Poly Adenosine Diphosphate-ribose Polymerase Activity Research Articles

109 (PB099) - A novel dual PARP-HDAC inhibitor for treatment of Ewing sarcoma

Background: Inhibition of poly-adenosine diphosphate-ribose polymerase (PARP) is an effective therapy against cancers with DNA damage repair (DDR) deficiencies, such as BRCA1 and BRCA2 defects. In preclinical studies, PARP inhibitors demonstrated potential therapeutic value in Ewing sarcoma (ES), but clinical trials with olaparib failed to show significant clinical benefit. A key regulatory event in DNA damage repair is acetylation and deacetylation of histones, controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Increased expression of HDACs correlate with more malignant phenotypes in sarcomas and inhibition of HDAC in pre-clinical models of ES inhibits tumor growth. HDAC inhibition combined with PARP inhibition has promising results in vitro, however clinically, combination therapies often require complicated sequential dosing protocols due to different pharmacokinetic profiles and overlapping toxicities, that severely limits clinical utility. Here, we report the efficacy of a novel bifunctional small-molecule compound designed to have both PARP and HDAC inhibition activity. Methods: PARP1 and PARP2 activity was measured using the Trevigen Universal Colorimetric PARP Assay Kit, the BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit, and by PARylation assays. HDAC activity was measured using HeLa cell nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50 s were determined using live cell imaging with an Incucyte S3 system and the CellTiter Glo viability assay. DNA damage was detected by western blot, immuno-fluorescence, and comet assays. Results: A representative compound from the kt-3000 series showed potent inhibition of PARP1 and PARP2 with IC50 values in the low nM range, comparable to FDA-approved PARP inhibitors. The compound also showed inhibition of HDAC enzymes with IC50 values in the low μM range, slightly lower than the FDA-approved HDAC inhibitor, vorinostat. Cell survival EC50 values were superior to olaparib in ES cell lines in vitro. Treatment with the kt-3000 compound also resulted in increased DNA damage and S and G2/M cell cycle arrest as compared to olaparib. Conclusion: Our kt-3000 compound shows potent inhibition of PARP1, PARP2, and HDAC, as well as induction of DNA damage and cell cycle arrest. Further development of these bifunctional single molecule inhibitors may offer a novel treatment opportunity for Ewing sarcoma and other solid tumors with limited responses to PARPi monotherapies. Conflict of interest: Board of Directors: J. Bacha and D. Brown are directors of Rakovina Therapeutics. Corporate-sponsored Research: S. Truong, B. Zhai, F. Ghaidi, L. Ramos, J. Joshi are employees of Rakovina Therapeutics. J. Langlands, M. Daugaard and W. Shen are consultants to Rakovina Therapeutics.

Abstract A024: In vitro efficacy of a novel dual PARP-HDAC inhibitor in ewing sarcoma

Abstract Introduction: Inhibition of poly-adenosine diphosphate-ribose polymerase (PARP) is an effective therapy against cancers with DNA damage repair (DDR) deficiencies, such as BRCA1 and BRCA2 defects. In preclinical studies, PARP inhibitors demonstrated potential therapeutic value in Ewing sarcoma (ES), though clinical trials with olaparib failed to show significant clinical benefit. While single agent therapy proved inefficacious in the clinical treatment of ES, combination therapies may show anti-tumour activity. A key regulatory event in DNA damage repair is acetylation and deacetylation of histones, controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Increased expression of HDACs have been correlated to more malignant phenotypes in sarcomas and inhibition of HDAC in ES has been shown to be effective in inhibiting tumor growth. HDAC inhibition combined with PARP inhibition has been shown to sensitize cells to treatment in vitro, however clinically, combination therapies often require sequential administration due to different pharmacokinetic profiles and overlapping toxicities, severely limiting clinical utility. Here, we evaluate the activity and efficacy of a novel bifunctional small-molecule compound designed to have both PARP and HDAC inhibiting activity. Methods: PARP1 activity was measured using the Trevigen Universal Colorimetric PARP Assay Kit and PARP2 activity was measured using the BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit. HDAC activity was measured using HeLa nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50s were determined using live cell imaging with an Incucyte S3 system and the CellTiter Glo viability assay. Accumulation of phospho-histone H2AX (pH2AX) was detected by western blot using anti-phospho histone H2AX (Ser139) antibody from Cell Signaling Technologies. Results: A representative compound from the kt-3000 series showed potent inhibition of PARP1 and PARP2 with IC50 values in the low nM range, comparable to FDA-approved PARP inhibitors. The compound also showed inhibition of HDAC enzymes with IC50 values in the low µM range, slightly lower than the FDA-approved HDAC inhibitor, vorinostat. Cell survival EC50 values were superior to olaparib in ES cell lines in vitro. Treatment with the kt-3000 compound also resulted in the increased accumulation of pH2AX by western blot and increased S and G2/M cell cycle arrest compared to olaparib. Conclusion: Our kt-3000 compound shows potent inhibition of PARP1, PARP2, and HDAC, as well as induction of DNA damage and cell cycle arrest. Further development of these bifunctional single molecule inhibitors may result in a novel treatment opportunity for Ewing sarcoma. Citation Format: Sarah Truong, Beibei Zhai, Fariba Ghaidi, Louise Ramos, Jay Joshi, Dennis Brown, Neil Sankar, John Langlands, Jeffrey Bacha, Wang Shen, Poul Sorensen, Mads Daugaard. In vitro efficacy of a novel dual PARP-HDAC inhibitor in ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A024.

Abstract P081: In vitro activity and efficacy of novel dual PARP-HDAC inhibitors

Abstract Introduction: Inhibition of poly adenosine diphosphate-ribose polymerase (PARP) is an effective treatment strategy against tumors with homologous recombination (HR) DNA repair deficiencies. Combination treatments utilizing PARP inhibitors (PARPi) in concert with inhibitors of key DNA repair or PARPi-resistance pathways, may expand the utility of PARPi beyond cancers harboring germ-line HR deficiencies. Acetylation and deacetylation of histones is an important regulatory event in the DNA damage response. Combining histone deacetylation inhibitors (HDACi) and PARPi have sensitized PARPi-resistant cells to treatment; however, combination regimens often require sequential administration to manage overlapping toxicities and accommodate diverse pharmacokinetics, constituting a significant limitation of these strategies in a clinical setting. Here, we test the activity of kt-3000 series, a novel bi-functional class of small molecules with dual PARP and HDAC inhibitor activity in HR-proficient cancer cells where PARPi have historically lacked single-agent activity. Methods: PARP enzyme activity was measured using the Trevigen Universal Colorimetric PARP Assay Kit. HDAC activity was measured using nuclear extracts of treated cells and the HDAC Fluorometric Activity Assay Kit from Cayman Chemical, as per the manufacturer’s protocol. PARP activity was measured by fluorescence in C41 cells following compound treatment for 2 hours and PARP-activation with 1M H2O2 by staining cells with an anti-PARP Ab followed by a FITC-coupled secondary antibodies. Cell survival EC50 values were obtained by treating cells with a range of inhibitor concentrations, then quantifying cell confluency after 72-hours of treatment based on images taken with an Incucyte S3 system. Results: kt-3000 series compounds are potent inhibitors of PARP and HDAC with IC50 values for PARP enzyme activity in the low nM range, comparable to those of FDA-approved PARP inhibitors (olaparib, rucaparib, niraparib, and talazoparib) and IC50 values for the inhibition of HDAC activity comparable to FDA-approved HDAC inhibitors (panabinostat, belinostat, and vorinostat). Fluorescent assays of PARP activity in C41 cells also resulted in IC50 values in the nM range and cell survival EC50 values of the dual inhibitors are comparable to FDA-approved PARP inhibitors alone and in similar range with FDA-approved HDAC inhibitors alone. Conclusion: Our novel dual PARP-HDAC inhibitors show potent inhibition of PARP activity in vitro comparable to FDA-approved PARP inhibitors and also show potent inhibition of HDAC activity. The potency and activity of kt-3000 series compounds exhibit potential superiority to FDA-approved PARP inhibitors and HDAC inhibitors, in formulation as a single molecule. Development of these multi-target inhibitors will target unmet medical needs in the treatment of HR-proficient cancer types with dysregulation of histone deacetylation. Citation Format: Sarah Truong, Fariba Ghaidi, Louise Ramos, Jay Joshi, Dennis Brown, Neil Sankar, John Langlands, Jeffrey Bacha, Wang Shen, Mads Daugaard. In vitro activity and efficacy of novel dual PARP-HDAC inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P081.

Poly(adenosine diphosphate ribose) polymerase inhibitors induce autophagy-mediated drug resistance in ovarian cancer cells, xenografts, and patient-derived xenograft models.

Poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors exhibit promising activity against ovarian cancers, but their efficacy can be limited by acquired drug resistance. This study explores the role of autophagy in regulating the sensitivity of ovarian cancer cells to PARP inhibitors. Induction of autophagy was detected by punctate LC3 fluorescence staining, LC3I to LC3II conversion on Western blot analysis, and electron microscopy. Enhanced growth inhibition and apoptosis were observed when PARP inhibitors were used with hydroxychloroquine, chloroquine (CQ), or LYS05 to block the hydrolysis of proteins and lipids in autophagosomes or with small interfering RNA against ATG5 or ATG7 to prevent the formation of autophagosomes. The preclinical efficacy of the combination of CQ and olaparib was evaluated with a patient-derived xenograft (PDX) and the OVCAR8 human ovarian cancer cell line. Four PARP inhibitors (olaparib, niraparib, rucaparib, and talazoparib) induced autophagy in a panel of ovarian cancer cells. Inhibition of autophagy with CQ enhanced the sensitivity of ovarian cancer cells to PARP inhibitors. In vivo, olaparib and CQ produced additive growth inhibition in OVCAR8 xenografts and a PDX. Olaparib inhibited PARP activity, and this led to increased reactive oxygen species (ROS) and an accumulation of γ-H2AX. Inhibition of autophagy also increased ROS and γ-H2AX and enhanced the effect of olaparib on both entities. Treatment with olaparib increased phosphorylation of ATM and PTEN while decreasing the phosphorylation of AKT and mTOR and inducing autophagy. PARP inhibitor-induced autophagy provides an adaptive mechanism of resistance to PARP inhibitors in cancer cells with wild-type BRCA, and a combination of PARP inhibitors with CQ or other autophagy inhibitors could improve outcomes for patients with ovarian cancer.

Iduna protects HT22 cells by inhibiting parthanatos: The role of the p53-MDM2 pathway

Traumatic brain injury (TBI) is common and often fatal in current times. The role of poly(adenosine diphosphate-ribose) polymerase (PARP)-induced cell death (parthanatos) in TBI has not been well studied. Our past study showed that oxidative stress-induced cell death includes parthanatos by confirming the occurrence of PARP activation and nuclear translocation of apoptosis-inducing factor (AIF). As oxidative stress plays a key role in pathological progression after TBI, we believe TBI may also be alleviated by the expression of Iduna, which is the only known endogenous regulator of parthanatos. Thus, a transection model in HT-22 cells was established for present study. Downregulation of Iduna aggravated the cell damage caused by mechanical cell injury, whereas upregulation of Iduna reduced mitochondrial dysfunction induced by mechanical cell injury but exerted no effect on apoptosis associated with mitochondrial dysfunction. By contrast, Iduna prevented parthanatos by reducing PARP activation and nuclear translocation of AIF. We also investigated 2 novel p53-MDM2 pathway inhibitors, AMG 232 and Nutlin-3, which substantially reduced the protective effects of Iduna. These findings indicate that Iduna might prevent TBI by specifically inhibiting parthanatos and promoting mitochondrial function, with the p53-MDM2 pathway playing a critical role.

PARP-1 and PARP-2 activity in cancer-induced cachexia: potential therapeutic implications.

Skeletal muscle dysfunction and mass loss is a characteristic feature in patients with chronic diseases including cancer and acute conditions such as critical illness. Maintenance of an adequate muscle mass is crucial for the patients' prognosis irrespective of the underlying condition. Moreover, aging-related sarcopenia may further aggravate the muscle wasting process associated with chronic diseases and cancer. Poly(adenosine diphosphate-ribose) polymerase (PARP) activation has been demonstrated to contribute to the pathophysiology of muscle mass loss and dysfunction in animal models of cancer-induced cachexia. Genetic inhibition of PARP activity attenuated the deleterious effects seen on depleted muscles in mouse models of oncologic cachexia. In the present minireview the mechanisms whereby PARP activity inhibition may improve muscle mass and performance in models of cancer-induced cachexia are discussed. Specifically, the beneficial effects of inhibition of PARP activity on attenuation of increased oxidative stress, protein catabolism, poor muscle anabolism and mitochondrial content and epigenetic modulation of muscle phenotype are reviewed in this article. Finally, the potential therapeutic strategies of pharmacological PARP activity inhibition for the treatment of cancer-induced cachexia are also being described in this review.

The NAD+/PARP1/SIRT1 Axis in Aging.

NAD+ levels decline with age in diverse animals from Caenorhabditis elegans to mice. Raising NAD+ levels by dietary supplementation with NAD+ precursors, nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN), improves mitochondrial function and muscle and neural and melanocyte stem cell function in mice, as well as increases murine life span. Decreased NAD+ levels with age reduce SIRT1 function and reduce the mitochondrial unfolded protein response, which can be overcome by NR supplementation. Decreased NAD+ levels cause NAD+-binding protein DBC1 to form a complex with PARP1, inhibiting poly(adenosine diphosphate-ribose) polymerase (PARP) catalytic activity. Old mice have increased amounts of DBC1-PARP1 complexes, lower PARP activity, increased DNA damage, and reduced nonhomologous end joining and homologous recombination repair. DBC1-PARP1 complexes in old mice can be broken by increasing NAD+ levels through treatment with NMN, reducing DNA damage and restoring PARP activity to youthful levels. The mechanism of declining NAD+ levels and its fundamental importance to aging are yet to be elucidated. There is a correlation of PARP activity with mammalian life span that suggests that NAD+/SIRT1/PARP1 may be more significant than the modest effects on life span observed for NR supplementation in old mice. The NAD+/PARP1/SIRT1 axis may link NAD+ levels and DNA damage with the apparent epigenomic DNA methylation clocks that have been described.

Pharmacological Reconditioning of Marginal Donor Rat Lungs Using Inhibitors of Peroxynitrite and Poly (ADP-ribose) Polymerase During Ex Vivo Lung Perfusion.

Donor lungs obtained after prolonged warm ischemia (WI) may be unsuitable for transplantation due to the risk of reperfusion injury, but could be reconditioned using ex-vivo lung perfusion (EVLP). Key processes of reperfusion injury include the formation of reactive oxygen species (ROS)/nitrogen species (RNS) and the activation of poly(adenosine diphosphate-ribose) polymerase (PARP). We explored whether rat lungs obtained after WI could be reconditioned during EVLP using the ROS/RNS scavenger Mn(III)-tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) or the PARP inhibitor 3-aminobenzamide (3-AB). Rat lungs obtained after 3 hours cold ischemia (CI group, control), or 1 hour WI plus 2 hours CI (WI group) were placed in an EVLP circuit for normothermic perfusion for 3 hours. Lungs retrieved after WI were treated or not with 3-AB (1 mg/mL) or MnTBAP (0.3 mg/mL), added to the perfusate. Measurements included physiological variables (lung compliance, vascular resistance, oxygenation capacity), lung weight gain, levels of proteins, lactate dehydrogenase, protein carbonyl (marker of ROS), 3-nitrotyrosine (marker of RNS), poly(adenosine diphosphate-ribose) (PAR, marker of PARP activation) and IL-6, in the bronchoalveolar lavage or the lung tissue, and histology. In comparison to the CI group, the lungs from the WI group displayed higher protein carbonyls, 3-nitrotyrosine, PAR, lactate dehydrogenase and proteins in bronchoalveolar lavage, lung weight gain, perivascular edema, as well as reduced static compliance, but similar oxygenation. All these alterations were markedly attenuated by 3-AB and MnTBAP. After EVLP, lungs obtained after WI exhibit oxidative stress, PARP activation, and tissue injury, which are suppressed by pharmacological inhibitors of ROS/RNS and PARP.

Apoptosis induced in MCF-7 human breast cancer cells by 2',4'-dihydroxy-6-methoxy-3,5-dimethylchalcone isolated from Eugenia aquea Burm f. leaves.

During a previous study that aimed to identify anticancer agents within primate-consumed plants, the present group identified that Eugenia aquea (E. aquea) possessed potential as a source of anticancer agents. The ethanol extract of E. aquea leaves exhibited strong inhibitory activity against the proliferation of the human breast adenocarcinoma MCF-7 cell line. The inhibition of proliferation was determined using an MTT assay. The present study was performed to isolate the active compound within the E. aquea leaves that generated the aforementioned activity, and resulted in the isolation of 2',4'-dihydroxy-6-methoxy-3,5-dimethylchalcone, which was identified through the analysis of spectroscopic data. This compound was examined for its inhibitory activity against the MCF-7 cell line using a MTT assay, and the ability of 2',4'-dihydroxy-6-methoxy-3,5-dimethylchalcone to induce apoptosis through the activation of the poly(adenosine diphosphate-ribose) polymerase (PARP) protein was also investigated. The results of the present study revealed that the isolated compound inhibited cell proliferation in a dose-dependent manner, possessed an IC50 of 74.5 µg/ml (250 µM) and promoted apoptosis via the activation of PARP. It was concluded that these results indicated a requirement for additional investigations into 2',4'-dihydroxy-6-methoxy-3,5-dimethylchalcone in order to provide a basis for the use of this compound in the management of cancer.

Inhibition of poly (adenosine diphosphate-ribose) polymerase attenuates lung-kidney crosstalk induced by intratracheal lipopolysaccharide instillation in rats

BackgroundAcute respiratory distress syndrome (ARDS) is a severe form of lung injury that frequently occurs during pneumonia and sepsis. Lung inflammation in ARDS patients may have deleterious effects on remote organs such as the kidney. The nuclear enzyme poly(adenosine diphosphate-ribose) polymerase (PARP) enhances the nuclear factor (NF)-κB-dependent transcription of inflammatory cytokines. This study was conducted to elucidate two questions: first, whether the activation of PARP and NF-κB mediates the renal inflammation secondary to the lipopolysaccharide (LPS)-induced acute lung inflammation; second, whether a PARP inhibitor, 3-aminobenzamide (3-AB), attenuates lung and kidney inflammation by inhibiting NF-κB-dependent proinflammatory cytokines.MethodsMale Sprague–Dawley rats were anesthetized, ventilated, and divided into three groups; a control group (n = 8); an LPS group (n = 12) intratracheally instilled with LPS (16 mg/kg), and an LPS + 3-AB group (n = 12) given the same dose of LPS by the same method followed by an intravenous injection of 3-AB (20 mg/kg). Hemodynamics, arterial blood gas, and the plasma levels of lactate, creatinine and potassium were measured at 0,1,2,3, and 4 h after treatment. The lung wet/dry ratio was measured at 4 h. The mRNA expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in the lung and kidney were measured by TaqMan real-time PCR. PARP and NF-κB in the lung and kidney were histologically examined by immunostaining and assigned expression scores.ResultsLPS induced metabolic acidosis, hypotension, hypoxemia, increased the lung wet/dry ratio, increased the plasma levels of creatinine and potassium, and increased the cytokine mRNA expressions in the lung and kidney. All of these effects were associated with strong expression of PARP and NF-κB. Treatment with 3-AB prevented the LPS-induced metabolic acidosis and hypotension, reduced the plasma levels of lactate, creatinine and potassium, reduced the cytokine mRNA expressions, reduced the expression of PARP and NF-κB, improved pulmonary edema and oxygenation and preserved renal function.ConclusionsThe PARP inhibition attenuated lung-kidney crosstalk induced by intratracheal LPS instillation, partly via an inhibition of NF-κB dependent proinflammatory cytokines.

Inhibition of Poly(ADP-Ribose) Polymerase Attenuates Acute Kidney Injury in Sodium Taurocholate-Induced Acute Pancreatitis in Rats

The aim of our present study was to investigate the efficacy of poly(adenosine diphosphate-ribose) polymerase (PARP) inhibition in the development of acute kidney injury in an experimental model of severe acute pancreatitis induced by retrograde infusion of sodium taurocholate into the bile-pancreatic duct. Severity of pancreatitis was evaluated by serum amylase, lipase, tumor necrosis factor α, interleukin-1β, interleukin-6, and histological grading. The following markers of renal dysfunction and injury were measured: serum creatinine level, urea nitrogen level, myeloperoxidase activity, and histology. Activation of PARP, intercellular adhesion molecule-1, and P-selectin protein in the kidney was studied using Western blot analysis. 3-Aminobenzamide attenuated the following: (1) serum amylase, lipase, and renal dysfunction; (2) serum concentrations of proinflammatory cytokines; (3) pancreatic and renal pathological injury; (4) renal myeloperoxidase activity; and (5) activation of PARP, intercellular adhesion molecule-1, and P-selectin in the kidney. Our results suggest that PARP activation may contribute to kidney injury and that PARP inhibitors may be beneficial in renal disorders associated with severe acute pancreatitis.

Poly(ADP‐Ribose) Polymerase Inhibition Improves Erectile Function by Activation of Nitric Oxide/Cyclic Guanosine Monophosphate Pathway in Diabetic Rats

Endothelial dysfunction-induced abnormalities of the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway in the corpus cavernosum are thought to be the main factors involved in the pathogenesis of diabetes-induced erectile dysfunction (ED). Recent studies have shown that the poly(adenosine diphosphate ribose) polymerase (PARP) pathway plays a critical role in diabetic endothelial dysfunction. The aim of this study is to determine whether activation of the PARP pathway is involved in diabetic cavernosal endothelial dysfunction and abnormalities of the NO/cGMP pathway. Male Sprague-Dawley rats were randomly divided into three groups: age-matched controls, diabetic controls (DM), and the 3-aminobenzamide (3-AB, a PARP inhibitor)-treated diabetic group (DM+3-AB). Diabetes was induced by intraperitoneal injection of streptozotocin. Eight weeks after inducing diabetes, the DM+3-AB group was treated with 3-AB for 4 weeks. Erectile function was assessed at 12 weeks after inducing diabetes by stimulating the cavernous nerve. Expression of poly(ADP-ribose), protein kinase B (Akt), phospho-Akt, endothelial nitric oxide synthase (eNOS), phospho-eNOS, and neuronal nitric oxide synthase (nNOS) were evaluated by Western blot. Cavernous NO generation and cGMP levels were also determined. The DM group showed impaired erectile function and significantly increased PARP activity. Expression of total eNOS and nNOS, phospho-Akt, and eNOS decreased significantly in the DM group compared with those in the control group. In addition, cavernous NO generation and cGMP levels decreased significantly in the DM group compared with those in the control group. Treatment with 3-AB restored erectile function and significantly reversed all molecular alterations except decreased nNOS expression. Overactivation of the PARP pathway in the corpus cavernosum of diabetic rats was involved in cavernosal endothelial dysfunction and abnormalities of the NO/cGMP pathway resulting in ED. These findings may be applied to develop novel therapies for patients with diabetic ED.

Abstract 679: Retrospective evaluation of the BRCA1 - PARP pathway in breast cancer tissues

Abstract Purpose: Poly(adenosine diphosphate-ribose) polymerase (PARP) and BRCA1 plays a key role in DNA repair and cellular stress response. Inhibitors of PARP show promising clinical activity in metastatic, triple-negative or BRCA-mutated breast cancer. However, a comprehensive analysis of the PARP activity / BRCA1 methylation profile in non-BRCA1 mutated tumors has not yet been assessed. Methods: We investigated cytosolic PARP activity and BRCA1 methylation profile in 155 surgical tumour samples from patients treated in our institution between January 2006 and November 2009 and evaluated their statistical association with classical breast cancer predictive and prognostic factors. DNA methylation patterns in the CpG islands of BRCA1 gene promoter were determined by a methylation specific PCR distinguishing unmethylated from methylated alleles in a given gene on the basis of sequence changes produced following bisulfite treatment of DNA. Cytosolic PARP activity was evaluated using a commercially available assay kit. Results: A total of 155 BC patients (50 HER-2 positive, 57 hormone receptors positive (HR+) / HER-2 negative and 48 triple negative tumors) were considered eligible for this study. Median age was 54 years (range 29-75 years). Only one tumor was classified as SBR score I and SBR Tubule formation score 1. No tumor was SBR Nuclear pleomorphism score 1. Considering these results, SBR scores I and II and SBR Tubule formation scores 1 and 2 were grouped for statistical analyses. Mean PARP activity (U/mg of cytosolic protein) was 12.2 (standard deviation 17.02), with a median of 7.0 (range 1.0 to 114.2). The only clearly associated parameter was SBR Mitotic count score. Bisulfite treatment was successfully performed for the whole 155 specimen. Eighteen tumors presented an hypermethylation of the BRCA1 gene promoter. The hypermethylation status was significantly associated with UPA and PAI-1 levels, SBR grade, SBR Mitotic count score, ER, PR and HER-2 negative status, and then conversely triple negative (ER, PR and HER-2 negative) profile. Twenty nine percent of the triple negative tumors presented and hypermethylated status, comparing to 5 and 2% for HR+/HER-2- and HER-2+ tumors respectively. No statistical association was found between a hypermethylation status and the PARP cytosolic activity. Conclusion: Cytosolic PARP activity is equally represented in TN, HER2+ and SBR II and III HR+ breast cancers. The only parameter clearly associated with the cytosolic PARP activity was the SBR Mitotic count score. In contrast, hypermethylation of the BRCA1 gene promoter appears restricted to a subgroup of TN tumors expressing an aggressive phenotype. No statistical association was found between a hypermethylation status and the PARP cytosolic activity. These results could allow a more accurate stratification of patients without BRCA1 famillial mutation in the future PARP inhibitors clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 679. doi:1538-7445.AM2012-679

Apoptotic effect of eugenol in human colon cancer cell lines

Eugenol, a natural compound available in honey and various plants extracts including cloves and Magnoliae flos, is exploited for various medicinal applications. Since most of the drugs used in the cancer are apoptotic inducers, the apoptotic effect and anticancer mechanism of eugenol were investigated against colon cancer cells. Antiproliferative effect was estimated using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay]. Earlier events like MMP (mitochondrial membrane potential), thiol depletion and lipid layer break were measured by using flow cytometry. Apoptosis was evaluated using PI (propidium iodide) staining, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assay and DNA fragmentation assay. MTT assay signified the antiproliferative nature of eugenol against the tested colon cancer cells. PI staining indicated increasing accumulation of cells at sub-G1-phase. Eugenol treatment resulted in reduction of intracellular non-protein thiols and increase in the earlier lipid layer break. Further events like dissipation of MMP and generation of ROS (reactive oxygen species) were accompanied in the eugenol-induced apoptosis. Augmented ROS generation resulted in the DNA fragmentation of treated cells as shown by DNA fragmentation and TUNEL assay. Further activation of PARP (polyadenosine diphosphate-ribose polymerase), p53 and caspase-3 were observed in Western blot analyses. Our results demonstrated molecular mechanism of eugenol-induced apoptosis in human colon cancer cells. This research will further enhance eugenol as a potential chemopreventive agent against colon cancer.

Renal hypoperfusion and impaired endothelium-dependent vasodilation in an animal model of VILI: the role of the peroxynitrite-PARP pathway

IntroductionMechanical ventilation (MV) can injure the lungs and contribute to an overwhelming inflammatory response, leading to acute renal failure (ARF). We previously showed that poly(adenosine diphosphate-ribose) polymerase (PARP) is involved in the development of ventilator-induced lung injury (VILI) and the related ARF, but the mechanisms underneath remain unclear. In the current study we therefore tested the hypothesis that renal blood flow and endothelial, functional and tissue changes in the kidney of rats with lipopolysaccharide (LPS)-induced lung injury aggravated by MV, is caused, in part, by activation of PARP by peroxynitrite.MethodsAnesthetized Sprague Dawley rats (n = 31), were subjected to intratracheal instillation of lipopolysaccharide at 10 mg/kg followed by 210 min of mechanical ventilation at either low tidal volume (6 mL/kg) with 5 cm H2O positive end-expiratory pressure or high tidal volume (19 mL/kg) with zero positive end-expiratory pressure in the presence or absence of a peroxynitrite decomposition catalyst, WW85 or a PARP inhibitor, PJ-34. During the experiment, hemodynamics and blood gas variables were monitored. At time (t) t = 0 and t = 180 min, renal blood flow was measured. Blood and urine were collected for creatinine clearance measurement. Arcuate renal arteries were isolated for vasoreactivity experiment and kidneys snap frozen for staining.ResultsHigh tidal volume ventilation resulted in lung injury, hypotension, renal hypoperfusion and impaired renal endothelium-dependent vasodilation, associated with renal dysfunction and tissue changes (leukocyte accumulation and increased expression of neutrophil gelatinase-associated lipocalin). Both WW85 and PJ-34 treatments attenuated lung injury, preserved blood pressure, attenuated renal endothelial dysfunction and maintained renal blood flow. In multivariable analysis, renal blood flow improvement was, independently from each other, associated with both maintained blood pressure and endothelium-dependent vasodilation by drug treatment. Finally, drug treatment improved renal function and reduced tissue changes.ConclusionsThe peroxynitrite-induced PARP activation is involved in renal hypoperfusion, impaired endothelium-dependent vasodilation and resultant dysfunction, and injury, in a model of lung injury.

Activation and Modulation of Cardiac Poly-Adenosine Diphosphate Ribose Polymerase Activity in a Rat Model of Brain Death

DNA damage during transplantation can activate poly-adenosine diphosphate ribose polymerase (PARP) resulting in the generation of polymers of adenosine diphosphate-ribose (PAR). Excessive linkage of PAR to nuclear proteins can induce cell death, thereby limiting the function of transplanted organs. This study uses a rat model of brain death to determine the profile of PARP activation and whether mechanisms that lead to cell death can be ameliorated by appropriate donor resuscitation. The expression of PAR-linked nuclear proteins within cardiac myocytes was greatly increased after the induction of donor brain death. Importantly, infusion of noradrenaline or vasopressin to normalize the chronic hypotension produced by brain death reduced the expression of PAR to a level below baseline. These data suggest that chronic hypotension after donor brain death has the potential to limit cardiac function through the activation of PARP; however, this early cause of graft damage can be mitigated by appropriate donor resuscitation.

Diabetes increases apoptosis and necrosis in both ischemic and nonischemic human myocardium: Role of caspases and poly–adenosine diphosphate–ribose polymerase

Diabetes is an important predictor of morbidity and mortality after cardiac surgery, but the reason is unclear. The aims of these studies, therefore, were to elucidate whether cell death is greater in ischemic and nonischemic diabetic human myocardium than in nondiabetic myocardium and to investigate the underlying mechanism. The right atrial appendages (n = 8 per group) of patients without diabetes and patients with type 1 and 2 diabetes were subjected to 90 minutes of simulated ischemia and 120 minutes reoxygenation. Tissue injury was measured by the release of creatine kinase into the media, and cellular apoptosis and necrosis were assessed in tissue by the terminal transferase deoxyuridine triphosphate nick-end labeling assay and propidium iodide staining. Initiator and effector caspases activations were also measured. Apoptosis and necrosis were greater in the type 2 and type 1 diabetes groups than in the nondiabetes group both in fresh tissue and after simulated ischemia-reoxygenation. Activation of effector caspases was also higher in the diabetes groups than in the nondiabetes group after simulated ischemia-reoxygenation. Caspase-3 inhibition reduced apoptosis in all study groups without influencing necrosis; however, poly-adenosine diphosphate-ribose polymerase inhibition resulted in a similar reduction in apoptosis and in necrosis in all groups, whereas caspase-2 inhibition did not. Diabetes increases both apoptosis and necrosis in human myocardium, both fresh and after being subjected to ischemia-reoxygenation, an effect that is mediated, at least in part, by caspase-3 and poly-adenosine diphosphate-ribose polymerase activation. These results may explain the increased cardiac-related morbidity and mortality associated with cardiac surgery in patients with diabetes.

Pathophysiological aspects of cyclophosphamide and ifosfamide induced hemorrhagic cystitis; implication of reactive oxygen and nitrogen species as well as PARP activation

Cyclophosphamide (CP) and ifosfamide (IF) are widely used antineoplastic agents, but their side-effect of hemorrhagic cystitis (HC) is still encountered as an important problem. Acrolein is the main molecule responsible of this side-effect and mesna (2-mercaptoethane sulfonate) is the commonly used preventive agent. Mesna binds acrolein and prevent its direct contact with uroepithelium. Current knowledge provides information about the pathophysiological mechanism of HC: several transcription factors and cytokines, free radicals and non-radical reactive molecules, as well as poly(adenosine diphosphate-ribose) polymerase (PARP) activation are now known to take part in its pathogenesis. There is no doubt that HC is an inflammatory process, including when caused by CP. Thus, many cytokines such as tumor necrosis factor (TNF) and the interleukin (IL) family and transcription factors such as nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) also play a role in its pathogenesis. When these molecular factors are taken into account, pathogenesis of CP-induced bladder toxicity can be summarized in three steps: (1) acrolein rapidly enters into the uroepithelial cells; (2) it then activates intracellular reactive oxygen species and nitric oxide production (directly or through NF-kappaB and AP-1) leading to peroxynitrite production; (3) finally, the increased peroxynitrite level damages lipids (lipid peroxidation), proteins (protein oxidation) and DNA (strand breaks) leading to activation of PARP, a DNA repair enzyme. DNA damage causes PARP overactivation, resulting in the depletion of oxidized nicotinamide-adenine dinucleotide and adenosine triphosphate, and consequently in necrotic cell death. For more effective prevention against HC, all pathophysiological mechanisms must be taken into consideration.

Valsartan improves resting skin blood flow in type 2 diabetic patients and reduces poly(adenosine diphosphate-ribose) polymerase activation

To examine the effect of a 12-week daily treatment with 160 mg of valsartan, an angiotensin II receptor blocker, on the microcirculation and macrocirculation of type 2 diabetic patients (T2DM) and healthy subjects. This was a prospective, randomized, double-blind, placebo-controlled crossover study. Thirteen T2DM with no severe complications and 13 healthy subjects completed the trial. Treatment with valsartan in T2DM improved the resting forearm skin blood flow and increased the resting brachial artery diameter but had no effects on arterial blood pressure, large vessel vascular reactivity, or carotid intima-media thickness. Resting skin blood flow increased by 60% (2%-90%; median and 25th-75th percentiles) during valsartan treatment and by only 2% (-22% to 27%) during placebo treatment (P < .05). No changes were observed in the nondiabetic subjects. Immunostaining studies of forearm skin biopsy samples from T2DM and healthy subjects showed that valsartan reduced poly(adenosine diphosphate-ribose) polymerase (PARP) activity in 50% (6/12) of the subjects. PARP activity remained unchanged in placebo-treated subjects (P < .02). In addition, valsartan treatment increased CD31 staining in 33% (4/12) of the subjects, whereas no change was noted in sequential skin biopsy samples of placebo-treated subjects (P = .057). Valsartan had no effect on the biochemical markers of endothelial cell activation and other cytokines, including CAMs, interleukin 6, tumor necrosis factor alpha, C-reactive protein, adiponectin, and plasma activator inhibitor 1. Valsartan increases the resting skin blood flow in T2DM, likely through reduction of PARP activity.

Molecular targets against mustard toxicity: implication of cell surface receptors, peroxynitrite production, and PARP activation

Despite many years of research into chemical warfare agents, cytotoxic mechanisms induced by mustards are not well understood. Reactive oxygen and nitrogen species (ROS and RNS) are likely to be involved in chemical warfare agents induced toxicity. These species lead to lipid peroxidation, protein oxidation, and DNA injury, and trigger many pathophysiological processes that harm the organism. In this article, several steps of pathophysiological mechanisms and possible ways of protection against chemical warfare agents have been discussed. In summary, pathogenesis of mustard toxicity is explained by three steps: (1) mustard binds target cell surface receptor, (2) activates intracellular ROS and RNS leading to peroxynitrite (ONOO(-)) production, and (3) the increased ONOO(-) level damages organic molecules (lipids, proteins, and DNA) leading to poly(adenosine diphosphate-ribose) polymerase (PARP) activation. Therefore, protection against mustard toxicity could also be performed in these ways: (1) blocking of cell surface receptor, (2) inhibiting the ONOO(-) production or scavenging the ONOO(-) produced, and (3) inhibiting the PARP, activated by ONOO(-) and hydroxyl radical (OH(*)) induced DNA damage. As conclusion, to be really effective, treatment against mustards must take all molecular mechanisms of cytotoxicity into account. Combination of several individual potent agents, each blocking one of the toxic mechanisms induced by mustards, would be interesting. Therefore, variations of combination of cell membrane receptor blockers, antioxidants, nitric oxide synthase inhibitors, ONOO(-) scavengers, and PARP inhibitors should be investigated.