poly(ADP-ribose) Polymerase Pathway Research Articles

The clinically active PARP inhibitor olaparib ameliorates doxorubicin-induced cardiotoxicity in both in vitro and in vivo model

Abstract Introduction Inhibition of poly (ADP-ribose) polymerase (PARP) has been shown to be cardioprotective in acute and chronic myocardial injury. Additionally, PARP inhibitor, Olaparib, is a clinically effective anti-cancer agent. Activation of PARP pathway has been suggested to be involved in cardiotoxicity arising from Doxorubicin (DOX, one of the most used anticancer drugs). It is thus, entirely possible that PARP inhibition could be a novel pathway to prevent DOX-induced cardiotoxicity (DIC). Purpose To determine the cardioprotective effects of Olaparib in preventing DOX-induced cardiotoxicity in in vitro and in vivo models. Methods In vitro: Human cardiomyocytes (HCMs) were treated with DOX at 1uM (EC50) +/- various doses (15.6nM, 1uM, 80uM, 100uM and 150uM) of Olaparib at 72hrs. Cell viability was assessed via CellTiter-Glo®. The mRNA expressions of treated HCMs were performed by qPCR. In vivo: Female C57BL/6 mice (6-8 weeks old) were administered: A) DOX at 5mg/kg/week for 6 weeks via IP injections, B) vehicle of 0.9% saline and 5% DMSO in PBS, C) Olaparib at 50mg/kg was administered 3 times/week, D) Olaparib at 50mg/kg was administered 3 times/week, followed by DOX treatments. Cardiac function was assessed by echocardiography at baseline and end of 6 weeks’ treatment. Results For the in vitro model, Doxorubicin induced marked reduction in HCMs cell viability. Concomitant Olaparib treatment at higher doses (80uM, 100uM, 150uM) significantly preserved DOX-induced cell viability after 72 hours of treatment. Molecular analysis in HCMs showed DOX-induced upregulation of mRNA gene expressions for apoptosis: Casp3, DNA damage: BBC3, and cardiac remodelling: TGF-β; all of which were reversed by Olaparib. In vivo: we found significant reduction of left ventricular function and increased ventricular wall thickness in DOX treated group after 6 weeks’ treatment compare to baseline [LVEF%: 52.4±1.3 vs.56.5±2.0, P=0.02; FS%:16.7±1.4 vs. 20.1±1.3, P=0.03; Total Wall thickness (mm):2.1±0.1 vs. 1.9±0.1, P<0.05]; whereas the mice pre-treated with Olaparib in combination with DOX showed preservation of cardiac functions between endpoint and baseline. Conclusion Olaparib protected HCMs against DIC and improved cardiac function in DIC mice model. Our findings indicate that Olaparib could provide cardioprotection in DOX-induced cardiotoxicity.

Pharmacological Modulation of TRPM2 Channels via PARP Pathway Leads to Neuroprotection in MPTP-induced Parkinson's Disease in Sprague Dawley Rats.

Transient receptor potential melastatin-2 (TRPM2) channels are cation channels activated by oxidative stress and ADP-ribose (ADPR). Role of TRPM2 channels has been postulated in several neurological disorders, but, it has not been explored in animal models of Parkinson's disease (PD). Thus, the role of TRPM2 and its associated poly (ADPR) polymerase (PARP) signaling pathways were investigated in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD rat model using TRPM2 inhibitor, 2-aminoethyl diphenyl borinate (2-APB), and PARP inhibitor, N-(6-Oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamino) acetamide hydrochloride (PJ-34). PD was induced by using a bilateral intranigral administration of MPTP in rats, and different parameters were evaluated. An increase in oxidative stress was observed, leading to locomotor and cognitive deficits in the PD rats. PD rats also showed an increased TRPM2 expression in the striatum and mid-brain accompanied by reduced expression of tyrosine hydroxylase (TH) in comparison to sham animals. Intraperitoneal administration of 2-APB and PJ-34 led to an improvement in the locomotor and cognitive deficits in comparison to MPTP-induced PD rats. These improvements were accompanied by a reduction in the levels of oxidative stress and an increase in TH levels in the striatum and mid-brain. In addition, these pharmacological interventions also led to a decrease in the expression of TRPM2 in PD in the striatum and mid-brain. Our results provide a rationale for the development of potent pharmacological agents targeting the TRPM2-PARP pathway to provide therapeutic benefits for the treatment of neurological diseases like PD.

515MO A phase I trial of durvalumab (Durv) in combination with olaparib (Ola) and capivasertib (Cap) in patients (pts) with advanced or metastatic cancers (Ca) (MEDIPAC)

Tumor-specific homologous recombination (HR) DNA defects and the phosphatidylinositol 3-kinase (PI3K)-AKT pathway are commonly implicated in tumorigenesis, and preclinical studies have shown synergistic effects of poly (ADP-ribose) polymerase (PARP) with PI3K-AKT inhibitors. Both pathways modulate the tumor immune microenvironment by regulation of tumor-infiltrating lymphocytes and reduction of Treg cells. We hypothesized that triplet combination of immunotherapy with PARP and PI3K-AKT pathway inhibition is tolerable and may demonstrate activity in pts with relevant pathway defects.

Abstract LB130: Combination of IGF-1R targeted alpha therapy with Olaparib results in synergistic efficacy against colorectal and lung cancer xenografts

Abstract Objectives: FPI-1434 is a targeted alpha-particle therapeutic consisting of an IGF-1R targeting antibody radiolabeled with Actinium-225. The primary mechanism of action for FPI-1434 is induction of double strand DNA breaks (DSB) in targeted tumors resulting in cell death. Poly (ADP-ribose) polymerase (PARP) is part of the cellular mechanism that repairs DSB. In cancer patients with genetic defects in DSB repair (eg. BRCA1/2), the PARP pathway becomes a primary repair system and its inhibition results in cell death. This mechanism has been leveraged as a therapy against DNA-repair deficient tumors leading to FDA-approval of PARP inhibitors (PARPi) including Olaparib. Treatment with PARPi to block repair of DNA damage driven by FPI-1434 may act synergistically to increase the lethal DNA damage load. To that end, Fusion has performed studies to combine FPI-1434 and Olaparib against tumor models with no pre-disposing defects in DNA repair. Methods: Colorectal (Colo-205) or lung (A549) cancer xenografts were established in Balb/c nude mice. For efficacy studies, a dose combination matrix for FPI-1434 and Olaparib was tested. FPI-1434 was dosed (i.v.) once at 20-200 nCi followed with Olaparib (i.p.) at doses of 0-50 mg/kg. Olaparib was dosed 24h after FPI-1434 using a 5 day on/2 day off schedule (5 mice/group). DSB formation (γH2AX) and apoptosis (cleaved caspase 3) were evaluated in treated tumors by IHC staining. Results: DSB formation and induction of apoptosis were detected in FPI-1434-treated tumors in a time and dose dependent manner. DSB formation was observed in all areas of tumor containing intact or apoptotic cancer cells confirming FPI-1434 mechanism. In tumor efficacy studies, Olaparib had no single-agent efficacy in the Colo-205 or A549 models. Single doses of FPI-1434 at 20 nCi had no effect on Colo-205 tumors, suppressed growth at 50 nCi and caused regression at 100 nCi. Combination efficacy was seen in the 20 and 50 nCi dose groups, including Olaparib. Doses of 20 nCi FPI-1434 and 25 mg/kg Olaparib had the strongest combined effect in this model. In the A549 model, single doses of FPI-1434 had no effect at 20 or 50 nCi, caused growth suppression at 100 nCi and regression at 200 nCi. Combination efficacy was seen in the 20, 50 and 100 nCi dose groups, including Olaparib. Doses of 50 nCi FPI-1434 and 25 mg/kg Olaparib had the strongest combined effect in this model. In general, the strongest efficacy was observed by combining ineffective single-agent doses of the two compounds. Strong efficacy of high dose FPI-1434 masked observable combination effects suggesting that FPI-1434 was the therapeutic driver. Conclusions: Olaparib co-dosing enhanced FPI-1434 efficacy which supports consideration of this combination for clinical use. Ineffective single-agent doses, when combined, resulted in synergistic efficacy in both Colo-205 and A549 models suggesting that the mechanism can be applied to multiple cancer types where predisposing mutations in DNA repair are lacking. The strongest combination effect appeared to occur at the low single-agent doses suggesting that PARP inhibition may potentiate efficacy at lower clinical doses of FPI-1434. Citation Format: Meiduo Hu, John Forbes, Ryan Simms, Yaryna Storozhuk, Eric Burak, John Valliant. Combination of IGF-1R targeted alpha therapy with Olaparib results in synergistic efficacy against colorectal and lung cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB130.

Update on the Role of Poly (ADP-Ribose) Polymerase Inhibitors in the DNA Repair-Deficient Pancreatic Cancers: A Narrative Review.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is the most common cancer found in the pancreas. It has a dismal prognosis and current therapeutic options, including surgical resection, provide only a temporary or limited response due to the development of treatment resistance.Methods: A narrative review of studies investigating poly (ADP-ribose) polymerase (PARP) pathway inhibitors in metastatic PDAC to highlight recent advances.Results: Mutations in BRCA genes confer a higher risk of PDAC, while germ line mutations are found in 4–7% of individuals harboring pancreatic cancer. Although solid tumors with defective DNA damage repair defect (DDR) genes such as BRCA show heightened sensitivity to platinum agents, tumors can exploit the PARP pathway as salvage pathways. Therefore, blocking this pathway will trigger cell death in vulnerable tumor cells with BRCA/DNA repair deficiency. Several drugs with inhibitory activity on the PARP pathway have been approved for breast and ovarian tumors harboring germ line or somatic BRCA mutations. Based on these results, the phase III POLO study showed a significant improvement in progression-free survival compared with placebo in BRCA mutant pancreatic tumors and highlighted the importance of germ line testing in everyone diagnosed with pancreatic cancer. In addition, expansion of the PARP inhibitor indication beyond BRCA mutations to other genes involved in DDR such as ATM and PALB2 merits attention.Conclusion: PARP inhibitors represent a safe and efficacious treatment for a subset of PDAC patients with BRCA mutations. Ongoing trials are evaluating PARP inhibitors in PDAC patients with non-BRCA DDR gene deficiencies as well as PARP inhibitors in combination with other agents, notably immune checkpoint inhibitors to expand the group of patients that derive benefit from this treatment.

Biochemical Evidence Indicates the Preventive Effect of Resveratrol and Nicotinamide in the Treatment of STZ-induced Diabetic Cataract

ABSTRACT Purpose High glucose level is a strong initiator of both oxidative stress and DNA damage to various cellular proteins. This activates the poly ADP-ribose polymerase (PARP) enzyme, which is responsible for disturbing physiological energy metabolic homeostasis. The present study aimed to elucidate the association between stress and the PARP pathway by using resveratrol (RSV) and nicotinamide (NAM, PARP inhibitor) to treat diabetic cataract. Method Albino rats were used for the experimental study. A single streptozotocin administration (55 mg/kg, i.p.) prompted diabetes in the animals. The experimental groups were the normal group (non-diabetic) and the diabetic groups: the diabetic control animals (group D), the diabetic animals treated with RSV at 40 mg/kg/day, i.p. (D+ RSV group), NAM at 100 and 300 mg/kg/day, i.p. (D+ NAM100, D+ NAM300 groups, respectively), and a combination of RSV and NAM i.p. (D+ RSV+NAM100 = Combi 1 group, D+ RSV+NAM300 = Combi 2 group). Glucose levels and the eyes were examined biweekly; various cataractogenic parameters in the lenses were examined after completion of the eight-week experimental protocol. Results Compared to diabetic control, RSV monotherapy significantly decreased hyperglycemia and other lenticular alterations. NAM at the high dose only showed beneficial effects without altering the blood glucose level, lenticular aldose reductase (AR) activity, and sorbitol content, primarily restored the lenticular NAD level and decreased oxidative stress in diabetic rats. These findings regarding NAM treatment indicate that a pathway other than the antioxidant defense system and the polyol pathway, which might be due to PARP inhibition, is involved in diabetic cataracts. Moreover, compared to RSV monotherapy, combination treatments were effective. Conclusion These results indicate that hyperglycemia and oxidative-osmotic-nitrosative stress play central roles in the pathophysiology of diabetic cataracts. Moreover, our study also revealed that concurrent treatment with the RSV and NAM may prove useful in the pharmacotherapy of diabetes and its secondary complications such as cataract.

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

Doxorubicin (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. Firstly, 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. In 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. Our 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.

Effect of Notch and PARP Pathways' Inhibition in Leukemic Cells.

Differentiation of blood cells is one of the most complex processes in the body. It is regulated by the action of transcription factors in time and space which creates a specific signaling network. In the hematopoietic signaling system, Notch is one of the main regulators of lymphocyte development. The aim of this study was to get insight into the regulation of Notch signalization and the influence of poly(ADP-ribose)polymerase (PARP) activity on this process in three leukemia cell lines obtained from B and T cells. PARP1 is an enzyme involved in posttranslational protein modification and chromatin structure changes. B and T leukemia cells were treated with Notch and PARP inhibitors, alone or in combination, for a prolonged period. The cells did not show cell proliferation arrest or apoptosis. Analysis of gene and protein expression set involved in Notch and PARP pathways revealed increase in JAGGED1 expression after PARP1 inhibition in B cell lines and changes in Ikaros family members in both B and T cell lines after γ-secretase inhibition. These data indicate that Notch and PARP inhibition, although not inducing differentiation in leukemia cells, induce changes in signaling circuits and chromatin modelling factors.

Identification of a novel truncating mutation in PALB2 gene by a multigene sequencing panel for mutational screening of breast cancer risk-associated and related genes.

Breast cancer (BC) is the most common neoplasm in women, with 5%-10% patients showing a familial predisposition, where germline mutations in BRCA1/BRCA2 genes are found in -20% of cases. Next-generation sequencing (NGS) is among the best available options for genetic screening, providing several benefits that include enhanced sensitivity and unbiased mutation detection. PALB2 (partner and localizer of BRCA2) is a cancer predisposing gene recently described that encodes a protein partner of BRCA2 involved in DNA double-strand break repair and cell cycle control. The DNA damage response represents a key cellular event, targeted by innovative anticancer therapies, including those based on poly (ADP-ribose) polymerase (PARP) inhibitors targeting PARP1 and PARP2 enzymes, activated by DNA damage and involved in single-strand break and base excision repair. Genomic DNA was isolated from 34 patient samples and four BC cell lines, as controls, and 27 breast cancer predisposing genes belonging to the BRCA1/BRCA2 and PARP pathways were sequenced by NGS. The panel described here allowed identification of several sequence variations in most investigated genes, among which we found a novel truncating mutation in PALB2. The NGS-based strategy designed here for molecular analysis of a customized panel of BC predisposing and related genes was found to perform effectively, providing a comprehensive exploration of all genomic sequences of the investigated genes. It is thus useful for BC molecular diagnosis, in particular for familiar cases where alterations in routinely investigated genes, such as BRCAs, result to be absent.

Abstract 1114: Combining anti-androgen therapy and PARP inhibition results in synergistic cytotoxicity of metastatic castration-resistant prostate cancer (mCRPC) cells

Abstract Androgen deprivation therapy is an efficient first line therapy for advanced prostate cancer. However, in most cases resistance inevitably develops resulting in metastatic castration-resistant prostate cancer (mCRPC). Abiraterone and enzalutamide are approved to treat mCRPC and act by inhibiting androgen biosynthesis and androgen receptor (AR) signaling, respectively. Poly (ADP-ribose) polymerase 1 (PARP1) is a key enzyme in the DNA damage repair pathway and has also been implicated in AR signaling cascade. Recent reports suggest significant efficacy combining anti-androgens and inhibitors of Poly (ADP-ribose) polymerase (PARP) particularly against cells harboring mutations in the DNA repair pathway. Here, we provide evidence of synergistic cytotoxicity combining PARP inhibition and androgen signaling ablation in the AR positive human CRPC cell line 22RV1 harboring mutation in the DNA repair gene BRCA2. The PARP inhibitor talazoparib (IC50 - 0.05 μM) was upto 20 times more potent than olaparib (IC50 – 1.0 μM) in inducing cell death in 22RV1 as seen by cell viability and clonogenic assays. When combined with AR antagonist enzalutamide (IC50 – 0.25 μM) or androgen synthesis inhibitor abiraterone (IC50 – 1.25 μM) both PARP inhibitors showed significant synergy, although talazoparib was more prominent owing to its superior potency. Higher concentrations of enzalutamide and abiraterone (>10μM) induced PARP cleavage and increased expression of γ-H2AX, a marker for DNA damage. Our results suggest that AR signaling inhibition involves PARP mediated apoptosis and highlight the crosstalk between PARP and AR pathways. In summary, combining anti-androgen therapy and PARP inhibitors carry significant therapeutic potential for CRPC patients harboring mutations in DNA repair pathway. Citation Format: Sahithi Pamarthy, Vinay Sagar, Rajita J. Vatapalli, Zachary Reinstein, Muhammad Zayd Ansari, Benedito A. Carneiro Filho, Francis J. Giles, Sarki A. Abdulkadir. Combining anti-androgen therapy and PARP inhibition results in synergistic cytotoxicity of metastatic castration-resistant prostate cancer (mCRPC) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1114. doi:10.1158/1538-7445.AM2017-1114

Protective effect of diethylcarbamazine inhibits NF-κB activation in isoproterenol-induced acute myocardial infarction rat model through the PARP pathway

The present study investigated the protective effect of diethylcarbamazine in inhibiting nuclear factor (NF)-κB activation in isoproterenol‑induced acute myocardial infarction (AMI) rats through the poly ADP ribose polymerase (PARP) pathway. Male albino Wistar rats were injected subcutaneously with isoproterenol (100mg/kg/day) for 2days to induce an AMI model. Diethylcarbamazine (50mg/kg) was administered by gavage for 12days prior to the isoproterenol-induced AMI. It was noted that diethylcarbamazine significantly inhibited AMI‑induced casein kinase and lactate dehydrogenase levels, and reduced the AMI‑induced wet heart weight to body weight ratio in AMI rats. Diethylcarbamazine treatment significantly weakened reactive oxygen species production and reduced the levels of tumor necrosis factor (TNF)‑α, interleukin‑6 and NF‑κB/p65 in AMI rats. Western blotting demonstrated that diethylcarbamazine significantly suppressed the AMI‑induced inducible nitric oxide synthase (iNOS), transforming growth factor (TGF)‑β1, cyclooxygenase‑2 (COX‑2) and PARP protein expression in AMI rats. The results demonstrated that the protective effect of diethylcarbamazine inhibited isoproterenol‑induced AMI through the suppression of inflammation, iNOS, TGF‑β1, COX‑2 and the PARP pathway, and revealed the clinical potential of diethylcarbamazine for therapeutic and clinical applications.

Inhibition of poly(ADP-ribose) polymerase may have preventive potential for varicocoele-associated testicular damage in rats.

Varicocele is ordinarily accompanied by testicular damage and male infertility. Several theories have been proposed to explain the detrimental effect of varicocele on testis tissue, including the possible effects of oxidative stress. The poly(ADP-ribose) polymerase (PARP) pathway has been established as a major downstream intracellular pathway of oxidative stress. Recently we have reported that PARP pathway has been activated in varicocele-induced rat testicular damage model. The aim of the present study was to investigate the possible protective effect of PARP inhibition in varicocele-associated testicular damage. Fifty male Wistar rats were divided into five groups: control, sham, varicocele-induced, varicocele-induced 1,5-isoquinolinediol (ISO, a PARP inhibitor)-treated, and ISO treated groups. The ISO-treated rats received intraperitoneal injections of 3mg/kg ISO daily for 13weeks. After 13weeks of varicocele induction, body and testes weights were investigated in all groups. Histopathology of testes were evaluated by light microscopy. Expressions of PAR, p53 and cytochrome c were detected by immunohistochemistry and cleaved PARP-1, PAR, p53 and cytochrome c by western blot. The degree of apoptosis was determined by TUNEL. Light microscopy revealed testicular damage comprising various degrees of seminiferous tubule degeneration in varicocele-induced rats and their testes weights decreased significantly, whereas ISO administration prevented it. Expressions of cleaved PARP-1, PAR, cytochrome c, and p53 increased significantly in varicocele-induced rats, whereas the level of these molecules were similar to controls in varicocele-induced rats treated with ISO. In conclusion, increased PARP activation in testes seems to be related with testicular damage and apoptosis associated with varicocele and pharmacological inhibition of this pathway might be an effective intervention to prevent varicocele-induced testicular injury.

Xanthohumol inhibits proliferation of laryngeal squamous cell carcinoma.

Xanthohumol is a flavonoid compound that exhibits antioxidant and anticancer effects, and is used to treat atherosclerosis. The aim of the present study was to investigate the effect of xanthohumol on the cell proliferation of laryngeal squamous cell carcinoma and to understand the mechanism of its action. The effects of xanthohumol on the cell viability and apoptosis rate of laryngeal squamous cell carcinoma SCC4 cells were assessed by Annexin V-fluorescein isothiocyanate/propidium iodide staining. In addition, the expression levels of pro-apoptotic proteins, caspase-3, caspase-8, caspase-9, poly ADP ribose polymerase (PARP) p53 and apoptosis-inducing factor (AIF), as well as anti-apoptotic markers, B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia 1 (Mcl-1), were analyzed by western blotting. The results revealed that treatment with 40 µM xanthohumol significantly inhibited the proliferation of SCC4 cells. Furthermore, xanthohumol treatment (40 µM) induced SCC4 cell apoptosis, as indicated by the significant increase in activity and expression of caspase-3, caspase-8, caspase-9, PARP, p53 and AIF. By contrast, the protein expression of Bcl-2 and Mcl-1 was significantly decreased following treatment with 40 µM xanthohumol. Taken together, the results of the present study indicated that xanthohumol mediates growth suppression and apoptosis induction, which was mediated via the suppression of Bcl-2 and Mcl-1 and activation of PARP, p53 and AIF signaling pathways. Therefore, future studies that investigate xanthohumol as a potential therapeutic agent for laryngeal squamous cell carcinoma are required.

Effects of Trans-Resveratrol on hyperglycemia-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase signaling in rat testis

Diabetes induces oxidative stress, DNA damage and alters several intracellular signaling pathways in organ systems. This study investigated modulatory effects of Trans-Resveratrol on type 1 diabetes mellitus (T1DM)-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase (PARP) signaling in rat testis. Trans-Resveratrol administration (5mg/kg/day, ip) to Streptozotocin-induced T1DM adult male Wistar rats from day 22–42 resulted in recovery of induced oxidative stress, abnormal spermatogenesis and inhibited DNA synthesis, and led to mitigation of 8-hydroxy-2'-deoxyguanosine formation in the testis and spermatozoa, and DNA double-strand breaks in the testis. Trans-Resveratrol aggravated T1DM-induced up-regulation of aminoacyl tRNA synthetase complex-interacting multifunctional protein 2 expression; however, it did not modify the up-regulated total PARP and down-regulated PARP1 expressions, but recovered the decreased SirT1 (Sirtuin 1) levels in T1DM rat testis. Trans-Resveratrol, when given alone, reduced the poly (ADP-ribosyl)ation (pADPr) process in the testis due to an increase in PAR glycohydrolase activity, but when given to T1DM rats it did not affect the pADPr levels. T1DM with or without Trans-Resveratrol did not induce nuclear translocation of apoptosis-inducing factor and the formation of 50 kb DNA breaks, suggesting to the lack of caspase-3-independent cell death called parthanatos. T1DM with or without Trans-Resveratrol did not increase necrotic cell death in the testis. Primary spermatocytes, Sertoli cells, Leydig cells and intra-testicular vessels showed the expression of PARP pathway related proteins. In conclusion, Trans-Resveratrol mitigates T1DM-induced sperm abnormality and DNA damage, but does not significantly modulate PARP signaling pathway, except the SirT1 expression, in the rat testis.

Recombinant tissue plasminogen activator enhances microparticle release from mouse brain-derived endothelial cells through plasmin

Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is currently the only approved pharmacological strategy for acute ischemic stroke. However, rt-PA exhibits vascular toxicity mainly due to endothelial damage. To investigate the mechanisms underlying rt-PA-induced endothelial alterations, we assessed the role of rt-PA in the generation of endothelial microparticles (EMPs), emerging biological markers and effectors of endothelial dysfunction.The mouse brain-derived endothelial cell line bEnd.3 was used. Cells were treated with rt-PA at 20, 40 or 80μg/ml for 15 or 24h, and EMPs were quantified in the culture media using Annexin-V staining coupled with flow cytometry. Rt-PA enhanced EMP release from bEnd.3 cells with a maximal increase at the 40μg/ml dose for 24h (+78% compared to controls). Using tranexamic acid and aprotinin we demonstrated that plasmin is responsible for rt-PA-induced EMP release. The p38 MAPK inhibitor SB203580 and the poly(ADP-ribose)polymerase (PARP) inhibitor PJ34 also reduced rt-PA-induced EMP production, suggesting that p38 MAPK and PARP are downstream intracellular effectors of rt-PA/plasmin. Rt-PA also altered through plasmin the morphology and the confluence of bEnd.3 cells. By contrast, these changes did not implicate p38 MAPK and PARP.This study demonstrates that rt-PA induces the production of microparticles by cerebral endothelial cells, through plasmin, p38 MAPK and PARP pathways. Determining the phenotype of these EMPs to clarify their role on the endothelium in ischemic conditions could thus be of particular interest.

TRPM2 channels in alveolar epithelial cells mediate bleomycin-induced lung inflammation

Lung inflammation is a major adverse effect of therapy with the antitumor drug bleomycin (BLM). Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable channel that is activated by oxidative stress through the production of ADP-ribose. We herein investigated whether TRPM2 channels contributed to BLM-induced lung inflammation. The intratracheal instillation of BLM into wild-type (WT) mice increased the number of polymorphonuclear leukocytes (PMNs) and inflammatory cytokine levels in the lung. Increases in inflammatory markers in WT mice were markedly reduced in trpm2 knockout (KO) mice, which demonstrated that the activation of TRPM2 channels was involved in BLM-induced lung inflammation. The expression of TRPM2 mRNA was observed in alveolar macrophages, alveolar epithelial cells, and lung fibroblasts. Actually, TRPM2 protein was expressed in lung tissues. Of these, TRPM2 channels in epithelial cells were activated by the addition of H2O2 following a BLM pretreatment, resulting in the secretion of macrophage inflammatory protein-2 (MIP-2). The H2O2-induced activation of TRPM2 by the BLM pretreatment was blocked by the poly(ADP-ribose) polymerase (PARP) inhibitors PJ34 and 3-aminobenzamide. The accumulation of poly(ADP-ribose) in the nucleus, a marker for ADP-ribose production, was strongly induced by H2O2 following the BLM pretreatment. Furthermore, administration of PRAP inhibitors into WT mice markedly reduced recruitment of inflammatory cells and MIP-2 secretion induced by BLM instillation. These results suggest that the induction of MIP-2 secretion through the activation of TRPM2 channels in alveolar epithelial cells is an important mechanism in BLM-induced lung inflammation, and the TRPM2 activation is likely to be mediated by ADP-ribose production via PARP pathway. TRPM2 channels may be new therapeutic target for BLM-induced lung inflammation.

Downregulation of NIT2 inhibits colon cancer cell proliferation and induces cell cycle arrest through the caspase-3 and PARP pathways.

Colorectal cancer, also known as colon cancer is the most devastating malignancy worldwide. Previous studies have reported that Nit2, a member of the nitrilase superfamily, is a potential tumor suppressor, although its function remains elusive in colon cancer. In the present study, we employed an RNA interference lentivirus system to silence endogenous NIT2 expression in the colon cancer cell line, HCT116. The knockdown efficiency was determined by RT-qPCR and western blot analysis. The depletion of NIT2 markedly inhibited colon cancer cell proliferation and colony formation and induced cell cycle arrest in the G0/G1 phase, as shown by MTT assay, colony formation assay and flow cytometric analysis, respectively. Further investigation with an intracellular signaling array demonstrated that the depletion of NIT2 triggered the apoptosis of colon cancer cells through the caspase-3 and poly(ADP-ribose) polymerase (PARP) pathways. Our findings suggest that NIT2 may be an oncogene in human colon cancer and may thus serve as a promising therapeutic target for the treatment of colon cancer.

Poly(ADP-ribose) polymerase inhibition improves endothelin-1-induced endothelial dysfunction in rat thoracic aorta

Aim. The aim of this study was to investigate whether poly(ADP-ribose) polymerase (PARP) inhibition improves endothelin-1 (ET-1)-induced endothelial dysfunction (ED).Methods. Isolated rat thoracic aorta rings were incubated with ET-1 (10 nmol/L) in the presence or absence of either polyethylene glycol–superoxide dismutase (PEG-SOD; a cell-permeable superoxide radical scavenger, 41 U/mL) plus apocynin (a NADPH oxidase inhibitor, 300 µmol/L) or PJ34 (an inhibitor of polyADP-ribose polymerase, 3 µmol/L) for 18 h. Isometric tension studies were performed in response to acetylcholine (ACh; an endothelium-dependent vasodilator), sodium nitroprusside (SNP; an endothelium-independent vasodilator), and phenylephrine (Phe). PARP-1 and PAR (an end-product of PARP activity) expressions were evaluated by both Western blot and immunohistochemistry.Results. Incubation of thoracic aorta rings with ET-1 resulted in a significant inhibition of the response to ACh, while SNP-induced relaxation was unaffected. The contractile response to Phe increased in arteries that were incubated with ET-1. PARP-1 and PAR expressions increased after ET-1 incubation. The diminished vasoreactivity as well as changes in expressions of PARP-1 and PAR in ET-1-incubated vessels were improved by both PEG-SOD plus apocynin and PJ34.Conclusion. Our studies demonstrate that ED induced by ET-1 seems to be effected via oxidative stress in the thoracic aorta endothelium with subsequent activation of the PARP pathway.

Review of Poly (ADP-ribose) Polymerase (PARP) Mechanisms of Action and Rationale for Targeting in Cancer and Other Diseases

Poly (ADP-ribose) polymerases (PARPs) are a family of related enzymes that share the ability to catalyze the transfer of ADP-ribose to target proteins. PARPs play an important role in various cellular processes, including modulation of chromatin structure, transcription, replication, recombination, and DNA repair. The role of PARP proteins in DNA repair is of particular interest, in view of the finding that certain tumors defective in homologous recombination mechanisms, may rely on PARP-mediated DNA repair for survival, and are sensitive to its inhibition. PARP inhibitors may also increase tumor sensitivity to DNA-damaging agents. Clinical trials of PARP inhibitors are investigating the utility of these approaches in cancer. The hyperactivation of PARP has also been shown to result in a specific programmed cell death pathway involving NAD+/ATP depletion, mu-calpain activation, loss of mitochondrial membrane potential, and the release of apoptosis inducing factor. Hyperactivation of the PARP pathway may be exploited to selectively kill cancer cells. Other PARP forms, including tankyrase 1 (PARP 5a), which plays an important role in enhancing telomere elongation by telomerase, have been found to be potential targets in cancer therapy. The PARP pathway and its inhibition thus offers a number of opportunities for therapeutic intervention in both cancer and other disease states.

Amelioration of diabetes-induced neurobehavioral and neurochemical changes by melatonin and nicotinamide: Implication of oxidative stress–PARP pathway

Diabetes associated hyperglycemia results in generation of reactive oxygen species which induces oxidative stress and initiate massive DNA damage leading to overactivation of poly (ADP-ribose) polymerase (PARP). In this study, we have elucidated the involvement of oxidative stress–PARP pathway using pharmacological interventions (melatonin, as an anti-oxidant and nicotinamide, as a PARP inhibitor) in diabetes-induced neurobehavioral and neurochemical alterations. Sprague–Dawley rats were rendered diabetic by a single intraperitoneal injection of streptozotocin. Behavioral and cognitive deficits were assessed after 8weeks of diabetes induction using a functional observation battery, passive avoidance and rotarod test. Acetylcholinesterase activity was significantly decreased in hippocampus of diabetic rats as compared to control rats. Diabetic animals showed significant increase in malondialdehyde levels and reduction in NAD levels in hippocampus. Glutamate and GABA levels were also altered in hippocampus of the diabetic animals. Two week treatment with melatonin (3 and 10mg/kg) and nicotinamide (300 and 1000mg/kg) alone and in combination significantly improved the neurobehavioral parameters which were altered in diabetes. Neurotransmitter (glutamate and GABA) levels were improved by these interventions. Our results emphasize that simultaneous inhibition of oxidative stress–PARP overactivation cascade can be beneficial in treatment of diabetes associated CNS changes.