Expression Of Poly ADP-ribose Polymerase Research Articles

Effect of exercise on telomere length and telomere proteins expression in mdx mice.

In Duchenne muscular dystrophy (DMD), telomere shortening has been postulated to contribute to the failure of regenerative activity promoting the premature senescence of satellite cells. The aim of the present study was to investigate the telomere length and the expression of telomeric repeat-binding factor-1 (TRF1), poly (ADP-ribose) polymerase-1 (PARP1) and mouse telomerase reverse transcriptase (MTERT) in gastrocnemius, tibialis anterior and diaphragm muscles of the murine model of DMD, the mdx mouse and whether a chronic protocol of forced exercise impacts on them. Our results confirmed a telomere shortening in mdx muscles, more evident in the diaphragm, in which exercise induced a greater shortening than in wild-type mice. Moreover, we showed for the first time in mdx an increased TRF1 and PARP1 expression and an augmented activity of MTERT, further enhanced by exercise. These results reinforce the hypothesis that a deregulation of mechanisms involved in telomere length occurs and may pave the way for the test of compounds targeting proteins modulating telomere maintenance as a novel strategy to treat dystrophinopathies.

Assessment of PARP-1 Distribution in Tissues of Cynomolgus Monkeys.

The poly(ADP-ribose) polymerase 1 (PARP-1) enzyme has received much attention in the last decade due to its promising role in cancer therapeutics. Despite the expanding use of PARP inhibitors in cancer therapy, little is known about PARP-1 tissue distribution. Our study provides a detailed survey of PARP-1 tissue and cellular distribution using well-preserved cynomolgus monkey organs and a well-characterized, highly specific monoclonal PARP-1 antibody. Overall, PARP-1 was detected in most organs, but its distribution was restricted to specific cells within each tissue, suggesting that PARP-1 expression is tightly regulated. The strongest expression was in the pituitary, the ovary, the male adrenal gland, and the thymus. One of the key findings of this study was the stronger expression of PARP-1 in proliferating cells rather than mature cells. This observation not only provides clues to the importance of PARP-1 in processes such as DNA replication and transcription in these cell types, but it also provides the basis for further investigation into the effects of its inhibition in the context of malignancy. Overall, this study greatly expands the current knowledge of PARP-1 tissue expression, enabling the identification of tissues where PARP inhibition may be most efficacious.

Poly (ADP-Ribose) polymerase-1 (PARP-1) overactivity plays a pathogenic role in bile acids-induced nephrotoxicity in cholestatic rats

Cholestatic liver disease is a clinical complication with a wide range of etiologies. The liver is the primary organ influenced by cholestasis. Other organs, rather than the liver (e.g., kidneys), could also be affected by cholestatic liver disease. Cholestasis-induced renal injury is known as cholemic nephropathy (CN). Although the structural and functional alterations of the kidney in cholestasis have been well described, the cellular and molecular mechanisms of CN are not well understood. Some studies mentioned the role of oxidative stress and mitochondrial impairment in CN. Several cellular targets, including proteins, lipids, and DNA, could be affected by oxidative stress. Poly (ADP-Ribose) polymerase-1 (PARP-1) is an enzyme that its physiological activity plays a fundamental role in DNA repair. However, PARP-1 overexpression is associated with enhanced oxidative stress and cell death. The current study was designed to evaluate the role of PARP-1 activity in the pathogenesis of CN. Bile duct ligated (BDL) rats were treated with nicotinamide (NA) as a PARP-1 inhibitor. Kidney, urine, and plasma samples were collected at scheduled time intervals (3, 7, 14, and 28 days after BDL surgery). Serum and urine biomarkers of kidney injury, markers of oxidative stress and DNA damage, PARP-1 expression and activity in the kidney tissue, inflammatory response, renal fibrosis markers, and kidney histopathological alterations were assessed. Significant changes in the serum and urine biomarkers of kidney injury were evident in the BDL rats. Markers of oxidative stress were increased, and tissue ATP levels and antioxidant capacity were decreased in the kidney of cholestatic animals. A significant increase in PARP-1 expression and activity was evident in BDL rats (3, 7, 14, and 28 days after BDL). Moreover, inflammatory response (IL-1β and TNF-α expression; and myeloperoxidase activity), renal tissue histopathological alterations, and kidney fibrosis (α-SMA and TGF-β expression, as well as collagen deposition) were detected in cholestatic animals. It was found that the PARP-1 inhibitor, NA (50 and 100 mg/kg, i.p), significantly mitigated cholestasis-induced renal injury. The positive effects of NA were more significant at a lower dose and the early stage of CN. These data indicate a pathogenic role for PARP-1 overexpression in CN.

Lipoic Acid Decreases the Expression of Poly ADP-Ribose Polymerase and Inhibits Apoptosis in Diabetic Rats.

PurposeTo study the effects of lipoic acid on poly ADP-ribose polymerase (PARP) expression and apoptosis in diabetic rats.Materials and MethodsSprague–Dawley rats (n=30) with high-fat diet- and streptozotocin-induced diabetes were randomly divided into two groups: diabetic model (DM) group and lipoic acid (LA) treatment group; another 10 rats were selected as normal controls (NC). The serum levels of 8-hydroxy-2ʹ-deoxyguanosine, nitrotyrosine, and 8-isoprostane; sciatic nerve cell apoptosis index; and PARP expression were detected in the rats, and morphological changes in the sciatic nerve were recorded.ResultsThe blood glucose level in the DM and LA groups was significantly higher than that of the NC group (P<0.01). Compared to the NC group, the DM group showed demyelinating changes to sciatic nerve fibers. PARP expression; serum levels of 8-hydroxy-2-deoxyguanosine, nitrotyrosine, and 8-isoprostane; and the apoptosis index of sciatic nerve cells were significantly higher than those of the NC group (P<0. 01). Following LA treatment, the above indices showed significant improvement (P<0.01).ConclusionLipoic acid may improve the symptoms of diabetic neuropathy by reducing PARP activity and inhibiting apoptosis.

Olaparib and temozolomide in desmoplastic small round cell tumors: a promising combination in vitro and in vivo

PurposeDesmoplastic small round cell tumors (DSRCTs) are highly malignant and very rare soft tissue sarcomas with a high unmet need for new therapeutic options. Therefore, we examined poly(ADP-ribose) polymerase 1 (PARP1) and Schlafen-11 (SLFN11) expression in DSRCT tumor tissue and the combination of PARP inhibitor olaparib with the alkylating agent temozolomide (TMZ) in a preclinical DSRCT model.MethodsPARP1 and SLFN11 have been described as predictive biomarkers for response to PARP inhibition. Expression of PARP1 and SLFN11 was assessed in 16 and 12 DSRCT tumor tissue samples, respectively. Effects of single-agent olaparib, and olaparib and TMZ combination treatment were examined using the preclinical JN-DSRCT-1 model. In vitro, single-agent and combination treatment effects on cell viability, the cell cycle, DNA damage and apoptosis were examined. Olaparib and TMZ combination treatment was also assessed in vivo.ResultsPARP1 and SLFN11 expression was observed in 100% and 92% of DSRCT tumor tissues, respectively. Olaparib treatment reduced cell viability and cell migration in a dose-dependent manner in vitro. Drug synergy between olaparib and TMZ was observed in vitro and in vivo. Combination treatment led to a cell-cycle arrest and induction of DNA damage and apoptosis, even when combined at low dosages.ConclusionWe show high PARP1 and SLFN11 expression in DSRCT tumor material and antitumor effects following olaparib and TMZ combination treatment in a preclinical DSRCT model. This suggests that olaparib and TMZ combination treatment could be a potential treatment option for DSRCTs.

PARP-1-regulated TNF-α expression in the dorsal root ganglia and spinal dorsal horn contributes to the pathogenesis of neuropathic pain in rats

Emerging evidence has implicated poly-(ADP-ribose) polymerase 1 (PARP-1), a transcriptional coregulator, in a variety of inflammatory diseases. In the current study, the role of PARP-1 in neuropathic pain and the underlying mechanisms were investigated. Neuropathic pain was determined by assessing the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) following lumbar 5 spinal nerve ligation (SNL) in male rates. Western blotting, qRT-PCR, immunohistochemistry, chromatin immunoprecipitation (ChIP), and Co-IP assays were performed to elucidate the mechanisms. The results showed that SNL resulted in a significant increase in the expression and activation of PARP-1 in the ipsilateral L4/5 dorsal root ganglia (DRG) and spinal dorsal horn, which occurred on day one, reached peak on day 7, and persisted more than 2 weeks after surgery. Double immunofluorescence staining revealed that PARP-1 was expressed exclusively in DRG A-type and C-type neurons. In the spinal cord, PARP-1 mainly colocalized with the neuronal marker NeuN and the astrocytic marker GFAP specifically in the superficial lamina. Prior intrathecal (i.t.) injection of PJ-34, a PARPs inhibitor, or Tiq-A, a specific PARP-1 inhibitor, dose-dependently prevented the reductions in PWT and PWL following SNL. Established neuropathic pain-like hypersensitivity was also attenuated with i.t. injection of PJ-34 and Tiq-A starting on day 7 following SNL, a timepoint at which neuropathic pain was fully established. SNL-induced mechanical allodynia and thermal hyperalgesia were also alleviated by i.t. injection of PARP-1 siRNA following a reduction in PARP-1 expression in the dorsal horn. Moreover, the SNL-induced increases in TNF-α protein and mRNA in the dorsal horn and DRG were dramatically suppressed by i.t. injection of Tiq-A or PARP-1 siRNA. The i.t. lipopolysaccharide (LPS)-induced increase in the production of TNF-α in the dorsal horn was also inhibited by prior to i.t. injection of PARP-1 siRNA. Results of ChIP assay showed that SNL-induced PARP-1 activation promoted the binding of NF-κB p65 with the TNF-α promoter in the dorsal horn and that PARP-1 inhibition reduced this binding and suppressed TNF-α expression. Co-IP assay revealed that SNL caused a significant increase in the level of histone H1 poly(ADP)–ribosylation. Together, these results indicate that PARP-1-regulated TNF-α expression in the DRG and spinal dorsal horn following SNL contributes to the development and maintenance of neuropathic pain. Targeting PARP-1 might be a promising therapeutic strategy for the treatment of the chronic pain.

Targeted PARP Inhibition Combined with FGFR1 Blockade is Synthetically Lethal to Malignant Cells in Patients with Pancreatic Cancer.

The role and therapeutic promise of poly-ADP ribose polymerase (PARP) inhibitors in anticancer chemotherapy are increasingly being explored, particularly in adjuvant or maintenance therapy, considering their low efficacy as monotherapy agents and their potentiating effects on concurrently administered contemporary chemotherapeutics. Against the background of increasing acquired resistance to FGFR1 inhibitors and our previous work, which partially demonstrated the caspase-3/PARP-mediated antitumor and antimetastatic efficacy of PD173074, a selective FGFR1 inhibitor, against ALDH-high/FGFR1-rich pancreatic ductal adenocarcinoma (PDAC) cells, we investigated the probable synthetic lethality and therapeutic efficacy of targeted PARP inhibition combined with FGFR1 blockade in patients with PDAC. Using bioinformatics-based analyses of gene expression profiles, co-occurrence and mutual exclusivity, molecular docking, immunofluorescence staining, clonogenicity, Western blotting, cell viability or cytotoxicity screening, and tumorsphere formation assays, we demonstrated that FGFR1 and PARP co-occur, form a complex, and reduce survival in patients with PDAC. Furthermore, FGFR1 and PARP expression was upregulated in FGFR1 inhibitor (dasatinib)-resistant PDAC cell lines SU8686, MiaPaCa2, and PANC-1 compared with that in sensitive cell lines Panc0403, Panc0504, Panc1005, and SUIT-2. Compared with the limited effect of single-agent olaparib (PARP inhibitor) or PD173074 on PANC-1 and SUIT-2 cells, low-dose combination (olaparib + PD173074) treatment significantly, dose-dependently, and synergistically reduced cell viability, upregulated cleaved PARP, pro-caspase (CASP)-9, cleaved-CASP9, and cleaved-CASP3 protein expression, and downregulated Bcl-xL protein expression. Furthermore, combination treatment markedly suppressed the clonogenicity and tumorsphere formation efficiency of PDAC cells regardless of FGFR1 inhibitor-resistance status and enhanced RAD51 and γ-H2AX immunoreactivity. In vivo studies have shown that both early and late initiation of combination therapy markedly suppressed tumor xenograft growth and increase in weight, although the effect was more pronounced in the early initiation group. In conclusion, FGFR1 inhibitor-resistant PDAC cells exhibited sensitivity to PD173074 after olaparib-mediated loss of PARP signaling. The present FGFR1/PARP-mediated synthetic lethality proof-of-concept study provided preclinical evidence of the feasibility and therapeutic efficacy of combinatorial FGFR1/PARP1 inhibition in human PDAC cell lines.

Effect of acupuncture on neurological function, cerebral infarction volume, thrombolysis time window and cerebral cell apoptosis signaling pathway in cerebral infarction rats

To observe the effect of acupuncture (Acupunct) on cerebral infarction volume and expression of poly ADP ribose polymerase 1 (PARP1), apoptosis-inducing factor (AIF) and endonuclease G (Endo-G) in the cerebral cortex tissue at different time-points after cerebral ischemia (CI) in acute cerebral infarction rats, so as to explore its underlying mechanisms in prolonging time window of thrombolysis. Forty-eight SD rats were randomly divided into sham operation, model, intravenous thrombolysis (IVT)-4.5 h, IVT-6 h, IVT-9 h, Acupunct＋IVT-4.5 h, Acupunct ＋IVT-6 h, Acupunct＋IVT-9 h groups (n＝6 in each group). The CI model was established by using modified autologous thromboembolism via the right common carotid artery. Two hours after modeling, rats of the Acupunct groups received Acupunct stimulation of "Shuigou" (GV26) and bilateral "Neiguan" (PC6) for 30 min. Thrombolysis was conducted by injection of recombinant human tissue-type plasminogen activator (rt-PA, 10 mg/kg) via caudal vein. The neurological deficit was assessed with reference to Bederson's methods. 2，3，5-triphenyltetrazolium chloride (TTC) staining was used to assess the cerebral infarction volume, and the expression of cerebral PARP1, AIF and Endo-G proteins detected by Western blot. Compared with the sham operation group, the neurological score and percentage of cerebral infarction volume, expression levels of PARP1, AIF and Endo-G proteins were significantly increased in the model group (P<0.01，P<0.05). After the intervention, modeling-induced increase of the aforementioned indexes was reversed in the IVT-4.5 h, Acupunct＋IVT-4.5 h and Acupunct＋IVT-6 h groups (P<0.05), except PARP1 expression of the Acupunct＋IVT 6 h group (P>0.05). The levels of neurological score, percentage of cerebral infarction volume, and AIF expression were significantly lower in both the Acupunct＋IVT 4.5 h and Acupunct＋IVT-6 h groups than in the simple IVT-4.5 h and simple IVT-6 h groups, respectively (P<0.05), and the expression levels of PARP1 and Endo-G proteins were obviously lower in the Acupunct＋IVT-4.5 h group than in the IVT-4.5 h group (P<0.05). Endo-G proteins were obviously lower in the Acupunct＋IVT-9 h group than in the IVT-9 h group (P<0.05). Acupuncture may improve neurological function, reduce cerebral infarction volume and prolong the time window of thrombolysis in CI rats, which may be associated with its effect in suppressing AIF/PARP1/ Endo-G signaling.

PLCε knockdown enhances the radiosensitivity of castration‑resistant prostate cancer via the AR/PARP1/DNA‑PKcs axis.

Radiotherapy (RT) has been used as a therapeutic option for treatment of prostate cancer (PCa) for a number of years; however, patients frequently develop RT resistance, particularly in castration-resistant PCa (CRPC), although the underlying mechanisms remain unknown. Understanding the underlying mechanism of RT resistance in CRPC may potentially highlight novel targets to improve therapeutic options for patients with PCa. In the present study, the expression levels of phospholipase Cε (PLCε), androgen receptor (AR) and DNA-dependent protein kinase catalytic subunit (PKcs) were examined in PCa tissue samples and PCa cells, and the effects of PLCε knockdown on AR and DNA damage repair (DDR)-related molecules were determined. The association between PLCε, AR and Poly (ADP-ribose) polymerase 1 (PARP1), as well as their respective roles in radiation resistance, were assessed using gene knockdown and pharmaceutical inhibitors or activators. A chromatin immunoprecipitation assay was used to determine the epigenetic regulatory effects of PLCε on PARP1. Animal experiments were performed to assess whether the mechanisms observed in vitro could be replicated in vivo. The expression levels of PLCε, AR and DNA-PKcs were significantly upregulated in PCa, particularly in CRPC. PLCε knockdown reduced the viability and increased apoptosis of cells subjected to radiation. Additionally, PLCε deficiency suppressed DDR progression by downregulating an AR and PARP1 positive feedback loop and the associated downstream molecules following radiation. PLCε depletion also increased the presence of histone H3 lysine 27 trimethylation in the PARP1 promoter region, suggesting increased methylation of the PARP1 gene and thus resulting in reduced expression of PARP1. In vivo, PLCε knockdown significantly potentiated the effects of radiation on tumor growth. Taken together, the results of the present study demonstrated that PLCε knockdown enhanced the radiosensitivity of CRPC by downregulating the AR/PARP1/DNA-PKcs axis.

Abstract P5-05-06: Umbelliferon derivatives for androgen receptor positive triple negative breast cancer

Abstract Purpose: Triple negative breast cancer (TNBC), which lacks hormone receptors and HER-2 over expression, has a poor prognosis due to the absence of targeted therapeutics. Gene expression profiling identified different subtypes of TNBC, several of which express androgen receptor (AR). AR inhibitors could provide a much-needed, less toxic, therapeutic intervention for patients with AR+ TNBC. However, clinical trials have shown modest activity of these agents in patients with metastatic AR+ TNBC to date. Conversion from AR antagonist to agonist is a well-recognized mechanism of resistance for AR inhibitors in the treatment of prostate cancer. To that end, we synthesized novel 7-substituted umbelliferon derivatives (UMBDs) with a distinct scaffold modified from available AR antagonists, designed to avoid such conversion. We have previously demonstrated that one of these agents, UMBD# 3 (compound 7a), demonstrates sub-micro molar inhibitory activity in a human prostate cancer with greater than 30 to 50 fold potency over the anti-androgens bicalutamide (Bica) and enzalutamide (Enza). In our current study, we tested the efficacy of UMBD# 3 in an AR+ TNBC cells that are sensitive or have acquired resistance to Bica. Methods: Bica-resistant cell lines were generated via multiple passaging of wild type (WT) MDA-MB-231 cells in media containing varying concentrations of Bica. Cell viability assays were conducted over a 72-hour period post seeding of AR+TNBC cell lines with AR antagonists Bica, Enza or UMBD#3. IC50 for respective drugs was determined via linear regression on normalized control values. Real time PCR and western blots were used to determine changes of AR at mRNA and protein level respectively, in response to the different agents. In order to isolate potential mechanistic pathway, we evaluated two proteins that are highly expressed in breast cancer, namely Poly (ADP-ribose) polymerase-1 (PARP-1) and Cadherin-11 (CAD-11) and assessed their expression in AR+ TNBC cells treated with UMBD#3. Results: The IC 50 for UMBD#3 was 6 and 4-fold lower than for Bica or Enza, respectively, in the WT AR+ TNBC cells (Bica, Enza and UMBD#3 IC50 were 136 ± 12.19, 91±12 and 20±7 µM respectively). The IC-50 for Bica in the Bica-resistant AR+ TNBC cells was 729 ± 42 µM, 6-fold higher than the WT cells. The IC50 for UMBD#3 in the Bica-resistant cells was 23.4±10 µM, similar to the WT cells. UMBD#3 caused a decrease in AR expression in the Wt and Bica-resistant calls. Expression of CAD-11 and PARP-1 were decreased in AR+ TNBC cells treated with UMBD#3 both at mRNA level and protein level. Conclusion: We show for the first time in vitro the anti-proliferative effect of a novel AR antagonist, UMBD#3, in WT and resistant AR+ TNBC. UMBD#3 appears to act via downregulation of AR with resultant decreased expression PARP and CAD-11. Given the apparent inherent resistance of AR+ TNBC to standard anti-androgens, further evaluation of this agent is warranted. Citation Format: Saswati Bhattacharya, Pam Westmark, Noah Siegel, Yuanqi Cai, Sahar Kandil, Andrew D Westwell, Christopher McGuigan, Ruth O'Regan. Umbelliferon derivatives for androgen receptor positive triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-06.

Role of poly (ADP-ribose) polymerase-1 in cadmium-induced cellular DNA damage and cell cycle arrest in rat renal tubular epithelial cell line NRK-52E

With the development of modern industry, the problem of cadmium (Cd) pollution cannot be ignored and its toxicity has caused great personal injury to humans. Poly (ADP-ribose) polymerase 1 (PARP-1) protein is a research hotspot in recent years, the research we have published shows that 5 μM of Cd-treated NRK-52E cells activated PARP-1, but the specific effects of PARP-1 on DNA damage and cell cycle is unclear. Therefore, the purpose of this study is to reveal the effect of Cd on DNA damage and cell cycle arrest in NRK-52E cells, in addition, to investigate the role of PARP-1 in mediating this effect. Western blotting, comet assay, QRT-PCR, immunofluorescence, and co-immunoprecipitation were used to detect DNA damage and cell cycle-associated protein expression. Flow cytometry was used to assess cell cycle distribution and the apoptosis rates. Results showed that after the increase in treatment time and Cd concentration, the degree of DNA damage was significantly increased, and a transition from G0/G1 to S phase arrest was observed. In addition, inhibition of PARP-1 expression exacerbated cell damage and cell cycle arrest when DNA damage was low, but attenuated cell damage and even cell cycle arrest when DNA damage was severe. These findings in this study indicate that Cd causes DNA damage in NRK-52E cells, leading to cell cycle arrest at different phases depending on the degree of DNA damage. Moreover, PARP-1 plays an important role in mediating this effect, when DNA damage is low, it functions in DNA repair, however, when DNA damage is severe, it aggravates cell damage and induces cell death.

Effects of Andrographolide on Intracellular pH Regulation, Cellular Migration, and Apoptosis in Human Cervical Cancer Cells †.

Cancer cells have been characterized with alkaline intracellular pH (pHi) values (≥7.2) to enable cancer proliferation, migration, and progression. The aim of the present study was to explore the concentration-dependent effects of Andrographolide, an active diterpenoid compound of herb Andrographis paniculata, on Na+/H+ exchanger isoform 1 (NHE1), cellular migration and apoptosis in human cervical cancer cells (HeLa). The pHi was detected by microspectrofluorometry method, and intracellular acidification was induced by NH4Cl prepulse technique. Viability and protein expression were determined by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and Western blot, respectively. Human normal endocervical cells (End1), ectocervical cells (Ect1), and HeLa were bought commercially. The resting pHi value of HeLa (≈7.47) was significantly higher than that of End1 and Ect1 (≈7.30), and shifted from alkaline to acidic following acid/base impacts. In HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid | N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) -buffered superfusate, NHE1 and V-ATPase co-existed functionally for acid extrusion in HeLa, while only NHE1 existed functionally in End/Ect1. Andrographolide (3–1000 μM) concentration-dependently inhibited NHE1 activity. Cell-migration and expressions of NHE1, V-ATPase, PARP (poly-ADP-ribose-polymerase), pro-Caspase-3, and Bcl-2 were significantly reduced by pretreating with Andrographolide (≥100 μM) for 24–48 h in HeLa. Andrographolide inhibited cell viability of End1-cells/Ect1 and HeLa (≥100 and ≥30 μM, respectively). The present findings implicate the promising clinical applications of Andrographolide on cervical cancer treatment.

Vitamin C supplementation had no side effect in non-cancer, but had anticancer properties in ovarian cancer cells.

Vitamin C (Vit C) has been widely used in the treatment and prevention of cancer. Nevertheless, the clinical results are still inconclusive. Using non-cancer (HOSEpiC) and cancer OVCAR-3 cells cultured in basal medium or in ovarian cancer-associated fibroblast (CAF)-supplemented medium, we estimated the dose-dependent effect of Vit C on sodium-ascorbate co-transporters (SVCT1, SVCT2) and glucose transporter (GLUT1) protein expression. Additionally, the action of Vit C on cell proliferation (alamarBlue), membrane permeability (LDH assay), caspase3 activity, the selected cell cycle and apoptosis pathway, poly(ADP-ribose) polymerase-1 (PARP) protein expression, and reactive oxygen species (ROS) activity was determined. We showed different effects of Vit C on the expression of the co-transporter in non-cancer and cancer cells. In non-cancer cells, Vit C, at a pharmacological concentration, increased SVCT2 and decreased GLUT1, while the opposite effect was noted in cancer cells. In cancer cells, Vit C, in a pharmacological dose, decreased cell proliferation through an inhibitory effect on cyclin-dependent kinase 2 (CDK2) (4.4-fold; p<0.01), mainly due to the stimulatory effect on the expression of cyclin-dependent kinase (CDK) inhibitors, such as p21 and p53 (3.2- and 2.8-fold, respectively; p<0.001), but not caspase pathway. The tumour microenvironment caused inefficiency of the lower doses of Vit C in ovarian cancer cells. At a pharmacological dose of 1mM, Vit C decreased PARP expression (1.5-fold; p<0.05). We suggest that it's nontoxic effects on non-cancer cells may be an indicator of its prophylactic use, while in a pharmacological dose Vit C should be considered a possible adjunctive drug in ovarian cancer. However, it is necessary to consider the effect of the CAF.

Preclinical and first-in-human-brain-cancer applications of [18F]poly (ADP-ribose) polymerase inhibitor PET/MR.

BackgroundWe report preclinical and first-in-human-brain-cancer data using a targeted poly (ADP-ribose) polymerase 1 (PARP1) binding PET tracer, [18F]PARPi, as a diagnostic tool to differentiate between brain cancers and treatment-related changes.MethodsWe applied a glioma model in p53-deficient nestin/tv-a mice, which were injected with [18F]PARPi and then sacrificed 1 h post-injection for brain examination. We also prospectively enrolled patients with brain cancers to undergo dynamic [18F]PARPi acquisition on a dedicated positron emission tomography/magnetic resonance (PET/MR) scanner. Lesion diagnosis was established by pathology when available or by Response Assessment in Neuro-Oncology (RANO) or RANO-BM response criteria. Resected tissue also underwent PARPi-FL staining and PARP1 immunohistochemistry.ResultsIn a preclinical mouse model, we illustrated that [18F]PARPi crossed the blood–brain barrier and specifically bound to PARP1 overexpressed in cancer cell nuclei. In humans, we demonstrated high [18F]PARPi uptake on PET/MR in active brain cancers and low uptake in treatment-related changes independent of blood–brain barrier disruption. Immunohistochemistry results confirmed higher PARP1 expression in cancerous than in noncancerous tissue. Specificity was also corroborated by blocking fluorescent tracer uptake with an excess unlabeled PARP inhibitor in patient cancer biospecimen.ConclusionsAlthough larger studies are necessary to confirm and further explore this tracer, we describe the promising performance of [18F]PARPi as a diagnostic tool to evaluate patients with brain cancers and possible treatment-related changes.

Epigallocatechin gallate alleviates neuronal cell damage against focal cerebral ischemia in rats.

Cerebral ischemia is a neurological disorder that causes permanent disability and is sometimes fatal. Epigallocatechin gallate (EGCG) is a natural polyphenol that exerts beneficial antioxidant and anti-inflammatory effects. The aim of this study was to investigate the neuroprotective effects of EGCG against cerebral ischemia. Middle cerebral artery occlusion was surgically initiated to induce focal cerebral ischemia in adult male rats. EGCG (50 mg/kg) or vehicle was intraperitoneally injected just prior to middle cerebral artery occlusion (MCAO) induction. Neuronal behavior tests were performed 24 hr after MCAO. Brain tissues were isolated to evaluate infarct volume, histological changes, apoptotic cell death, and caspase-3 and poly ADP-ribose polymerase (PARP) levels. MCAO injury led to serious functional neurological deficits and increased infarct volume. Moreover, it induced histopathological lesions and increased the numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the cerebral cortex. However, EGCG improved MCAO-induced neurological deficits and reduced infarct volume, alleviated histopathological changes, and decreased TUNEL-positive cells in the cerebral cortex of MCAO rats. Western blot analysis showed increases of caspase-3 and PARP expression levels in MCAO rats with vehicle, whereas EGCG administration alleviated these increases after MCAO injury. These results demonstrate that EGCG exerts a neuroprotective effect by regulating caspase-3 and PARP proteins during cerebral ischemia. In conclusion, we suggest that EGCG acts as a potent neuroprotective agent by modulating the apoptotic signaling pathway.

Poly(ADP-ribose) polymerase-1 depletion enhances the severity of inflammation in an imiquimod-induced model of psoriasis.

Poly(ADP-ribose) polymerase-1 (PARP1) is a pro-inflammatory protein, whose pro-inflammatory properties were demonstrated in human. The pro-inflammatory properties of PARP1 were shown in Th1- and Th2-mediated inflammatory pathologies, but not Th17-mediated inflammation. Thus, we studied the role of PARP1 in the imiquimod-induced model of psoriasis. To our surprise, in imiquimod-induced psoriasis, PARP1 acted as an anti-inflammatory factor and its genetic deletion exacerbated symptoms. We showed that in the absence of PARP1, the epidermis thickened and the number of TUNEL-positive cells decreased in the epidermis. These data indicate programmed cell death is decreased in keratinocytes. Changes in involucrin expression suggest that keratinocyte differentiation is hampered. Furthermore, epidermal expression of IL6 increased in the psoriasiform lesions of PARP1 knockout mice, suggesting that the inflammatory response is also derailed in the absence of PARP1. Finally, we showed that PARP1 expression is reduced in human psoriatic lesions compared with control skin samples. In imiquimod-treated HPV-KER keratinocytes, PARP inhibition recapitulated the in vivo findings, namely keratinocyte hyperproliferation; furthermore, the mRNA expression of psoriasis-associated cytokines (IL6, IL1β, IL8, IL17 and IL23A) was also induced. The inhibition of TRPV1 abrogated the effects of the combined imiquimod+PARP inhibitor treatment.

PARP-1 Targeted Alpha-emitting Radiotherapeutics: An Examination of Potential Toxicity

Radiolabeling poly-ADP ribose polymerase 1 (PARP-1) inhibitors potentially enables targeted delivery of alpha-emitting isotopes directly to cancer cells overexpressing PARP-1. However, there is concern for bone marrow toxicity due to its intrinsically high PARP-1 expression. Therefore, we examined bone marrow toxicity at therapeutically relevant doses of an astatine-211 labeled PARP-1 inhibitor, [211At]MM4. Male and female C57BL6 mice received 0, 12, 24, and 36 MBq/kg of [211At]MM4 and then were sacrificed after 24 hours, 2 weeks, and 4 weeks. The red marrow-containing femur was harvested and was analyzed with colony formation assay, immunofluorescent (IF) microscopy, histopathology, and immunohistochemistry (IHC). Peripheral blood samples were analyzed for complete blood count (CBC). Additionally, we performed [211At]MM4 biodistribution assay on another set of 20 C57BL6 mice at post-injection time points of 5, 30, 60, 180, and 360 minutes. With the biodistribution data, OLINDA/EXM v1.1 was used for radiation dosimetry calculations in reference to human pediatric models. The CBC revealed significant lymphopenia only in the 2-week group treated with 24 MBq/kg. A slight decrease in lymphocyte percentage was observed in the 24 MBq/kg mice at 2 weeks and the 36 MBq/kg at 2 and 4 weeks. Significant neutrophilia was observed in all 2-week groups, as well as the 4-week group treated at 36 MBq/kg. Colony formation assay revealed no reduction, but rather significant increase in granulocyte, macrophage, and burst forming unit-erythroid colonies. Histopathology revealed maintained bone marrow cellularity across the treatment groups. IF and IHC demonstrated heterogeneous PARP-1 expression in the bone marrow. The biodistribution and subsequent dosimetry calculations found significantly higher, but nonlethal organ radiation dose per injected activity levels in the red bone marrow, spleen, and thyroid in both 1 and 5-year-old human models. Overall, the results suggest that doses up to 36 MBq/kg of [211At]MM4 can be administered to C57BL6 mouse models without producing significant systemic or bone marrow toxicity. These results provide promising developments for understanding toxicity associated with the alpha-emitting compound [211At]MM4 in vivo. Future studies using fractionated dosing regimens in pre-clinical tumor models will evaluate therapeutic efficacy and further evaluate associated toxicities.

Evidence for Decreased Nucleolar PARP-1 as an Early Marker of Cognitive Impairment.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear protein that regulates gene expression through poly(ADP)-ribosylation, resulting in the loosening of chromatin structure. PARP-1 enzymatic activity has been shown to be necessary for the expression of several genes required for memory formation and consolidation. Previously, we showed that nucleolar PARP-1 is significantly decreased in hippocampal pyramidal cells in Alzheimer's disease (AD). We proposed that the displacement of PARP-1 from the nucleolus results in downregulation of new rRNA expression and ribosome biogenesis, leading to cognitive impairment. To further investigate the relationship between nucleolar PARP-1 and memory impairment, we examined PARP-1 expression in the hippocampi of individuals with mild cognitive impairment (MCI) compared to control and AD cases. We used immunohistochemical techniques to examine the nucleolar distribution of PARP-1 in the Cornu Ammonis (CA region) of the hippocampus. PARP-1 positive cells were then scored for the presence or absence of PARP-1 in the nucleolus. We found a significant decrease of PARP-1 staining in the nucleolar compartment of hippocampal pyramidal cells in MCI compared with Control and AD. When the four CA (CA1-4) regions were considered separately, only the CA1 region showed significant differences in nucleolar PARP-1 with Control > AD > MCI cases. Categorization of nucleolar PARP-1 into “distinct” and “diffuse” groups suggest that most of the changes occur within the distinct group. In addition, measurements of the nucleolar diameter of nucleolar PARP-1 positive cells in CA2 and CA4 showed Control > MCI. Thus, MCI cases had a lower percentage of PARP-1 nucleolar positive cells in CA1 and smaller nucleolar diameters in CA2 and CA4, compared to Control. Our data suggest that disruption of nucleolar form and function is an early and important step in the progression of cognitive impairment.

MicroRNA-577 inhibits cardiomyocyte apoptosis induced by myocardial infarction via targeting PARP1.

The aim of this study was to investigate whether microRNA-577 could inhibit myocardial infarction (MI)-induced cardiomyocyte apoptosis by regulating poly ADP-ribose polymerase 1 (PARP1). MI model was successfully established in mice by ligation of the left anterior descending coronary artery (LAD). The expression levels of microRNA-577 and PARP1 in myocardial tissues of mice were examined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). MI model in cells was established by hypoxia pre-treatment in primary cardiomyocytes and MCM cells. Subsequently, the expression levels of microRNA-577 and PARP1 in hypoxia preconditioning cardiomyocytes were determined as well. Meanwhile, Caspase3 activity in cardiomyocytes overexpressing microRNA-577 or PARP1 was detected using a relative commercial kit. Furthermore, the binding relationship between microRNA-577 and PARP1 was examined by Dual-Luciferase reporter gene assay. Infarct size/risk region and risk region/LV in MI group were (62.1±2.2)% and (57.6±1.9)%, respectively. Both of the above two indexes in the MI group were significantly higher than those of the control group. The serum level of LDH in MI mice increased by 2.8-fold when compared with controls. Meanwhile, the expressions of microRNA-577 and PARP1 in myocardial tissues of MI mice were markedly down-regulated in a time-dependent manner. Compared with normoxia preconditioning cardiomyocytes, microRNA-577 expression in hypoxia preconditioning MCM cells and primary cardiomyocytes was remarkably decreased. Dual-Luciferase reporter gene assay confirmed that microRNA-577 could bind to PARP1. After transfection of microRNA-577 mimics, the expression of PARP1 was significantly down-regulated. Moreover, microRNA-577 over-expression inhibited caspase3 expression in hypoxia preconditioning cells, which could be reversed by PARP1 up-regulation. Similarly, microRNA-577 over-expression markedly decreased infarct size, risk region and serum level of LDH in MI mice, which could be reversed by PAPR1 over-expression. MicroRNA-577 inhibits MI-induced cardiomyocyte apoptosis by degrading PARP1.

PARP-1 deregulation in multiple sclerosis.

BackgroundPoly (ADP-ribose) polymerase 1 (PARP-1) plays pivotal roles in immune and inflammatory responses. Accumulating evidence suggests PARP-1 as a promising target for immunomodulation in multiple sclerosis and natalizumab-associated progressive multifocal leukoencephalopathy.ObjectiveThis study explores expression of PARP-1 and downstream effectors in multiple sclerosis and during natalizumab treatment.MethodsTranscriptional expressions were studied by real-time reverse transcriptase polymerase chain reaction on CD4+T/CD8+T/CD14+/B cells and peripheral blood mononuclear cells from healthy volunteers, untreated and natalizumab-treated non-progressive multifocal leukoencephalopathy and progressive multifocal leukoencephalopathy multiple sclerosis patients.ResultsPARP-1 expression was higher in CD4+T, CD8+T and B cells from untreated patients compared to healthy volunteers. Natalizumab treatment restored deregulated PARP-1 expression in T cells but not in B cells. Sustained upregulation of PARP-1 was associated with decreased expression of downstream PARP-1 factors such as TGFBR1/TGFBR2/BCL6 in B cells. Notably, a higher expression of PARP-1 was detected in progressive multifocal leukoencephalopathy patients.ConclusionsGiven the importance of PARP-1 in inflammatory processes, its upregulation in multiple sclerosis lymphocyte populations suggests a potential role in the immune pathogenesis of multiple sclerosis. Strikingly higher PARP-1 expression in progressive multifocal leukoencephalopathy cases suggests its involvement in progressive multifocal leukoencephalopathy disease pathomechanisms. These results further support the value of PARP-1 inhibitors as a potential novel therapeutic strategy for multiple sclerosis and natalizumab-associated progressive multifocal leukoencephalopathy.