Pathophysiological role of poly(ADP-ribose) polymerase (PARP) activation during acetaminophen-induced liver cell necrosis in mice.

DNA fragmentation in hepatocytes occurs early after acetaminophen (AAP) overdose in mice. DNA strandbreaks can induce excessive activation of poly(ADP-ribose) polymerases (PARP), which may lead to oncotic necrosis. Based on controversial findings with chemical PARP inhibitors, the role of PARP-1 activation in AAP hepatotoxicity remains unclear. To investigate PARP-1 activation and evaluate a pathophysiological role of PARP-1, we used both PARP inhibitors (3-aminobenzamide; 5-aminoisoquinolinone) and PARP gene knockout mice (PARP-/-). Treatment of C3Heb/FeJ mice with 300 mg/kg AAP resulted in DNA fragmentation and alanine aminotransferase (ALT) release as early as 3 h, with further increase of these parameters up to 12 h. Few nuclei of hepatocytes stained positive for poly-ADP-ribosylated nuclear proteins (PAR) as indicator for PARP-1 activation at 4.5 h. However, the number of PAR-positive cells and staining intensity increased substantially at 6 and 12 h. Pretreatment with 500 mg/kg 3-aminobenzamide before AAP attenuated hepatic glutathione depletion and completely eliminated DNA fragmentation and liver injury. Delayed treatment several hours after AAP was still partially protective. On the other hand, liver injury was not attenuated in PARP-/- mice compared to wild-type animals. Similarly, the specific PARP-1 inhibitor 5-aminoisoquinolinone (5 mg/kg) was not protective. However, 3-aminobenzamide attenuated liver injury in WT and PARP-/- mice. In summary, PARP-1 activation is a consequence of DNA fragmentation after AAP overdose. However, PARP-1 activation is not a relevant event for AAP-induced oncotic necrosis. The protection of 3-aminobenzamide against AAP-induced liver injury was due to reduced metabolic activation and potentially its antioxidant effect but independent of PARP-1 inhibition.     

Decrease of the inflammatory response and induction of the Akt/protein kinase B pathway by poly-(ADP-ribose) polymerase 1 inhibitor in endotoxin-induced septic shock

The lack of efficacy of anti-inflammatory drugs, anti-coagulants, anti-oxidants, etc. in critically ill patients has shifted interest towards developing alternative treatments. Since inhibitors of the nuclear enzyme poly-(ADP-ribose) polymerase (PARP) were found to be beneficial in many pathophysiological conditions associated with oxidative stress and PARP-1 knock-out mice proved to be resistant to bacterial lipopolysaccharide (LPS)-induced septic shock, PARP inhibitors are candidates for such a role. In this study, the mechanism of the protective effect of a potent PARP-1 inhibitor, PJ34 was studied in LPS-induced (20mg/kg, i.p.) septic shock in mice. We demonstrated a significant inflammatory response by magnetic resonance imaging in the dorsal subcutaneous region, in the abdominal regions around the kidneys and in the inter-intestinal cavities. We have found necrotic and apoptotic histological changes as well as obstructed blood vessels in the liver and small intestine. Additionally, we have detected elevated tumor necrosis factor-alpha levels in the serum and nuclear factor kappa B activation in liver of LPS-treated mice. Pre-treating the animals with PJ34 (10mg/kg, i.p.), before the LPS challenge, besides rescuing the animals from LPS-induced death, attenuated all these changes presumably by activating the phosphatidylinositol 3-kinase-Akt/protein kinase B cytoprotective pathway.     

New inhibitors of poly(ADP-ribose) polymerase (PARP)

Poly(ADP-ribose) polymerase-1 (PARP-1), the most prominent member of the PARP family, is a DNA-binding protein that is activated by nicks in DNA occurring during inflammation, ischaemia, neurodegeneration or cancer therapy. Activated PARP-1 consumes NAD⁺ that is cleaved into nicotinamide and ADP-ribose and polymerises the latter onto nuclear acceptor proteins. This highly energy consuming process is pivotal for the maintenance of genomic stability although over-activation can culminate in cell dysfunction and necrosis. Therefore, PARP-1 is regarded as a promising target for the development of drugs useful in various forms of inflammation, ischaemia–reperfusion injury and as an adjunct in cancer therapy. This review summarises the structural classes of known PARP-1 inhibitors, with a focus on new inhibitors published for this target, between 2002 and July 2004. The chemistry and biological data disclosed in these patent applications are discussed in light of new structural knowledge of the catalytic domain of the PARP family and recent work with potent inhibitors demonstrating the effects of PARP inhibition in various animal disease models.     

Role of poly(ADP-ribose) polymerase activation in endotoxin-induced cardiac collapse in rodents

Reactive oxygen and nitrogen species are overproduced in the cardiovascular system during circulatory shock. Oxidant-induced cell injury involves the activation of poly(ADP-ribose) polymerase (PARP). Using a dual approach of PARP-1 suppression, by genetic deletion or pharmacological inhibition with the new potent phenanthridinone PARP inhibitor PJ34 [the hydrochloride salt of N-(oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide], we studied whether the impaired cardiac function in endotoxic shock is dependent upon the PARP pathway. Escherichia coli endotoxin (lipopolysaccharide, LPS) at 55 mg/kg, i.p., induced a severe depression of the systolic and diastolic contractile function, tachycardia, and a reduction in mean arterial blood pressure in both rats and mice. Treatment with PJ34 significantly improved cardiac function and increased the survival of rodents. In addition, LPS-induced depression of left ventricular performance was significantly less pronounced in PARP-1 knockout mice (PARP(-/-)) as compared with their wild-type littermates (PARP(+/+)). Thus, PARP activation in the cardiovascular system is an important contributory factor to the cardiac collapse and death associated with endotoxin shock.     

Brain-derived neurotrophic factor prevents phencyclidine-induced apoptosis in developing brain by parallel activation of both the ERK and PI-3K/Akt pathways

Phencyclidine is an N-methyl d-aspartate receptor (NMDAR) blocker that has been reported to induce neuronal apoptosis during development and schizophrenia-like behaviors in rats later in life. Brain-derived neurotrophic factor (BDNF) has been shown to prevent neuronal death caused by NMDAR blockade, but the precise mechanism is unknown. This study examined the role of the phosphatidylinositol-3 kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways in BDNF protection of PCP-induced apoptosis in corticostriatal organotypic cultures. It was observed that BDNF inhibited PCP-induced apoptosis in a concentration-dependent fashion. BDNF effectively prevented PCP-induced inhibition of the ERK and PI-3K/Akt pathways and suppressed GSK-3β activation. Blockade of either PI-3K/Akt or ERK activation abolished BDNF protection. Western blot analysis revealed that the PI-3K inhibitor LY294002 prevented the stimulating effect of BDNF on the PI-3K/Akt pathway, but had no effect on the ERK pathway. Similarly, the ERK inhibitor PD98059 prevented the stimulating effect of BDNF on the ERK pathway, but not the PI-3K/Akt pathway. Co-application of LY294002 and PD98059 had no additional effect on BDNF-evoked activation of Akt or ERK. However, concurrent exposure to PD98059 and LY294002 caused much greater inhibition of BDNF-evoked phosphorylation of GSK-3β at serine 9 than did LY294002 alone. Finally, either BDNF or GSK-3β inhibition prevented PCP-induced suppression of cyclic-AMP response element binding protein (CREB) phosphorylation. These data demonstrate that the protective effect of BDNF against PCP-induced apoptosis is mediated by parallel activation of the PI-3K/Akt and ERK pathways, most likely involves inhibition of GSK-3β and activation of CREB.     

苯并咪唑类聚腺苷二磷酸核糖聚合酶抑制剂的合成及初步活性研究

目的 设计合成一系列苯并咪唑类衍生物,并测定其聚腺苷二磷酸核糖聚合酶(PARP)抑制活性.方法 以3-硝基邻苯二甲酸酐为基本原料,经开环、Hofmann重排、酰胺化或酯化、还原得到邻二氨基苯化合物,再与相应的苯甲醛及其衍生物环合得到目标分子;采用体外抑酶试验初步筛选目标分子的PARO抑制活性.结果与结论 合成了22个苯...     

Marine compounds for the therapeutic treatment of neurological disorders

Background: During the last few years an increasing number of poly(ADP-ribose) polymerase (PARP) inhibitors have been appearing in the context of cancer therapy. This is mainly due to a better knowledge of the best-characterized member of the PARP family of enzymes, PARP-1, further reinforced by the recognition of the clinical benefits arising from its inhibition. Objective/method: The aim of this review is to give the reader an update on PARP inhibition in cancer therapy, by covering both the scientific (SciFinder^® search) and the patent literature (Chemical Abstract^®/Derwent^® search) published recently (2005 – 2008). Conclusions: More patient-compliant orally available PARP-1 inhibitor clinical candidates, along with their possible use as single agents in specific, molecularly defined cancer indications, increase the expectations for this therapeutic approach. The growing understanding of the biological role of other PARPs, such as Tankyrase 1, may be of interest as new potential targets. Besides the classical NAD-mimicking pharmacophore, additional compounds, which either do not resemble nicotinamide or exploit different binding sites, are emerging.     

Selective PARP-2 inhibitors increase apoptosis in hippocampal slices but protect cortical cells in models of post-ischaemic brain damage

Background and purpose:

Poly(ADP-ribose) polymerases (PARP)-1 and PARP-2 play complementary tasks in the maintenance of genomic integrity, but their role in cell death or survival processes is rather different. A recently described series of selective PARP-2 inhibitors (UPF-1035, UPF-1069) were used to study the role of PARP-1 and PARP-2 in post-ischaemic brain damage.

Experimental approach:

We evaluated post-ischaemic brain damage in two different in vitro models: rat organotypic hippocampal slices exposed to oxygen-glucose deprivation (OGD) for 20–30 min, a model characterized by apoptosis-like cell death and mouse mixed cortical cell cultures exposed to 60 min OGD, a model in which cells die with mostly necrosis-like features.

Key results:

In organotypic hippocampal slices, PARP-2 inhibition with UPF-1069 (0.01–1 µmol·L⁻¹) caused a concentration-dependent exacerbation (up to 155%) of OGD-induced CA1 pyramidal cell death. Higher concentrations, acting on both PARP-1 and PARP-2, had no effect on OGD injury. In mouse mixed cortical cells exposed to OGD, on the contrary, UPF-1069 (1–10 µmol·L⁻¹) significantly reduced post-ischaemic damage.

Conclusion and implications:

Selective PARP-2 inhibitors increased post-OGD cell death in a model characterized by loss of neurons through a caspase-dependent, apoptosis-like process (hippocampal slice cultures), but they reduced post-OGD damage and increased cell survival in a model characterized by a necrosis-like process (cortical neurons). UPF-1069 may be a valuable tool to explore the function of PARP-2 in biological systems and to examine the different roles of PARP isoenzymes in the mechanisms of cell death and survival.     

The JNK,ERK and p53 pathways play distinct roles in apoptosis mediated by the antitumor agents vinblastine,doxorubicin, and etoposide

Assessment of specific apoptosis and survival pathways implicated in anticancer drug action is important for understanding drug mechanisms and modes of resistance in order to improve the benefits of chemotherapy. In order to better examine the role of mitogen-activated protein kinases, including JNK and ERK, as well as the tumor suppressor p53, in the response of tumor cells to chemotherapy, we compared the effects on these pathways of three structurally and functionally distinct antitumor agents. Drug concentrations equal to 50 times the concentration required to reduce cell proliferation by 50% were used. Vinblastine, doxorubicin, or etoposide (VP-16) induced apoptotic cell death in KB-3 carcinoma cells, with similar kinetic profiles of PARP cleavage, caspase 3 activation, and mitochondrial cytochrome c release. All three drugs strongly activated JNK, but only vinblastine induced c-Jun phosphorylation and AP-1 activation. Inhibition of JNK by SP600125 protected cells from drug-induced cytotoxicity. Vinblastine caused inactivation of ERK whereas ERK was unaffected in cells exposed to doxorubicin or VP-16. Inhibition of ERK signaling by the MEK inhibitor, U0126, potentiated the cytotoxic effects of vinblastine and doxorubicin, but not that of VP-16. Vinblastine induced p53 downregulation, and chemical inhibition of p53 potentiated vinblastine-induced cell death, suggesting a protective effect of p53. In contrast, doxorubicin and VP-16 induced p53, and inhibition of p53 decreased drug-induced cell death, suggesting a pro-apoptotic role for p53. These results highlight the differential roles played by several key signal transduction pathways in the mechanisms of action of key antitumor agents, and suggest ways to specifically potentiate their effects in a context-dependent manner. In addition, the novel finding that JNK activation can occur without c-Jun phosphorylation or AP-1 activation has important implications for our understanding of JNK function.     

Functions of epidermal growth factor receptor in cisplatin response of thyroid cells

Epidermal growth factor receptor (EGFR) signal transduction pathway has been reported to play a vital role in the biologic progression of several tumours and as targets for therapeutic intervention. We have investigated the role of EGFR in the thyroid PC Cl3 cells response to the chemo-therapeutic agent cisplatin. It was found that cisplatin provoked (1) the activation (phosphorylation) and internalization of EGFR, (2) the phosphorylation of mitogen-activated protein kinase (MAPK)/p38, (3) the activation of PKC-?, (4) the enhancement of matrix metalloproteinase-2 (MMP-2) expression and activity, (5) the generation of reactive oxygen species (ROS) and (6) the activation of the apoptotic intrinsic pathway. Inhibition or down regulation of EGFR reduced (1) the phosphorylation of MAPK/p38, (2) the cisplatin-provoked activation of PKC-?, and (3) the activation of caspase-7 and PARP cleavage and the overall cells sensitivity to cisplatin. PKC-? inhibition achieved by siRNA blocked MAPK/p38 activation and significantly increased the cell resistance to cisplatin. Finally, when the cisplatin-induced ROS generation was blocked by using NAD(P)H oxidase inhibitors, a decrease in cisplatin-induced MMP-2 enhancement, MAPK/p38 and EGFR activation, and caspase-7 proteolysis occurred.In conclusion, these findings supported a model in which cisplatin provokes an oxidant-induced MMP-2-dependent EGFR transactivation responsible for the induction of cell apoptosis, a process ascribable to the intracellular signalling of PKC-? and MAPK/p38.     

Poly(ADP-ribose) polymerase signaling of topoisomerase 1-dependent DNA damage in carcinoma cells

A molecular approach to enhance the antitumour activity of topoisomerase 1 (TOP1) inhibitors relies on the use of chemical inhibitors of poly(ADP-ribose)polymerases (PARP). Poly(ADP-ribosyl)ation is involved in the regulation of many cellular processes such as DNA repair, cell cycle progression and cell death. Recent findings showed that poly(ADP-ribosyl)ated PARP-1 and PARP-2 counteract camptothecin action facilitating resealing of DNA strand breaks. Moreover, repair of DNA strand breaks induced by poisoned TOP1 is slower in the presence of PARP inhibitors, leading to increased toxicity.In the present study we compared the effects of the camptothecin derivative topotecan (TPT), and the PARP inhibitor PJ34, in breast (MCF7) and cervix (HeLa) carcinoma cells either PARP-1 proficient or silenced, both BRCA1/2^+/+ and p53^+/+.HeLa and MCF7 cell lines gave similar results: (i) TPT-dependent cell growth inhibition and cell cycle perturbation were incremented by the presence of PJ34 and a 2 fold increase in toxicity was observed in PARP-1 stably silenced HeLa cells; (ii) higher levels of DNA strand breaks were found in cells subjected to TPT + PJ34 combined treatment; (iii) PARP-1 and -2 modification was evident in TPT-treated cells and was reduced by TPT + PJ34 combined treatment; (iv) concomitantly, a reduction of soluble/active TOP1 was observed. Furthermore, TPT-dependent induction of p53, p21 and apoptosis were found 24–72 h after treatment and were increased by PJ34 both in PARP-1 proficient and silenced cells. The characterization of such signaling network can be relevant to a strategy aimed at overcoming acquired chemoresistance to TOP1 inhibitors.     

Critical role of PI3-kinase/Akt activation in the PARP inhibitor induced heart function recovery during ischemia-reperfusion

Poly(ADP-ribose) polymerase (PARP) inhibitors protect hearts from ischemia-reperfusion (IR)-induced damages by limiting nicotinamide adenine dinucleotide (NAD+) and ATP depletion, and by other, not yet elucidated mechanisms. Our preliminary data suggested that PARP catalyzed ADP-ribosylations may affect signaling pathways in cardiomyocytes. To clarify this possibility, we studied the effect of a well-characterized (4-hydroxyquinazoline) and a novel (carboxaminobenzimidazol-derivative) PARP inhibitor on the activation of phosphatidylinositol-3-kinase (PI3-kinase)/Akt pathway in Langendorff-perfused hearts. PARP inhibitors promoted the restoration of myocardial energy metabolism (assessed by 31P nuclear magnetic resonance spectroscopy) and cardiac function compared to untreated hearts. PARP inhibitors also attenuated the infarct size and reduced the IR-induced lipid peroxidation, protein oxidation and total peroxide concentration. Moreover, PARP inhibitors facilitated Akt phosphorylation and activation, as well as the phosphorylation of its downstream target glycogen synthase kinase-3beta (GSK-3beta) in normoxia and, more robustly, during IR. Blocking PI3-kinase by wortmannin or LY294002 reduced the PARP inhibitor-elicited robust Akt and GSK-3beta phosphorylation upon ischemia-reperfusion, and significantly diminished the recovery of ATP and creatine phosphate showing the importance of Akt activation in the recovery of energy metabolism. In addition, inhibition of PI3-kinase/Akt pathway decreased the protective effect of PARP inhibitors on infarct size and the recovery of heart functions. All these data suggest that contrary to the original view, which considered preservation of NAD+ and consequently ATP pools as the exclusive underlying mechanism for the cytoprotective effect of PARP inhibitors, the activation of PI3-kinase/Akt pathway and related processes are at least equally important in the cardioprotective effects of PARP inhibitors during ischemia-reperfusion.     

Mechanisms of cell death induced by 2-chloroadenosine in leukemic B-cells

2-chloroadenosine (2-CAdo) is an adenosine deaminase-resistant analogue of adenosine, widely used as an adenosine receptor agonist. This compound has been shown to induce apoptosis in several cell types either via activation of adenosine receptors or via intracellular metabolism. However, the molecular mechanisms of 2-CAdo-induced apoptosis are unclear. Here, we analyzed the effects of 2-CAdo in the leukemia cell line EHEB. 2-CAdo was found to induce apoptosis in EHEB cells, as shown by caspase-3 activation, DNA fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage and phosphatidylserine exposure. Cytotoxicity of 2-CAdo was completely suppressed by 5-iodotubercidin, an adenosine kinase inhibitor, indicating that apoptosis induced by 2-CAdo was the result of its intracellular metabolism. Accordingly, we found that 2-CAdo was efficiently converted into 2-chloroATP. In parallel, a decrease of intracellular ATP concentration as well as a general inhibition of macromolecular synthesis, involving DNA, RNA and protein synthesis, was observed. Moreover, 2-CAdo induced cytochrome c release into the cytosol, indicating activation of the intrinsic pathway of apoptosis. This was found associated with a decline in Mcl-1 protein level and p53-independent. Inhibition of AMP deaminase by coformycin markedly prevented ATP depletion, and also significantly reduced 2-CAdo cytotoxicity and caspase-3 activation. In conclusion, our data show that intracellular metabolism of 2-CAdo can lead to activation of the intrinsic pathway of apoptosis and that ATP depletion, in addition to the accumulation of the triphosphate analogue, contributes to 2-CAdo-induced apoptosis.     

Doxorubicin-induced testicular damage is related to PARP-1 signaling molecules in mice

BackgroundDoxorubicin (DOX), is a chemotherapeutic agent, which evokes oxidative stress and cell apoptosis in testicular tissue. It is known that the activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP), leading to apoptosis induced by DOX. The aim of the present study is to investigate whether PARP pathway has a role in DOX-induced testicular damage and infertility utilizing pharmacological PARP-1 inhibitor, PJ34, and PARP-1 knockout mice model.MethodsFirstly, we assessed the activation of PARP pathway after DOX-induction at various hours by immunohistochemistry and western blot analysis. Secondly, we evaluated the role of PARP pathway in DOX-induced testicular damage, sperm motility, and fertility with pharmacological inhibition of PARP by using PJ34. Finally, we aimed to correlate a functional relationship between PARP-1 and DOX using PARP-1 knockout mice in DOX-induced testicular damage. Doxorubicin levels in plasma and testis tissue were also assessed.ResultsIn DOX-induced group; PARP-1, PAR and apoptotic pathway protein expressions, increased significantly. In DOX + PJ34 group; PAR, cytochrome c, and AIF levels decreased significantly. Testicular weights, sperm motility, and mean the number of pups per litter decreased in DOX-induced group after 28 days, however they were similar to the control group in DOX-PJ34 group. In PARP-1 KO group, seminiferous tubule morphology was impaired significantly after 28 days of DOX-administration.ConclusionsOur study indicates that DOX-induced testicular damage may be related to over-activation of PARP-1. PJ34 application was effective in preventing severe testicular damage caused by DOX-injection and may be considered for fertility protection against DOX-induced testicular damage.     

New inhibitors of poly(ADP-ribose) polymerase and their potential therapeutic targets

Poly(ADP-ribose) polymerase (PARP) is a DNA-binding protein that is activated by nicks in the DNA molecule. It regulates the activity of various enzymes, including itself, that are involved in the control of DNA metabolism. Evidence obtained with both benzamide and isoquinolinone PARP inhibitors and the PARP-1(^-/-) phenotype, clearly indicate that PARP plays an important role in NO/ROS-induced cell damage during inflammation, ischaemia and neurodegeneration. PARP is involved in the maintenance of genomic stability and PARP inhibition may also potentiate the cytotoxic action of agents used in cancer therapy. Benzamides, although not very potent (IC₅₀ ~ 20 – 50 μM) PARP inhibitors, have been widely used to probe PARP functions, because of their lack of toxicity both in vitro and in vivo, even at high doses. In the early 1990s, a new class of very potent PARP inhibitors (i.e., at least 100-fold more potent thatn benzamide), the dihydroisoquinolinones, benzamide derivatives with the carbamoyl group constrained into the antiorientation, was discovered. At the same time, a large structure–activity surevey identified over 13 chemical classes of PARP inhibitors, the most potent calss sharing a common structural feature, the presence of a carbonyl group built into a polyaromatic heterocyclic skeleton or a carbamoyl group attached to an aromatic ring. Recently, a better knowledge of the PARP catalytic domain and the use of its crystal structure have led to the design and synthesis of the tricyclic lactam indoles, active at low nanomolar concentrations, and with favourable physical properties and in vivo characteristics. In the last few years the interest in PARP as a therapeutic target has been rapidly growing. This article reviews the patents filed for new PARP inhibitors over the last three years, up to February 2002, and their development status.     

Benzo(a)pyrene-caused increased G1-S transition requires the activation of c-Jun through p53-dependent PI-3K/Akt/ERK pathway in human embryo lung fibroblasts

Benzo(a)pyrene (B(a)P) is a potent lung carcinogen mainly derived from tobacco smoking and environmental contamination, however, the molecular mechanisms by which it accelerates the cell cycle progression and induces the abnormal cell proliferation are still far away from understood. Our current analysis of human embryo lung fibroblasts (HELF) showed that B(a)P exposure was able to promote cell cycle G₁–S phase transition. This effect was correlated with c-Jun activation because inhibition of c-Jun by its dominant negative mutant (TAM67) reversed B(a)P action on cell cycle with the down-regulation of expression of cyclin D1, pRb and E2F1. Further study found that overexpression of dominant negative mutants of, PI-3K or Akt, dramatically reduced B(a)P-induced the activation of c-Jun and extracellular signaling regulated kinase (ERK), but not c-Jun NH2 terminal kinase (JNK). Inhibition of p53 by either its inhibitor pifithrin- or p53 siRNA markedly increased B(a)P-induced the activation of c-Jun, Akt and ERK in this context. Take together, our results indicate that c-Jun activation by p53-dependent PI-3K/Akt/ERK pathway is responsible for B(a)P-induced cell cycle alternations in human embryo lung fibroblasts.