Levels Of Poly ADP-ribose Polymerase Research Articles

Poly(ADP-Ribose) Polymerase Inhibition Sensitizes Colorectal Cancer-Initiating Cells to Chemotherapy.

Colorectal cancer (CRC) remains a leading killer in the U.S. with resistance to treatment as the largest hurdle to cure. Colorectal cancer-initiating cells (CICs) are a self-renewing tumor population that contribute to tumor relapse. Here, we report that patient-derived CICs display relative chemoresistance compared with differentiated progeny. In contrast, conventional cell lines failed model therapeutic resistance. CICs preferentially repaired chemotherapy-induced DNA breaks, prompting us to interrogate DNA damage pathways against which pharmacologic inhibitors have been developed. We found that CICs critically depended on the key single-strand break repair mediator, poly(ADP-ribose) polymerase (PARP), to survive treatment with standard-of-care chemotherapy. Small molecule PARP inhibitors (PARPi) sensitized CICs to chemotherapy and reduced chemotherapy-treated CIC viability, self-renewal, and DNA damage repair. Although PARPi monotherapy failed to kill CICs, combined PARPi therapy with chemotherapy attenuated tumor growth in vivo. Clinical significance of PARPi for CRC patients was supported by elevated PARP levels in colorectal tumors compared with normal colon, with further increases in metastases. Collectively, our results suggest that PARP inhibition serves as a point of fragility for CICs by augmenting therapeutic efficacy of chemotherapy. Stem Cells 2019;37:42-53.

Ketogenic Diet Modulates NAD+-Dependent Enzymes and Reduces DNA Damage in Hippocampus.

The ketogenic diet’s (KD) anti-seizure effects have long been documented. Recently, its therapeutic potential in multiple neurodegenerative and neurodevelopmental disorders has emerged. Yet experimental evidence for a fundamental mechanism underlying beneficial effects across numerous diseases remains lacking. We previously showed that feeding rats a KD produced an early (within 2 days) and persistent elevation of hippocampal nicotinamide adenine dinucleotide+ (NAD+), an essential metabolic coenzyme and signaling molecule. NAD+ is a marker of cellular health and a substrate for enzymes implicated in longevity and DNA damage repair such as sirtuins and poly-ADP ribose polymerase-1 (PARP-1). As a result, activation of NAD+-dependent enzymes’ downstream pathways could be the origin of KD’s broad beneficial effects. Here rats were fed ad libitum regular chow or KD for 2 days or 3 weeks and the levels of hippocampal sirtuins, PARP-1, and the oxidative DNA damage marker 8-hydroxy-2’-deoxyguanosine were quantified. We found a significant immediate and persistent increase in the collective activity of nuclear sirtuin enzymes, and a significant augmentation of Sirt1 mRNA at 2 days. Levels of PARP-1 and 8-hydroxy-2’-deoxyguanosine decreased after 2 days of treatment and further declined at 3 weeks. Our data show that a KD can rapidly modulate energy metabolism by acting on NAD+-dependent enzymes and their downstream pathways. Thus, therapy with a KD can potentially enhance brain health and increase overall healthspan via NAD+-related mechanisms that render cells more resilient against DNA damage and a host of metabolic, epileptic, neurodegenerative, or neurodevelopmental insults.

Alpinetin inhibits proliferation and migration of ovarian cancer cells via suppression of STAT3 signaling.

Natural bioactive components are increasingly being applied in cancer research. Alpinetin is a type of natural flavonoid primarily derived from Alpiniakatsumadai Hayata, which exhibits anti‑bacterial and anti‑inflammatory properties. Therefore, it may possess anticancer potential and be employed therapeutically for different diseases. The aim of the present study was to investigate the anticancer effects of alpinetin on the SKOV3 ovarian cancer cell line. The effect of alpinetin treatment on SKOV3 cell proliferation, apoptosis, spheroid and colony formation were measured using Cell Counting kit‑8, cell apoptosis, 3D spheroid and colony formation assays, respectively. Analysis of the cell cycle was performed using flow cytometry. Western blot analysis was used to determine the protein expression levels of B‑cell lymphoma (Bcl)‑2, Bcl‑2‑associated X protein, cleaved caspase‑3, cleaved poly (ADP‑ribose) polymerase (PARP), cyclin D1, cyclin‑dependent kinase (CDK)4, CDK6, signal transducer and activator of transcription (STAT) 3, phosphorylated (p)‑STAT3, c‑myc, survivin, tissue inhibitor of metalloproteinase (TIMP)‑1, TIMP‑2, matrix metalloproteinase (MMP)‑2 and MMP‑9. In addition, a wound healing assay was used to determine cancer cell migration. The results revealed alpinetin suppressed cell viability and induced apoptosis of SKOV3 cells in a dose‑ and time‑dependent manner, and cells were arrested in the G1 phase. Alpinetin treatment upregulated protein expression levels of Bax, cleaved caspase‑3 and PARP, and downregulated protein expression levels of Bcl‑2, cyclin D1, CDK4 and CDK6. Alpinetin also inhibited cell migration, through increased protein expression levels of TIMP‑1 and TIMP‑2, and decreased protein expression levels of MMP‑2 and MMP‑9. Alpinetin also significantly suppressed colony and spheroid formation by SKOV3 cells. In addition, the STAT3 pathway was suppressed as demonstrated by downregulation of p‑STAT3 and reduced expression of downstream factors, including c‑myc and survivin. Overall, these results indicated that alpinetin may have anticancer effects on human ovarian cancer by inhibiting the STAT3 signaling pathway.

Poly(ADP-ribose) polymerases-1 inhibitor MRL-45696 alleviates DNA damage after myocardial ischemia-reperfusion in diabetic rats

To study the protective effect of MRL-45696, an inhibitor of poly(ADP-ribose) polymerase-1 (PARP-1), against DNA damage after myocardial ischemia/reperfusion (I/R) in diabetic rats. Rat models of type 2 diabetes mellitus were established by high-fat feeding and a single peritoneal dose of streptozotocin. Forty diabetic rats were randomized equally into diabetic group, sham-operated group, sham-operated group with MRL-45696 treatment, I/R injury model group and I/R injury group with MRL-45696 treatment. The rats in MRL-45696-treated groups were subjected to daily intragastric administration of MRL-45696 (50 mg/kg) for 7 consecutive days, after which sham operation was performed or myocardial I/R injury was induced by ligation of the left anterior descending coronary artery for 30 min followed by reperfusion for 120 min. The range of myocardial infarction, plasma cardiac troponin I (cTnI), serum creatine kinase (CK), lactate dehydrogenase (LDH) activity, malondialdehyde (MDA), superoxide dismutase (SOD) activity, and cardiac myocyte apoptosis were detected. The levels of γ-H2AX, cleaved caspase-3, PARP-1, and PAR were detected with Western blotting, and the level of NAD was detected using colorimetry. The infarct size was significantly smaller in MRL-45696 treatment group than in I/R injury group (P < 0.05). In I/R model group, the levels of cTnI, CK, and LDH in the plasma or serum and MDA, γ-H2AX, cleaved caspase-3 and apoptotic rate in the cardiac myocytes were significantly higher than those in the other groups (P < 0.05), and SOD activity was significantly decreased (P < 0.05). Compared with I/R model group, the rats with MRL- 45696 treatment showed significantly decreased levels of cTnI, CK, LDH, MDA, γ-H2AX, cleaved caspase-3, PARP- 1, PAR expression and cell apoptosis with significantly increased levels of SOD and NAD (P < 0.05). MRL-45696 can inhibit excessive activation of PARP-1, increase intracellular level of NAD and inhibit cardiac myocyte apoptosis to alleviate myocardial I/R-induced DNA damage and reduce myocardial infarct size in diabetic rats.

Lycium barbarum polysaccharides exert an antioxidative effect on rat chondrocytes by activating the nuclear factor (erythroid-derived 2)-like 2 signaling pathway.

IntroductionOxidative stress is the main cause of osteoarthritis (OA). Lycium barbarum polysaccharides (LBP) have antioxidant properties. Thus, the potential effect of LBP on H2O2-stimulated chondrocytes was examined.Material and methodsThe cell viability was detected by CCK-8. The reactive oxygen species (ROS) production and apoptosis rates were determined by flow cytometric analysis. The DNA damage was detected by comet assay. Real-time polymerase chain reaction (qPCR) and Western blot assays were performed to examine the expression of histone 2A family member X (γH2AX), checkpoint kinase 1 (Chk1), poly ADP-ribose polymerase (PARP), cysteinyl aspartate specific proteinase (caspase)-3/8/9, and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and its antioxidant-response element (ARE) dependent factors including heme oxygenase-1 (HO-1) and quinine oxidoreductase-1 (NQO-1).ResultsCompared to the H2O2 group, LBP inhibited the ROS production and DNA damage caused by H2O2 (p < 0.05), respectively. LBP inhibited the mRNA and protein expressions of γH2AX and Chk1 (p < 0.05). Meanwhile, LBP significantly decreased apoptosis (p < 0.05). And LBP inhibited the expression levels of PARP and Caspase-3/8/9 (p < 0.05). Moreover, LBP increased the expression of Nrf2, HO-1and NQO-1 (p < 0.05). Furthermore, the depletion of Nrf2 that mediated by RNA interference reversed the apoptosis and DNA damage inhibition effect of LBP (p < 0.05).ConclusionsLBP protected chondrocytes through inhibiting DNA damage and apoptosis caused by H2O2, in which the Nrf2/ARE signaling pathway played a positive role. It provided an inspiration for clinical application – developing LBP as a therapeutic agent and Nrf2 as a promising candidate.

Assessment of Poly(ADP-ribose) Polymerase1 (PARP1) expression and activity in cells purified from blood and milk of dairy cattle

Poly(ADP-ribosyl)ation (PAR) is a post-translational protein modification catalysed by enzyme member of the poly(ADP-ribose) polymerases (PARPs) family. The activation of several PARPs is triggered by DNA strand breakage and the main PARP enzyme involved in this process is PARP1. Besides its involvement in DNA repair, PARP1 is involved in several cellular processes including transcription, epigenetics, chromatin re-modelling as well as in the maintenance of genomic stability. Moreover, several studies in human and animal models showed PARP1 activation in various inflammatory disorders. The aims of the study were (1) to characterize PARP1 expression in bovine peripheral blood mononuclear cells (PBMC) and (2) to evaluate PAR levels as a potential inflammatory marker in cells isolated from blood and milk samples following different types of infection, including mastitis. Our results show that (i) bovine PBMC express PARP1; (ii) lymphocytes exhibit higher expression of PARP1 than monocytes; (iii) PARP1 and PAR levels were higher in circulating PBMCs of infected cows; (iv) PAR levels were higher in cells isolated from milk with higher Somatic Cell Counts (SCC > 100,000 cells/mL) than in cells from milk with low SCCs. In conclusion, these findings suggest that PARP1 is activated during mastitis, which may prove to be a useful biomarker of mastitis.

A2BAR activation attenuates acute lung injury by inhibiting alveolar epithelial cell apoptosis both in vivo and in vitro.

The epithelial barrier of the lung is destroyed during acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) due to the apoptosis of alveolar epithelial cells (AECs). Therefore, treatments that block AEC apoptosis might be a therapeutic strategy to ameliorate ALI. Based on recent evidence, A2B adenosine receptor (A2BAR) plays an important role in ALI in several different animal models, but its exact function in AECs has not been clarified. We investigated the role of A2BAR in AEC apoptosis in a mouse model of oleic acid (OA)-induced ALI and in hydrogen peroxide (H2O2)-induced AEC (A549 cells and MLE-12 cells) injury. Mice treated with BAY60-6583, a selective A2BAR agonist, showed lower AEC apoptosis rates than mice treated with OA. However, the role of BAY60-6583 in OA-induced ALI was attenuated by a specific blocker of A2BAR, PSB1115. A2BAR activation decreased H2O2-induced cell apoptosis in vitro, as characterized by the translocation of apoptotic proteins, the release of cytochrome c, and the activation of caspase-3 and poly (ADP ribose) polymerase 1 (PARP-1). In addition, apoptosis was required for the phosphorylation of ERK1/2, p38, and JNK. Importantly, compared with cells transfected with the A2BAR-siRNA, an ERK inhibitor or p38 inhibitor exhibited decreased apoptotic ratios and cleaved caspase-9 and cleaved PARP-1 levels, whereas the JNK inhibitor displayed increases in these parameters. In conclusion, A2BAR activation effectively attenuated OA-induced ALI by inhibiting AEC apoptosis and mitigated H2O2-induced AEC injury by suppressing the p38 and ERK1/2-mediated mitochondrial apoptosis pathway.

PARP-1 Overexpression as an Independent Prognostic Factor in Adult Non-M3 Acute Myeloid Leukemia.

Poly (ADP-ribose) polymerase-1 (PARP-1) plays an important role in the repair of damaged DNA and has prognostic significance in a variety of human malignancies. However, little is known about its expression levels and clinical implication in patients with acute myeloid leukemia (AML). Quantitative reverse transcription-polymerase chain reaction was done to evaluate PARP-1 expression levels in the bone marrow of 65 patients with non-M3 AML and 54 healthy counterparts. The correlation of PARP-1 expression with clinicopathological features of non-M3 AML patients was also analyzed. Non-M3 AML patients have higher PARP-1 expression than the healthy controls (p < 0.01). Patients with adverse cytogenetic risk have higher PARP-1 expression than other cytogenetic risk groups (p = 0.004). The PARP-1 median expression level divided AML patients into PARP-1 low-expressed and PARP-1 high-expressed groups. High expression levels of PARP-1 were associated with worse overall survival (OS) (p = 0.01) and relapse-free survival (RFS) (p = 0.005). Moreover, multivariate analysis revealed that high PARP-1 expression was an independent risk factor for both OS and RFS. Our results suggest that PARP-1 overexpression may define an important risk factor in non-M3 AML patients and PARP-1 is a potential therapeutic target for AML treatment.

Inhibitory effect of baicalein combined with gemcitabine in human pancreatic cancer cell lines.

Pancreatic cancer is an aggressive disease with a particularly poor prognosis contributing to a substantial percentage of cancer-associated mortality rates. In the present study, the combination treatment of baicalein (BAI) and gemcitabine (GEM) was investigated to examine whether it inhibited the growth of the human CFPAC-1 pancreatic cancer cell line in vitro and in vivo. The cytotoxic interactions between BAI and GEM in human pancreatic cancer cell lines were determined using MTT assays, and the effect of the two agents on apoptosis was detected using Hoechst 33258 staining and annexin V/7-AAD. The protein levels of Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), caspase-3, poly ADP ribose polymerase (PARP) and survivin were detected using western blot analysis. Furthermore, the expression levels of Bax, Bcl-2, caspase-3 and survivin in tumor tissues were detected using immunohistochemistry. The results demonstrated that following GEM treatment, the growth of CFPAC-1 cells and xenografts in nude mice were inhibited, and the expression levels of Bcl-2 and survivin were downregulated, whilst the expression levels of Bax, caspase-3 and PARP were upregulated. These effects were enhanced with the use of BAI in combination with GEM. The mechanism underlying the anti-tumor effect of BAI combined with GEM may be associated with the induction of cell apoptosis and the inhibition of proliferation. To the best of our knowledge, this is the first evidence of the efficacy of BAI against pancreatic cancer and may provide the potential clinical evidence for the use of this drug combination for the treatment of patients with pancreatic cancer.

Cancer Imprints an Increased PARP-1 and p53-Dependent Resistance to Oxidative Stress on Lymphocytes of Patients That Later Develop Alzheimer's Disease.

We have proposed that a common biological mechanism deregulated in opposite directions might explain the inverse epidemiological association observed between Alzheimer's disease (AD) and cancer. Accordingly, we showed that lymphocytes from AD patients have an increased susceptibility, whereas those from survivors of a skin cancer, an increased resistance to oxidative death induced by hydrogen peroxide (H2O2), compared to healthy controls (HC). We investigated the susceptibility to H2O2-induced death of lymphocytes in survivors of any type of cancer and in cancer survivors who later developed AD (Ca&AD). We also explored the involvement of Poly [ADP-ribose] polymerase-1 (PARP-1) and p53 pathways in the process, since both are involved in the increased susceptibility to death of AD lymphocytes. Lymphocytes from 11 cancer and 13 Ca&AD patients, and 12 HC were submitted to increasing concentrations of H2O2 for 20 h. Cell death was determined by flow cytometry, in the presence or absence of PARP-1 inhibition (3-aminobenzamide, 3-ABA), or p53 inhibition (pifithrin-α) or stabilization (Nut-3). PARP-1 and p53 mRNA levels were determined by Real-Time PCR. Lymphocytes from cancer and Ca&AD patients showed increased survival compared to HC, without differences between them, opposite to the increased susceptibility to death previously shown in AD. PARP-1 inhibition provided marked protection from H2O2-induced death in the two groups of patients, significantly greater than in HC. Pharmacological inhibition of p53 increased lymphocyte survival in Ca&AD patients, contrary to the effect previously reported in HC and AD. PARP-1 and p53 mRNA levels were elevated in Ca&AD lymphocytes compared with controls. In all, these results show that cancer imprints an increased resistance to H2O2-induced death in lymphocytes that persists after AD development, and is dependent on both PARP-1 and p53. p53 inhibition showed a differential role in cancer and Ca&AD compared to HC and AD lymphocytes, that could explain the inverse susceptibility to oxidative death in cancer and AD. These results are in agreement with the hypothesis of a common biological mechanism in AD and cancer. The similar cell death susceptibility and cell death pattern observed in cancer and Ca&AD lymphocytes suggests that cancer history leaves long term effects on lymphocyte cell death susceptibility.

JWA deficiency induces malignant transformation of murine embryonic fibroblast cells.

The present study aimed to investigate the effects of JWA knockout (JWA-/-) on malignant transformation of murine embryonic fibroblast (MEF) cells using a conditional JWA-/- mouse model. Once MEF cells were prepared, the potential role of JWA-/- on proliferation, migration, invasion and colony formation of MEF cells was investigated by cytological examination. The effects of JWA-/- on the regulation and protein expression levels of epithelial-mesenchymal transition (EMT)-related proteins in MEF cells, including poly(ADP-ribose) polymerase-1 (PARP-1), vimentin, β-catenin and E-cadherin, were investigated using western blot analysis. The tumorigenicity of JWA deficiency was explored using nude mouse xenografts and subcutaneous inoculation of MEF cells exhibiting JWA-/-. JWA-/- was able to increase cell proliferation, migration, invasion and colony formation in the malignant transformation of MEF cells. The protein expression levels of PARP-1, vimentin and β-catenin were upregulated, whereas E-cadherin was downregulated in JWA-/- MEF cells. The tumor formation was observed in mice following subcutaneous inoculation of MEF with JWA-/-, whereas no tumor was formed in the mice treated with functional JWA MEF cells. In conclusion, the present findings suggest that JWA-/- has important roles in cell proliferation, migration, invasion and colony formation and is able to induce the malignant transformation of MEF cells. The expression levels of EMT-related proteins changed and tumorigenicity increased in JWA-/- MEF cells compared with cells with functional JWA. The present findings indicate that JWA may function as an anti-oncogene in tumorigenesis.

Omi/HtrA2 Regulates a Mitochondria-Dependent Apoptotic Pathway in a Murine Model of Septic Encephalopathy

Background/Aims: the pathogenesis of sepsis-associated encephalopathy (SAE) is multifactorial, involving neurotransmitter alterations, inflammatory cytokines, oxidative damage, mitochondrial dysfunction, apoptosis, and other factors. Mitochondria are major producers of reactive oxygen species, resulting in cellular injury. Omi/HtrA2 is a proapoptotic mitochondrial serine protease involved in caspase-dependent cell death; it is translocated from mitochondria to the cytosol after an apoptotic insult. We previously found that UCF-101, a specific inhibitor of Omi/HtrA2, has neuroprotective effects on cerebral oxidative injury and cognitive impairment in septic rats. In this study, the mechanisms and molecular pathways underlying these effects were investigated. Methods: Male Sprague–Dawley rats were subjected to cecal ligation and puncture (CLP) or sham-operated laparotomy and were administered vehicle or UCF-101 (10 µmol/kg). The hippocampus was isolated for subsequent analysis. Omi/HtrA2 expression in the mitochondria or cytosol was evaluated by immunofluorescence or western blotting. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was utilized to evaluate levels of apoptosis, and western blotting was used to evaluate apoptosis-related proteins, such as cleaved caspase-3, caspase-9, and poly (ADP-ribose) polymerase (PARP). Tight junction expression was assessed by immunofluorescence and western blotting. Mitochondrial function, inflammatory cytokines, and oxidative stress were also assayed. In addition, a wet/dry method was used to evaluate brain edema and Evans blue extravasation was used to evaluate blood–brain barrier (BBB) integrity. Results: After CLP treatment, the hippocampus exhibited a mild increase in Omi/HtrA2 expression; cytosolic Omi/HtrA2 expression increased significantly, whereas mitochondrial Omi/HtrA2 expression was reduced, indicating that CLP-induced oxidative stress resulted in the translocation of Omi/HtrA2 from mitochondria to the cytosol. Hippocampal cleaved caspase-3, caspase-9, and PARP levels were significantly higher in animals treated with CLP than in sham-operated animals, while XIAP expression was lower. Treatment with UCF-101 prevented the mobilization of Omi/HtrA2 from mitochondria to the cytosol, attenuated XIAP degradation, and decreased cleaved caspase-3, caspase-9, and PARP expression as well as apoptosis. UCF-101 also reversed the decreased mitochondrial complex I, II, and III respiration and the reduced ATP caused by CLP. In addition, UCF-101 treatment resulted in a significant improvement in BBB integrity, as demonstrated by increased occludin, claudin-5, and zonula occludens 1 levels and reduced Evans blue extravasation. No significant effects of UCF-101 on brain edema were found. Inflammatory cytokines and oxidative stress were significantly higher in the CLP-treated group than in the sham-operated group. However, the inhibition of Omi/HtrA2 by UCF-101 significantly alleviated these responses. Conclusion: Our data indicated that Omi/ HtrA2 regulates a mitochondria-dependent apoptotic pathway in a murine model of septic encephalopathy. Inhibition of Omi/HtrA2 by UCF-101 leads to neuroprotection by inhibiting the cytosolic translocation of Omi/HtrA2 and antagonizing the caspase-dependent apoptosis pathway. Therapeutic interventions that inhibit Omi/HtrA2 translocation or protease activity may provide a novel method to treat SAE.

Curcumin regulates intracellular calcium release and inhibits oxidative stress parameters, VEGF, and caspase-3/-9 levels in human retinal pigment epithelium cells.

In this study, we aimed to observe whether curcumin (cur), a polyphenolic compound derived from the dietary spice turmeric, a yellow substance obtained from the root of the plant Curcuma longa Linn, has any protective effect against blue light irradiation in human retinal pigment epithelium (ARPE-19) cells. For this purpose, we evaluated the intracellular calcium release mechanism, poly ADP ribose polymerase (PARP), procaspase-3/-9 protein expression levels, caspase activation, and reactive oxygen species levels. ARPE-19 cells were divided into four main groups, such as control, cur, blue light, and cur + blue light. Results were evaluated by Kruskal-Wallis and Mann-Whitney U tests as post hoc tests. The cells in cur and cur + blue light samples were incubated with 20μM cur. Blue light exposure was performed for 24h in an incubator. Lipid peroxidation and cytosolic-free Ca2+ [Ca2+]i concentrations were higher in the blue light exposure samples than in the control samples; however, their levels were determined as significantly lower in the cur and cur + blue light exposure samples than in the blue light samples alone. PARP and procaspase-3 levels were significantly higher in blue light samples. Cur administration significantly decreased PARP and procaspase-3 expression levels. Reduced glutathione and glutathione peroxidase values were lower in the blue light exposure samples, although they were higher in the cur and cur + blue light exposure samples. Caspase-3 and -9 activities were lower in the cur samples than in the blue light samples. Moreover, vascular endothelial growth factor (VEGF) levels were significantly higher in the blue light exposure samples. In conclusion, cur strongly induced regulatory effects on oxidative stress, intracellular Ca2+ levels, VEGF levels, PARP expression levels, and caspase-3 and -9 values in an experimental oxidative stress model in ARPE-19 cells.

Abstract 68: Functional interactions of HPV and PARP-1 in head and neck cancer

Abstract Though the overall incidence of head and neck cancer has been declining over the last several decades, there has been a concomitant and worrisome rise in human-papilloma virus (HPV)-associated head and neck squamous cell carcinoma (HNSCC). HPV infection is a necessary but not sufficient condition for cancer development. Unlike in cervical cancer, where several co-factors including smoking and co-infection with other agents are well established, how HPV drives malignant transformation in head and neck cancer and which cellular factors are required are not well characterized. Recent evidence from our group has shown that poly (ADP-ribose) polymerase-1 (PARP-1) is overexpressed and overactive in HPV-positive (HPV(+)), but not HPV-negative (HPV(-)), oropharyngeal squamous cell carcinoma (OPSCC). The significance of PARP-1 overexpression and hyperactivation in cancer is only beginning to emerge. High levels of PARP-1 are found in a fraction of hepatocellular and breast cancers and correlate with poor prognosis. Recently, PARP-1 was implicated in prostate cancer progression. This study explores the role of PARP-1 in HPV infection and HNSCC carcinogenesis. Biochemical methods were used to determine PARP activity in vitro, in head and neck tumors, and during infection with HPV16 pseudovirus (PsV) or overexpression of HPV16 proteins in epithelial cells. The response to PARP inhibition was evaluated with clonogenic survival assays. HPV L1 mRNA in HNSCC was detected using quantitative real-time PCR and HPV L1 protein levels were determined by immunohistochemistry. Here, we show that the HPV16 major capsid protein L1 directly binds PARP-1 and activates poly(ADP-ribosyl)ation in the absence of DNA damage in vitro and in HPV(-) cells. Using PARP-1 knockout cells, we found that PARP-1 expression and enzymatic activity supports initial HPV infection. Furthermore, HPV(-) cells were sensitized to PARP inhibition both after L1 overexpression and after infection with HPV16 PsV particles. In conclusion, we report functional interactions between the viral capsid and cellular protein, signifying a vital role played by PARP-1 in HPV infection and carcinogenesis. Importantly, our data provide a mechanistic explanation for pharmacological PARP inhibition as an intriguing therapeutic opportunity for HPV(+) head and neck cancers. Citation Format: Cyril S. Gary, Michael Hajek, Asel Biktasova, Andrew Sewell, Yarbrough G. Wendell, Natalia Issaeva. Functional interactions of HPV and PARP-1 in head and neck cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 68.

Sodium Thiosulfate Preconditioning Ameliorates Ischemia/Reperfusion Injury in Rat Hearts Via Reduction of Oxidative Stress and Apoptosis

Sodium thiosulfate (STS) has of late been proven efficacious in models of urolithiasis and vascular calcification. However, its cardiovascular effects on ischemia reperfusion injury (IR) have not been revealed. Being an antioxidant and calcium chelator, it is assumed to play a vital role in IR as ROS production and calcium overload are major perpetrators of IR injury. The cardioprotective effect of STS was evaluated in vitro using H9C2 cardiomyocytes and in vivo using both isolated rat heart and intact left anterior descending artery (LAD) occlusion models of ischemia reperfusion injury. Finally, in silico tools were utilized to establish its possible mode of action. Myocardial injury markers and expression of apoptotic proteins were studied along with myocardial histopathology. STS of 1mM recovered H9C2 cells from glucose oxidase/catalase-induced apoptosis. The isolated rat heart treated with STS prior to IR injury improved its hemodynamics and reduced the infarct size to 9%. This was supported by the absence of derangement of cardiac fibers from H&E stained section of LAD-occluded rats. Plasma troponin levels decreased by 15% compared to IR and the myocardium showed diminished apoptotic proteins. An in silico docking analysis revealed higher binding affinity of STS for caspase-3 with a binding energy of -60.523kcal/mol for the complex. The effectiveness of STS as a cardioprotective agent is attributed to the reduction of apoptosis by binding to the active site of caspase-3 in silico, which was substantiated by the reduced expression of caspase-3 and poly ADP ribose polymerase levels.

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

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

Expression of Caspase Signaling Components in the Outer Membranes of Chronic Subdural Hematomas

Chronic subdural hematoma (CSDH) is fundamentally treatable through surgery, although CSDH recurs in some cases. We have observed several cases of spontaneous resolution of CSDH outer membranes, including in trabecular CSDH, after trepanation surgery. In this study, we examined the expression of molecules involved in caspase signaling in CSDH outer membranes. Eight patients whose outer membranes were obtained successfully during trepanation surgery were included in this study. The expression of Fas; Fas-associated death domain (FADD); tumor necrosis factor receptor type 1-associated death domain (TRADD); receptor-interacting protein (RIP); caspases 3, 7, 8, and 9; poly-(ADP-ribose) polymerase (PARP); DNA fragmentation factor 45 (DFF45) and β-actin was examined by Western blot analysis. The expression levels of PARP, caspase-3, and cleaved caspase-3 were also examined by immunohistochemistry. Fas; FADD; TRADD; RIP; caspases 3, 7, 8, and 9; PARP, and DFF45 were detected in nearly all samples. Caspase-3 and PARP were localized in the endothelial cells of vessels and in fibroblasts in CSDH outer membranes. In addition, cleaved caspase-3 was detected in fibroblasts. We detected molecules of the caspase signaling pathway in CSDH outer membranes. In particular, cleaved caspase-3 was detected, which suggests that apoptosis may occur within these membranes. Thus, during the growth of CSDH outer membranes, the caspase signaling pathway may be restrained. Once the pathway is activated, gradual resolution of CSDH outer membranes may occur. Therefore, these molecules may be novel therapeutic targets for intractable CSDH.

Abstract 3852: Validation of synthetic lethality of PARP and NAMPT dual inhibition in a small cell lung cancer PDX model

Abstract Small cell lung cancer (SCLC) is a highly malignant cancer type with a 5-year survival rate of less than 10%. Different from non-small cell lung cancer (NSCLC), no effective target therapies have been approved to treat SCLC. Poly (ADP) ribose polymerase (PARP) overexpression in SCLC has prompted great efforts to evaluate the role of PARP inhibitors in clinic. To maximize their therapeutic value it is urgent in need to explore the best combination strategies between PARP inhibitors and other pathway modulators. In this study we identified a SCLC patient-derived xenograft (PDX) model with high PARP level and low nicotinamide phosphoribosyltransferase (NAMPT) level. Furthermore, synergistic effect of PARP and NAMPT dual inhibition was demonstrated, supporting translational research of PARP inhibitors and NAMPT inhibitors in this model. The mRNA expression of PARP as well as its 30 most-studied synthetic lethality genes was compared between SCLC and NSCLC in both PDXs and cancer cell lines. We found that NAMPT levels decreased most significantly in SCLC compared with NSCLC across the 30 synthetic lethality genes of PARP. As NAMPT is the rate-limiting enzyme for the synthesis of the PARP substrate β-NAD+, we hypothesized that low β-NAD+ level due to low NAMPT level might render SCLC sensitive to PARP inhibition. LU-01-0547, a SCLC PDX model with high PARP and low NAMPT expression, was identified here. To test our hypothesis, we investigated the preclinical efficacy of ABT888, a PARP inhibitor, with or without FK866, a NAMPT inhibitor, in this subcutaneous xenograft model. The single-agent efficacy of ABT888 was demonstrated (TGI=96%@100 mg/kg, BID). At a sub-optimal dose (50 mg/kg, BID), treatment of ABT888 alone produced little activity in the same model, while its combination with a NAMPT inhibitor, FK866, significantly boosted the antitumor response indicating the synergistic effect (TGI=117%, ORR=6/6, CR=4/6). When nicotinic acid (NA) was administered to promote β-NAD+ biosynthesis via the de novo pathway other than the NAMPT-mediated transformation, the PARP inhibitor’s effectiveness was greatly antagonized (TGI=70% vs. 13%), suggesting β-NAD+ level might correlate with PARP inhibitor sensitivity. In conclusion, this study identified a SCLC PDX model with high PARP and low NAMPT expression. Synergistic inhibition of PARP and NAMPT in this model was demonstrated superior to either treatment alone in efficacy, which warranted its future application in drug discovery. To our knowledge, this was the first in vivo evidence of synthetic lethality in SCLC PDX, supporting future clinical test of combination of PARP and NAMPT inhibitors in SCLC patients. Citation Format: Zhixiang Zhang, Dongfang Li, Chen Chen, Bo Zhang, Xuzhen Tang, Hao Ye, Qingyang Gu, Qunsheng Ji. Validation of synthetic lethality of PARP and NAMPT dual inhibition in a small cell lung cancer PDX model [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 3852. doi:10.1158/1538-7445.AM2017-3852

MKP-1 suppresses PARP-1 degradation to mediate cisplatin resistance.

Understanding the mechanisms of platinum compound resistance, including cisplatin resistance, has important implications for improving cancer treatments. Previous studies identified a potential role for mitogen-activated protein kinase phosphatase-1 (MKP-1) in cisplatin resistance. This work focuses on the regulation of poly(ADP-ribose) polymerase-1 (PARP-1) expression by MKP-1. We found that MKP-1 overexpression stimulates PARP-1 and poly(ADP-ribose) (PAR) protein expression and cisplatin resistance while its downregulation suppresses PARP-1 and PAR protein expression and cisplatin resistance. Silencing MKP-1 promoted PARP-1 ubiquitination, which decreased PARP-1 protein levels. We also found that silencing c-Jun N-terminal kinase 1/2 (JNK1/2) decreased PARP-1 ubiquitination while increasing total PARP-1 protein levels. Furthermore, we showed that acquired cisplatin resistant ovarian cancer cells expressed the high levels of MKP-1 and PARP-1 proteins, and that silencing MKP-1 or PARP-1 increased cisplatin sensitivity in resistant cells. Notably, the pharmacologic inhibition of PARP activity restored cisplatin sensitivity in MKP-1 overexpressing cells. Thus, this work indicates that suppression of JNK1/2 activity by MKP-1 maintains PARP-1 levels and suggests that MKP-1-mediated cisplatin resistance can be bypassed by PARP-1 inhibition.

Protective Functions of PJ34, a Poly(ADP-ribose) Polymerase Inhibitor, Are Related to Down-Regulation of Calpain and Nuclear Factor-κB in a Mouse Model of Traumatic Brain Injury

Poly(ADP-ribose) polymerase (PARP), calpain, and nuclear factor-κB (NF-κB) are reported to participate in inflammatory reactions in pathologic conditions and are involved in traumatic brain injury. The objective of this study was to investigate whether PARP participates in inflammation related to calpain and NF-κB in a mouse model of controlled cortical impact (CCI). PJ34 (10 mg/kg), a selective PARP inhibitor, was administered intraperitoneally 5 minutes and 8 hours after experimental CCI. We then performed a histopathologic analysis, and we measured calpain activity and protein levels in all animals. The cytosolic, mitochondria, and nuclear fractions were prepared and used to determine the levels of PARP, calpastatin, NF-κB p65, inhibitory-κB-α, tumor necrosis factor-α, interleukin-1β, intracellular adhesion molecule-1, inducible nitric oxide synthase, and cyclooxygenase-2. We then measured blood-brain barrier disruption using electron microscopy at 6 and 24 hours after CCI. Treatment with PJ34 markedly reduced the extent of both cerebral contusion and edema, improved neurologic scores, and attenuated blood-brain barrier damage resulting from CCI. Our data showed that the cytosolic and nuclear fractions of calpain and NF-κB were up-regulated in the injured cortex and that these changes were reversed by PJ34. Moreover, PJ34 significantly enhanced the calpastatin and inhibitory-κB levels and decreased the levels of inflammatory mediators. PARP inhibition by PJ34 suppresses the overactivation of calpain and the production of inflammatory factors that are caused by NF-κB activation and attenuates neuronal cell death in a mouse model of CCI.