreatic/periampullary cancers.

Periampullary cancer (PAC) is highly aggressive with no effective adjuvant therapy or prognostic markers. Recently, poly (ADP-ribose) polymerases-1 (PARP-1) have emerged as a target in solid cancers, and its relationship with epithelial-mesenchymal transition (EMT) has been observed. However, the relationship between PARP-1 and EMT in PAC has not explored well. We assessed the prognostic significance of PARP1 in 190 PACs patients and correlated it with EMT markers, including FGF8, FGFR4, MMP2, MMP3, Snail, and ZEB1. Immunohistochemistry for PARP-1 and EMT markers was performed using a tissue microarray. PARP-1 and FGF8 expression were associated with better survival unlike other solid cancers (P = 0.006 and P = 0.003), and MMP3 and ZEB1 expression were associated with poor prognosis in multivariate and survival analyses (P = 0.009 and P < 0.001). In addition, PARP-1 is related negatively to Snail but not related with other EMT markers, implying an independent mechanism between PARP-1 and EMT in PACs. PARP-1 and FGF8 are independent good survival markers in PACs unlike other solid cancers. PARP-1 and FGF8 in PACs could not be related to the EMT pathway but must be rather understood in light of similar cancer-protective roles. Further studies are required on EMT-associated immune markers in PACs.

Nicaraven attenuates the acquired radioresistance of established tumors in mouse models via PARP inhibition.

Nicaraven has been reported to inhibit the activity of poly (ADP-ribose) polymerase (PARP). In this study, we investigated the probable ability of nicaraven to attenuate cancer radioresistance during fractionated radiotherapy. Tumor models were established in C57BL/6 mice and BALB/c nude mice by subcutaneous injection of Lewis mouse lung carcinoma cancer cells and A549 human lung cancer cells, respectively. When the tumors had grown to approximately 100 mm3, we initiated fractionated radiotherapy. Nicaraven or saline was administered immediately after each irradiation exposure. Compared to saline treatment, nicaraven administration significantly induced gamma-H2AX foci formation and cell apoptosis in tumors at 1 or 3days after an additional challenge exposure to 10Gy and inhibited tumor growth during the short-term follow-up period, suggesting increased radiosensitivity of cancer cells. Moreover, the expression of PARP in tumor tissue was decreased by nicaraven administration. Our data suggest that nicaraven likely attenuates the acquired radioresistance of cancers through PARP inhibition.

NAMPT/NAD+/PARP1 Pathway Regulates CFA-Induced Inflammatory Pain via NF-κB Signaling in Rodents.

Emerging evidence has implicated nicotinamide adenine dinucleotide (NAD+) metabolism in various inflammatory diseases. In the study, the role of NAD+ metabolism in Complete Freund's Adjuvant (CFA)-evoked inflammatory pain and the underlying mechanisms are investigated. The study demonstrated that CFA induced upregulation of nicotinamide phosphoribosyltransferase (NAMPT) in dorsal root ganglia (DRG) without significant changes in the spinal cord. Inhibition of NAMPT expression by intrathecal injection of NAMPT siRNA alleviated CFA-induced pain-like behavior, decreased NAD+ contents in DRG, and lowered poly-(ADP-ribose) polymerase 1 (PARP1) activity levels. These effects are all reversed by the supplement of nicotinamide mononucleotide (NMN). Inhibition of PARP1 expression by intrathecal injection of PARP1 siRNA alleviated CFA-induced pain-like behavior, while elevated NAD+ levels of DRG. The analgesic effect of inhibiting NAMPT/NAD+/PARP1 axis can be attributed to the downregulation of the NF-κB/IL-1β inflammatory pathway. Double immunofluorescence staining showed that the expression of NAMPT/NAD+/PARP1 axis is restricted to DRG neurons. In conclusion, PARP1 activation in response to CFA stimulation, fueled by NAMPT-derived NAD+, mediates CFA-induced inflammatory pain through NF-κB/IL-1β inflammatory pathway.

PARP1-targeted fluorescence molecular endoscopy as novel tool for early detection of esophageal dysplasia and adenocarcinoma

BackgroundEsophageal cancer is one of the 10 most common cancers worldwide and its incidence is dramatically increasing. Despite some improvements, the current surveillance protocol with white light endoscopy and random untargeted biopsies collection (Seattle protocol) fails to diagnose dysplastic and cancerous lesions in up to 50% of patients. Therefore, new endoscopic imaging technologies in combination with tumor-specific molecular probes are needed to improve early detection. Herein, we investigated the use of the fluorescent Poly (ADP-ribose) Polymerase 1 (PARP1)-inhibitor PARPi-FL for early detection of dysplastic lesions in patient-derived organoids and transgenic mouse models, which closely mimic the transformation from non-malignant Barrett’s Esophagus (BE) to invasive esophageal adenocarcinoma (EAC).MethodsWe determined PARP1 expression via immunohistochemistry (IHC) in human biospecimens and mouse tissues. We also assessed PARPi-FL uptake in patient- and mouse-derived organoids. Following intravenous injection of 75 nmol PARPi-FL/mouse in L2-IL1B (n = 4) and L2-IL1B/IL8Tg mice (n = 12), we conducted fluorescence molecular endoscopy (FME) and/or imaged whole excised stomachs to assess PARPi-FL accumulation in dysplastic lesions. L2-IL1B/IL8Tg mice (n = 3) and wild-type (WT) mice (n = 2) without PARPi-FL injection served as controls. The imaging results were validated by confocal microscopy and IHC of excised tissues.ResultsIHC on patient and murine tissue revealed similar patterns of increasing PARP1 expression in presence of dysplasia and cancer. In human and murine organoids, PARPi-FL localized to PARP1-expressing epithelial cell nuclei after 10 min of incubation. Injection of PARPi-FL in transgenic mouse models of BE resulted in the successful detection of lesions via FME, with a mean target-to-background ratio > 2 independently from the disease stage. The localization of PARPi-FL in the lesions was confirmed by imaging of the excised stomachs and confocal microscopy. Without PARPi-FL injection, identification of lesions via FME in transgenic mice was not possible.ConclusionPARPi-FL imaging is a promising approach for clinically needed improved detection of dysplastic and malignant EAC lesions in patients with BE. Since PARPi-FL is currently evaluated in a phase 2 clinical trial for oral cancer detection after topical application, clinical translation for early detection of dysplasia and EAC in BE patients via FME screening appears feasible.

An antihypertensive drug-AT1 inhibitor attenuated BRCA development promoted by chronic psychological stress via Ang II/PARP1/FN1 pathway

Chronic psychological stress contributes to the occurrence of cancer and activates the renin-angiotensin system (RAS). However, the mechanisms by which RAS promotes the progression of breast cancer (BRCA) under chronic psychological stress are remain unknown. In this study, we observed elevated levels of Angiotensin II (Ang II) in both serum and BRCA tissue under chronic stress, leading to accelerated BRCA growth in a mouse model. An antihypertensive drug, candesartan (an AT1 inhibitor), effectively attenuated Ang II-induced cell proliferation and metastasis. Utilizing mass spectrometry and weighted gene co-expression network analysis (WGCNA), we identified fibronectin 1 (FN1) as the hub protein involved in chronic stress-Ang II/AT1 axis. Focal adhesion pathway was identified as a downstream signaling pathway activated during the progression of chronic stress. Depletion of FN1 significantly attenuated Ang II-induced proliferation and metastasis of BRCA cells. Poly (ADP-ribose) polymerase 1 (PARP1) was found to bind to the DNA promoter of FN1, leading to the transcription of FN1. Ang II upregulated PARP1 expression, resulting in increased FN1 levels. Recombinant FN1 partially restored the progress of BRCA malignancy induced by the Ang II/PARP1 pathway. In vivo, candesartan reversed the progressive effect of chronic psychological stress on BRCA. In clinical samples, Ang II levels in both serum and tumor tissues are higher in stressed patients compared to control patients. Serum Ang II levels were positively correlated with chronic stress indicators. In conclusion, our study demonstrated that chronic psychological stress accelerates the malignancy of BRCA, and the AT1 inhibitor candesartan counteracts these effects by suppressing the Ang II-AT1 axis and the downstream PARP1/FN1/focal adhesion pathway.

Exposure to fluoride exacerbates the cognitive deficit of diabetic patients living in areas with endemic fluorosis, as well as of rats with type 2 diabetes induced by streptozotocin via a mechanism that may involve excessive activation of the poly(ADP ribose) polymerase-1/P53 pathway

Epidemiology has shown that fluoride exposure is associated with the occurrence of diabetes. However, whether fluoride affects diabetic encephalopathy is unclear. Elderly diabetic patients in areas with endemic (n = 169) or no fluorosis (108) and controls (85) underwent Montreal Cognitive Assessment. Sprague-Dawley rats receiving streptozotocin and/or different fluoride doses were examined for spatial learning and memory, brain morphology, blood-brain barrier, fasting blood glucose and insulin. Cultured SH-SY5Y cells were treated with 50 mM glucose and/or low- or high-dose fluoride, and P53-knockdown or poly-ADP-ribose polymerase-1 (PARP-1) inhibition. The levels of PARP-1, P53, poly-ADP-ribose (PAR), apoptosis-inducing factor (AIF), and phosphorylated-histone H2A.X (ser139) were measured by Western blotting. Reactive oxygen species (ROS), 8-hydroxydeguanosine (8-OHdG), PARP-1 activity, acetyl-P53, nicotinamide adenine dinucleotide (NAD+), activities of mitochondrial hexokinase1 (HK1) and citrate synthase (CS), mitochondrial membrane potential and apoptosis were assessed biochemically. Cognition of diabetic patients in endemic fluorosis areas was poorer than in other regions. In diabetic rats, fasting blood glucose, insulin resistance and blood-brain barrier permeability were elevated, while spatial learning and memory and Nissl body numbers in neurons declined. In these animals, expression and activity of P53 and PARP-1 and levels of NAD+, PAR, ROS, 8-OHdG, p-histone H2A.X (ser139), AIF and apoptosis content increased; whereas mitochondrial HK1 and CS activities and membrane potential decreased. SH-SY5Y cells exposed to glucose exhibited changes identical to diabetic rats. The changes in diabetic rats and cells treated with glucose were aggravated by fluoride. P53-knockout or PARP-1 inhibition mitigated the effects of glucose with/without low-dose fluoride. Elevation of diabetic encephalopathy was induced by exposure to fluoride and the underlying mechanism may involve overactivation of the PARP-1/P53 pathway.

Generation of Bispecific Antibodies by Functionalized Poly-ADP-Ribose Polymers.

Bispecific antibodies have drawn considerate research interests for therapeutic development. Numerous genetic and chemical methods are established to produce bispecific antibodies with varied formats. This protocol describes a novel approach to the synthesis of bispecific antibodies by utilizing chemically functionalized poly-ADP-ribose polymers derived from post-translational poly-ADP-ribosylation. Basic Protocol 1 includes experimental procedures for expressing and purifying recombinant full-length human poly-ADP-ribose polymerase 1 (PARP1) as well as monoclonal antibodies targeting T-cell CD3 and breast cancer tumor-associated human epidermal growth factor receptor 2 (HER2) molecules. Basic Protocol 2 details methods for enzymatic preparation of functionalized poly-ADP-ribose polymers by PARP1 and chemical conjugation of antibody molecules for bispecific antibody production. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of PARP1 and antibodies Basic Protocol 2: PARP1 auto-modification and antibody conjugation.

The Relationship Between Poly-ADP-Ribose Polymerase-1 (PARP-1) Expression with Clinicopathological Characteristics of Nasopharyngeal Carcinoma in Bali, Indonesia

Background: According to epidemiological research conducted in Bali, nasopharyngeal carcinoma (NPC) is the most common cancer among males, accounting for 4.9% of total cases reported between 2017 and 2019. NPC is typically detected at stage IV, indicating the presence of distant metastasis by malignant cells. However, research on the role of Poly-ADP-Ribose Polymerase-1 (PARP-1) in this type of cancer is still limited. Therefore, this research aims to investigate the relationship between PARP-1 and the clinicopathological aspects of NPC.Methods: This research employed an analytical observational design with a cross-sectional approach. From January 2018 to July 2022, biopsy tissue samples were collected from NPC patients, strictly following predefined inclusion and exclusion criteria. The clinical data of these patients were extracted from electronic medical records. Subsequently, the collected samples were subjected to immunohistochemical staining for PARP-1. The interpretation of PARP-1 results was conducted using the H-Score method. Data were analyzed using statistical software, specifically SPSS version 25.Results: The results showed that high PARP-1 expression was detected in 18 samples (62.1%), while the low aspect was observed in 11 samples (37.9%). Independent T-test analysis was conducted for sex, age, and clinical stage. No significant difference was found in the mean PARP-1 H-score among groups based on gender, age, and lymph node enlargement, with p-values of 0.68, 0.71, and 0.74, respectively. However, a statistically significant association was found between PARP-1 expression and clinical stage (p=0.02, 95% CI 1.45 – 63.50). Multivariate analysis showed that sex, age, lymph node enlargement, and clinical stage accounted for 22.1% of the variation in PARP-1 expression, with the clinical stage demonstrating a significant correlation (p=0.02). Conclusions: The clinicopathological data of NPC indicate a male-to-female ratio of 3:1. It was discovered that clinical stages IVA and IVB were the most commonly observed stages. Statistically significant differences were found in the mean and proportion of PARP-1 H-scores across different clinical stages. Future reviews need to include other histological subtypes and employ a prospective research design to evaluate the relationship between PARP-1 and NPC.

Folic acid supplementation rescues bladder injury in fetal rats with myelomeningocele.

Bladder dysfunction has been linked to the progression of renal failure in children with neurogenic bladder (NB) dysfunction. The purpose of this study was to determine whether bladder injuries in fetal rats with myelomeningocele (MMC) may be treated with folic acid. Pregnant Sprague-Dawley rats were randomly divided into three groups. On the 10th day of gestation, pregnant rats were intragastrically injected with all-trans retinoic acid (ATRA) (60 mg/kg) to induce MMC fetal rats. The same amount of olive oil was put into the control group to create normal fetal rats. The rats in the rescue group were given folic acid (40 mg/kg) by gavage 0.5 and 12 hr after ATRA therapy. Bladders were obtained via cesarean section on embryonic day E20.5 and examined for MMC. The histology of the fetuses was examined using hematoxylin and eosin staining, and immunohistochemistry (IHC) was utilized to determine the expression of α-smooth muscle actin (α-SMA) and neuron-specific nuclear-binding protein (NeuN). Furthermore, the levels of neuromuscular development-related and apoptotic proteins were determined by western blotting. The incidence of MMC in the model group was 60.6% (20/33) while it was much lower in the rescue group (21.4%). In comparison to the model group, the weight and crown-rump length of the fetal rats in the rescue group were significantly improved. IHC revealed that there was no significant difference in the expression of α-SMA and NeuN between the control and ATRA groups, while the expression levels decreased significantly in the MMC group. Western blot analysis showed that there was no significant difference between the model and ATRA groups, but the expression of the α-SMA protein and the β3-tubulin was much lower in the MMC group than in the control group. After the administration of folic acid, the α-SMA and β3-tubulin proteins considerably increased in the folic acid-rescued MMC group and folic acid-rescued ATRA group. Meanwhile, in the control group, the expression of cleaved caspase-3 in the bladder tissue was significantly higher, and the expression of poly (ADP-ribose) polymerase (PARP) protein was significantly lower compared to the control group. Folic acid therapy reduced cleaved caspase-3 expression while increasing PARP expression in comparison to the MMC group. NB in MMC fetal rats is associated with the reduction of bladder nerve and smooth muscle-related protein synthesis. However, folic acid therapy can help improve these functional deficiencies. Folic acid also exhibits strong anti-apoptotic properties against NB in MMC fetal rats.

Preclinical studies of a PARP targeted, Meitner-Auger emitting, theranostic radiopharmaceutical for metastatic ovarian cancer

Advanced ovarian cancer currently has few therapeutic options. Poly(ADP-ribose) polymerase (PARP) inhibitors bind to nuclear PARP and trap the protein-inhibitor complex to DNA. This work investigates a theranostic PARP inhibitor for targeted radiopharmaceutical therapy of ovarian cancer in vitro and PET imaging of healthy mice in vivo. Methods[77Br]RD1 was synthesized and assessed for pharmacokinetics and cytotoxicity in human and murine ovarian cancer cell lines. [76Br]RD1 biodistribution and organ uptake in healthy mice were quantified through longitudinal PET/CT imaging and ex vivo radioactivity measurements. Organ-level dosimetry following [76/77Br]RD1 administration was calculated using RAPID, an in-house platform for absorbed dose in mice, and OLINDA for equivalent and effective dose in human. ResultsThe maximum specific binding (Bmax), equilibrium dissociation constant (Kd), and nonspecific binding slope (NS) were calculated for each cell line. These values were used to calculate the cell specific activity uptake for cell viability studies. The half maximal effective concentration (EC50) was measured as 0.17 (95 % CI: 0.13–0.24) nM and 0.46 (0.13–0.24) nM for PARP(+) and PARP(−) expressing cell lines, respectively. The EC50 was 0.27 (0.21–0.36) nM and 0.30 (0.22–0.41) nM for BRCA1(−) and BRCA1(+) expressing cell lines, respectively. When measuring the EC50 as a function of cellular activity uptake and nuclear dose, the EC50 ranges from 0.020 to 0.039 Bq/cell and 3.3–9.2 Gy, respectively. Excretion through the hepatobiliary and renal pathways were observed in mice, with liver uptake of 2.3 ± 0.4 %ID/g after 48 h, contributing to estimated absorbed dose values in mice of 19.3 ± 0.3 mGy/MBq and 290 ± 10 mGy/MBq for [77Br]RD1 and [76Br]RD1, respectively. Conclusion[77Br]RD1 cytotoxicity was dependent on PARP expression and independent of BRCA1 status. The in vitro results suggest that [77Br]RD1 cytotoxicity is driven by the targeted Meitner-Auger electron (MAe) radiotherapeutic effect of the agent. Further studies investigating the theranostic potential, organ dose, and tumor uptake of [76/77Br]RD1 are warranted.

Synthesis and Degradation of Poly(ADP-ribose) in Zebrafish Brain Exposed to Aluminum.

Poly(ADPribosyl)ation is a post-translational protein modification, catalyzed by poly(ADP-ribose) polymerase (PARPs) enzymes, responsible for ADP-ribose polymer synthesis (PAR) from NAD+. PAR turnover is assured by poly(ADPR) glycohydrolase (PARGs) enzymes. In our previous study, the altered histology of zebrafish brain tissue, resulting in demyelination and neurodegeneration also with poly(ADPribosyl)ation hyperactivation, was demonstrated after aluminum (Al) exposure for 10 and 15 days. On the basis of this evidence, the aim of the present research was to study the synthesis and degradation of poly(ADP-ribose) in the brain of adult zebrafish exposed to 11 mg/L of Al for 10, 15, and 20 days. For this reason, PARP and PARG expression analyses were carried out, and ADPR polymers were synthesized and digested. The data showed the presence of different PARP isoforms, among which a human PARP1 counterpart was also expressed. Moreover, the highest PARP and PARG activity levels, responsible for the PAR production and its degradation, respectively, were measured after 10 and 15 days of exposure. We suppose that PARP activation is related to DNA damage induced by Al, while PARG activation is needed to avoid PAR accumulation, which is known to inhibit PARP and promote parthanatos. On the contrary, PARP activity decrease at longer exposure times suggests that neuronal cells could adopt the stratagem of reducing polymer synthesis to avoid energy expenditure and allow cell survival.

B-cell lymphoma-extra large is a promising drug target in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive skin tumour with neuroendocrine differentiation. Immunotherapies are effective in the treatment of patients with advanced-stage MCC, but for patients whose tumours cannot be controlled by the immune system, alternative approaches are urgently needed. To identify overexpressed oncogenes as potential drug targets for MCC. NanoString platform, digital droplet polymerase chain reaction (ddPCR) and fluorescence in situ hybridization (FISH) assays were used to determine copy number variations (CNVs); BCL2L1 and PARP1 mRNA expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR), B-cell lymphoma extra-large (Bcl-xL) and poly (ADP-ribose) polymerase 1 (PARP1) protein by immuno-blot. Specific Bcl-xL inhibitors and a PARP1 inhibitor were used alone or in combination to test their antitumour effect. Screening for CNVs in 13 classic Merkel cell polyomavirus (MCPyV)-positive and MCPyV-negative MCC cell lines revealed BCL2L1 gains and amplifications, confirmed by ddPCR in 10 cell lines. By ddPCR and FISH, we demonstrated that BCL2L1 gains are present in tumour tissue. BCL2L1 copy number gains were associated with increased Bcl-xL mRNA and protein expression. However, high Bcl-xL expression was not restricted to MCC cells harbouring a BCL2L1 gain/amplification, suggesting additional epigenetic means of regulation. The functional relevance of Bcl-xL in MCC cells was demonstrated by the fact that specific Bcl-xL inhibitors (A1331852 and WEHI-539) led to the induction of apoptosis. Owing to the strong expression and activation of PARP1 in MCC cell lines, we next tested the combination of Bcl-xL inhibitors with the PARP1 inhibitor olaparib, which showed synergistic antitumour effects. Bcl-xL, which is highly expressed in MCC, appears to be an attractive therapeutic target for the treatment of this tumour, especially as the effect of specific Bcl-xL inhibitors is synergistically enhanced by simultaneous PARP inhibition.

Isometamidium chloride alters redox status, down-regulates p53 and PARP1 genes while modulating at proteomic level in Drosophila melanogaster

As trypanocide, several side effects have been reported in the use of Isometamidium chloride. This study was therefore, designed to evaluate its ability to induce oxidative stress and DNA damage using D. melanogaster as a model organism. The LC50 of the drug was determined by exposing the flies (1–3 days old of both genders) to six different concentrations (1 mg, 10 mg, 20 mg, 40 mg, 50 mg and 100 mg per 10 g of diet) of the drug for a period of seven days. The effect of the drug on survival (28 days), climbing behavior, redox status, oxidative DNA lesion, expression of p53 and PARP1 (Poly-ADP-Ribose Polymerase-1) genes after five days exposure of flies to 4.49 mg, 8.97 mg, 17.94 mg and 35.88 mg per 10 g diet was evaluated. The interaction of the drug in silico with p53 and PARP1 proteins was also evaluated. The result showed the LC50 of isometamidium chloride to be 35.88 mg per 10 g diet for seven days. Twenty-eight (28) days of exposure to isometamidium chloride showed a decreased percentage survival in a time and concentration-dependent manner. Isometamidium chloride significantly (p < 0.05) reduced climbing ability, total thiol level, Glutathione-S-transferase, and Catalase activity. The level of H2O2 was significantly (p < 0.05) increased. The result also showed significant (p < 0.05) reduction in the relative mRNA levels of p53 and PARP1 genes. The in silico molecular docking of isometamidium with p53 and PARP1 proteins showed high binding energy of −9.4 Kcal/mol and −9.2 Kcal/mol respectively. The results suggest that isometamidium chloride could be cytotoxic and a potential inhibitor of p53 and PARP1 proteins.

Mollugin ameliorates murine allergic airway inflammation by inhibiting Th2 response and M2 macrophage activation

Mollugin, isolated from Rubia cordifolia L, is a pharmacological compound with anti-inflammatory activity. This study aimed to investigate whether mollugin protects mice against shrimp tropomyosin (ST)-induced allergic airway inflammation. Mice were sensitized with ST combined with Al(OH)3 administered intraperitoneally (i.p.) once weekly for 3 wk followed by ST challenge for 5 d. Mice were i.p.-administered daily with mollugin for 7 d. Results showed that mollugin attenuated ST-induced infiltration of eosinophils and epithelial mucus secretion in the lung tissues and suppressed lung eosinophil peroxidase activity. Additionally, mollugin lowered the Th2 cytokine, IL-4 and IL-5, production and downregulated the mRNA levels of Il-4, Il-5, Il-13, eotaxin, Ccl-17, Muc5ac, arginase-1, Ym-1, and Fizz-1 in the lung tissues. Network pharmacology was employed to predict core targets, and the molecular docking approach was used to verify the compound targets. The results of the molecular docking study of mollugin into p38 MAPK or poly(ADP-ribose) polymerase 1 (PARP1) binding sites revealed that its mechanism was possibly similar to that of SB203580 (a p38 MAPK inhibitor) or olaparib (a PARP1 inhibitor). Immunohistochemistry analysis revealed that mollugin mitigated ST-induced elevation of arginase-1 expression and macrophage levels in the lungs and bronchoalveolar lavage fluid, respectively. Furthermore, arginase-1 mRNA level and phosphorylation of p38 MAPK were inhibited in IL-4-stimulated peritoneal macrophages. In ST-stimulated mouse primary splenocytes, mollugin notably inhibited IL-4 and IL-5 production and downregulated PARP1 and PAR protein expression. According to our findings, mollugin ameliorated allergic airway inflammation by inhibiting Th2 response and macrophage polarization.

PARP-1 Expression Influences Cancer Stem Cell Phenotype in Colorectal Cancer Depending on p53

Poly(ADP-ribose) polymerase-1 (PARP-1) is a protein involved in multiple physiological processes. Elevated PARP-1 expression has been found in several tumours, being associated with stemness and tumorigenesis. In colorectal cancer (CRC), some controversy among studies has been described. In this study, we analysed the expression of PARP-1 and cancer stem cell (CSC) markers in CRC patients with different p53 status. In addition, we used an in vitro model to evaluate the influence of PARP-1 in CSC phenotype regarding p53. In CRC patients, PARP-1 expression correlated with the differentiation grade, but this association was only maintained for tumours harbouring wild-type p53. Additionally, in those tumours, PARP-1 and CSC markers were positively correlated. In mutated p53 tumours, no associations were found, but PARP-1 was an independent factor for survival. According to our in vitro model, PARP-1 regulates CSC phenotype depending on p53 status. PARP-1 overexpression in a wild type p53 context increases CSC markers and sphere forming ability. By contrast, those features were reduced in mutated p53 cells. These results could implicate that patients with elevated PARP-1 expression and wild type p53 could benefit from PARP-1 inhibition therapies, meanwhile it could have adverse effects for those carrying mutated p53 tumours.

Association between CHFR and PARP-1, and Their Roles in Regulation of Proliferation and Apoptosis of B Cell Lymphoma

Background. Aberrant methylation of checkpoint with forkhead and ring finger domains (CHFR) was found in B-cell non-Hodgkin lymphoma (NHL), whereas its role in carcinogenesis is not clear. CHFR can control poly (ADP-ribose) polymerase levels by causing its degradation. The study was aimed to explore the roles and mechanisms of CHFR in the pathogenesis of B-cell NHL. Methods. Short hairpin ribonucleic acid (ShRNAs) targeting CHFR and poly (ADP-ribose) polymerase 1 (PARP-1) were transduced into Raji cells, and real-time polymerase chain reaction (PCR) and western blotting were carried out to determine their expression. Afterwards, the CCK-8 assay and flow cytometry were used to evaluate the cell growth and apoptosis. Tumor size and weight were determined using a xenograft model, and decitabine (5-Aza-dC) was used to further determine the methylation status of CHFR through a methylation specificity-PCR assay. Results. 5-Aza-dC-treatment promoted the expression of CHFR and decreased the expression of PARP-1 at both messenger ribonucleic acid (mRNA) and protein levels. 5-Aza-dC also accelerated Raji-cell apoptosis and restrained its growth in vitro and in vivo ( P &lt; 0.05 ). These results were contrary to those observed in the shRNA-CHFR group but consistent with those observed in the shRNA-PARP-1 group. The expression profiles of CHFR and PARP-1 in the xenograft model were consistent with those in the cellular model. Treatment with 5-Aza-dC led to demethylation of CHFR in nude mice. Besides, there may be a negative correlation between CHFR and PARP-1 in B-cell NHL cells. Conclusion. Our findings indicated that 5-Aza-dC could lead to the demethylation of the CHFR promoter and suppress Raji cell growth.

Preclinical evaluation of a brain penetrant PARP PET imaging probe in rat glioblastoma and nonhuman primates.

Currently, there are multiple active clinical trials involving poly(ADP-ribose) polymerase (PARP) inhibitors in the treatment of glioblastoma. The noninvasive quantification of baseline PARP expression using positron emission tomography (PET) may provide prognostic information and lead to more precise treatment. Due to the lack of brain-penetrant PARP imaging agents, the reliable and accurate in vivo quantification of PARP in the brain remains elusive. Herein, we report the synthesis of a brain-penetrant PARP PET tracer, (R)-2-(2-methyl-1-(methyl-11C)pyrrolidin-2-yl)-1H-benzo[d]imidazole-4-carboxamide ([11C]PyBic), and its preclinical evaluations in a syngeneic RG2 rat glioblastoma model and healthy nonhuman primates. We synthesized [11C]PyBic using veliparib as the labeling precursor, performed dynamic PET scans on RG2 tumor-bearing rats and calculated the distribution volume ratio (DVR) using simplified reference region method 2 (SRTM2) with the contralateral nontumor brain region as the reference region. We performed biodistribution studies, western blot, and immunostaining studies to validate the in vivo PET quantification results. We characterized the brain kinetics and binding specificity of [11C]PyBic in nonhuman primates on FOCUS220 scanner and calculated the volume of distribution (VT), nondisplaceable volume of distribution (VND), and nondisplaceable binding potential (BPND) in selected brain regions. [11C]PyBic was synthesized efficiently in one step, with greater than 97% radiochemical and chemical purity and molar activity of 148 ± 85MBq/nmol (n = 6). [11C]PyBic demonstrated PARP-specific binding in RG2 tumors, with 74% of tracer binding in tumors blocked by preinjected veliparib (i.v., 5mg/kg). The in vivo PET imaging results were corroborated by ex vivo biodistribution, PARP1 immunohistochemistry and immunoblotting data. Furthermore, brain penetration of [11C]PyBic was confirmed by quantitative monkey brain PET, which showed high specific uptake (BPND > 3) and low nonspecific uptake (VND < 3mL/cm3) in the monkey brain. [11C]PyBic is the first brain-penetrant PARP PET tracer validated in a rat glioblastoma model and healthy nonhuman primates. The brain kinetics of [11C]PyBic are suitable for noninvasive quantification of available PARP binding in the brain, which posits [11C]PyBic to have broad applications in oncology and neuroimaging.

An electrostatic force-independent dephosphorylation-driven chemiluminescent biosensor for sensitive and rapid detection of poly(ADP-ribose) polymerase-1 in human breast tissues

Poly (ADP-ribose) polymerase-1 (PARP-1) is a multi-domain nuclear enzyme that mediates gene transcription and DNA repair. Accurate detection of PARP-1 is essential to clinical diagnostic. However, conventional PARP-1 assays rely on electrostatic force with the involvement of lengthy and complex construction processes and false positive. Herein, we construct an ultrasensitive chemiluminescent biosensor for rapid detection of PARP-1 in human breast tissues by integrating PARP-1-directed hyperbranched poly(ADP-ribose) (PAR) formation with alkaline phosphatase (ALP)-3-(2′-spiroadamantyl)-4-methoxy-4- (3′’-phosphoryloxyphenyl)-1,2-dioxetane (AMPDD)-mediated chemiluminescence system. Upon activation by dsDNA, PARP-1 cleaves nicotinamide adenine dinucleotide (NAD+), initiating the repeated polymerization of biotinylated ADP-ribose to form the dsDNA- PAR-biotin complex. The subsequent assembly of dsDNA-PAR-biotin complexes onto AuNPs through Au-S covalent bond results in the construction of the AuNPs-dsDNA-biotin nanostructure. AuNPs-dsDNA-biotin nanostructure subsequently captures SA-ALP to form the AuNPs-dsDNA-ALP nanostructure. After centrifugation and separation, the AuNPs-dsDNA-ALP nanostructure initiates the dephosphorylation of AMPPD to produce a high chemiluminescent signal. In this assay, only PARP-1 activated by dsDNA can cleave NAD+, efficiently eliminating background signal. The introduction of frozen method greatly shortens the assay time. Benefiting from high precision of the PARP-1-catalyzed NAD+ cleavage, high efficiency of biotinylated ADP-ribose polymerization reaction, and high signal-to-noise ratio of ALP-AMPPD chemiluminescence system, this biosensor can rapidly and sensitively detect PARP-1 with a detection limit of 2.94 × 10−7 U/μL, accurately screen PARP-1 inhibitors, and quantify PARP-1 at single-cell level. Most importantly, it can differentiate PARP-1 expression between breast cancer patient tissues and healthy person tissues, with promising applications in clinical diagnosis and biomedical research.