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Embryo implantation is a highly complex process involving many regulatory factors, including several micro RNAs (miRNAs/miRs). One miRNA present in the stromal cells of normal endometrium is miR-149, which targets poly (ADP-ribose) polymerase 2 (PARP-2), a gene involved in endometrial receptivity for trophoblast implantation. However, the precise role of miR-149 in the endometrial receptivity during blastocyst implantation is still unknown. We studied miR-149-dependent PARP-2 regulation during trophoblast attachment to endometrial epithelial cells. Using FISH, we found that miR-149 is expressed in mouse endometrial epithelial and stromal cells at implantation and inter-implantation sites. Endometrial receptivity for embryo implantation and attachment is inhibited by the upregulation of miR-149 in the endometrium. Our RT-PCR analysis revealed downregulation of miR-149 in the implantation region of the uterus during the receptive stage (Day 5, 0500 h, p.c.) in the mouse. Under in-vitro conditions, miR-149 overexpression in human endometrial epithelial cells (hEECs) abrogated the human trophoblastic cells spheroid and mouse blastocyst attachment. Subsequently, miR-149 also regulates transformed human endometrial stromal cell (T-hESCs) decidualization by downregulating PARP-2 and upregulating caspase-8 proteins. Overexpression of miR-149 in hEECs and downregulated PARP-2 protein expression, reconfirming that PARP-2 is a downstream target of miR-149 in endometrial cells as well. miR-149 is also able to alter the expression of caspase-8, another PARP-2 regulator. In conclusion, our data indicate that miR-149 is one of the regulators of endometrial receptivity and decidualization for trophoblast implantation, and it exerts the effects by acting on the downstream targets PARP-2 and caspase-8., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Glioma is a primary malignancy of the central nervous system (CNS). As biomedicine advances, an efficient molecular target is urgently needed for the diagnosis and treatment of glioma. Meanwhile, several studies have demonstrated that glioma development is closely related to immunity. PARP9 is an inactive mono-ADP-ribosyltransferase belonging to the poly-ADP ribosyltransferase (ARTD) family. In this article, we aimed to reveal the relationship between PARP9 and glioma and explore the potential prognostic value and immunotherapeutic targetability of PARP9 in glioma., Methods: PARP9 transcript levels were analyzed with TCGA and GEO databases. The clinicopathological information of patients with glioma in the TCGA database and gene expression profiles were analyzed to determine the relationship between the expression of PARP9 and clinicopathologic characteristics. Kaplan-Meier survival analysis, univariate Cox regression analysis, and multivariate Cox regression analysis were used for survival analysis. Gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) were used for bioinformatics analysis. Correlation analysis explored the relationships between PARP9, infiltrating inflammatory immune cells, and immune checkpoint molecules., Results: PARP9 is highly expressed in glioma, and high expression of PARP9 is associated with poor prognosis and advanced clinicopathological features. Bioinformatics analysis showed that some immune-related pathways were closely associated with high expression of PARP9. Correlation analysis indicated that PARP9 was closely related to inflammatory and immune responses, high immune cell infiltration, and immune checkpoint molecules., Conclusions: PARP9 may serve as an unfavorable prognosis predictor for glioma and a potential immunotherapeutic target., (© 2020 The Authors. CNS Neuroscience & Therapeutics Published by John Wiley & Sons Ltd.)

Background: In the United States endometrial carcinoma is the most common female gynecologic malignancy. An average of more than 60,000 new cases of endometrial carcinomas have been diagnosed yearly over the past 5 years, with a higher incidence occurring in the central Appalachian states of Ohio and West Virginia. In the U.S., the national average of newly diagnosed endometrial carcinomas is 26.8 in every 100,000 women, while in the states of Ohio and West Virginia the average is 30.5 and 31.1 in every 100,000 women, respectively. This notable increase in the incidence of endometrial carcinomas may be due a variety of elevated risk factors including but not limited to: tobacco use, obesity, and genetic predisposition of the predominant demographic. The American Cancer Society estimates that approximately 55,000 new cases of endometrial carcinoma will be diagnosed in 2020 yet, this disease is widely considered understudied and under-represented in mainstream cancer research circles., Methods: The aim of this study was to quantitate the co-expression of two DNA repair proteins poly-ADP-ribose polymerase 1 and 2 (Parp-1 and Parp-2) by enzyme- linked immuno-sorbent assay (ELISA) in 60 endometrioid endometrial tumor samples and compare their expression to matched non-malignant endometrial tissue from the same corresponding donors from central Appalachia., Results: We found that Parp-1 was significantly overexpressed in endometrial carcinoma relative to corresponding normal tissue. This overexpression implicates Parp inhibition therapy as a possible treatment for the disease. Our results also found a protective effect of native Parp-2 expression in non-malignant endometrial tissue with each 1 ng/mL increase in PARP-2 concentration in normal tissue was associated with a 10 % reduction in the hazard of tumor progression (HR = 0.90; p = 0.039) and a 21 % reduction in the hazard of death (HR = 0.79; p = 0.044)., Conclusions: This study demonstrated the over-expression of the druggable target Parp-1 in endometrial adenocarcinoma and observed a strong negative correlation of native Parp-2 expression and disease progression via the quantification of the Parp proteins using enzyme- linked immuno-sorbent assay (ELISA) assays., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Published by Elsevier GmbH.)

The aryl hydrocarbon receptor (AhR) is a cytoplasmic receptor/transcription factor that modulates several cellular and immunological processes following activation by pathogen-associated stimuli, though its role during virus infection is largely unknown. Here, we show that AhR is activated in cells infected with mouse hepatitis virus (MHV), a coronavirus (CoV), and contributes to the upregulation of downstream effector TCDD-inducible poly(ADP-ribose) polymerase (TiPARP) during infection. Knockdown of TiPARP reduced viral replication and increased interferon expression, suggesting that TiPARP functions in a proviral manner during MHV infection. We also show that MHV replication induced the expression of other genes known to be downstream of AhR in macrophages and dendritic cells and in livers of infected mice. Further, we found that chemically inhibiting or activating AhR reciprocally modulated the expression levels of cytokines induced by infection, specifically, interleukin 1β (IL-1β), IL-10, and tumor necrosis factor alpha (TNF-α), consistent with a role for AhR activation in the host response to MHV infection. Furthermore, while indoleamine 2,3-dioxygenase (IDO1) drives AhR activation in other settings, MHV infection induced equal expression of downstream genes in wild-type (WT) and IDO1 -/- macrophages, suggesting an alternative pathway of AhR activation. In summary, we show that coronaviruses elicit AhR activation by an IDO1-independent pathway, contributing to upregulation of downstream effectors, including the proviral factor TiPARP, and to modulation of cytokine gene expression, and we identify a previously unappreciated role for AhR signaling in CoV pathogenesis. IMPORTANCE Coronaviruses are a family of positive-sense RNA viruses with human and agricultural significance. Characterizing the mechanisms by which coronavirus infection dictates pathogenesis or counters the host immune response would provide targets for the development of therapeutics. Here, we show that the aryl hydrocarbon receptor (AhR) is activated in cells infected with a prototypic coronavirus, mouse hepatitis virus (MHV), resulting in the expression of several effector genes. AhR is important for modulation of the host immune response to MHV and plays a role in the expression of TiPARP, which we show is required for maximal viral replication. Taken together, our findings highlight a previously unidentified role for AhR in regulating coronavirus replication and the immune response to the virus., (Copyright © 2020 American Society for Microbiology.)

Backgroud: Recently long non-coding RNAs (lncRNAs) have attracted attention in several biomedical fields. The purpose of this study is to investigate the profile of myocardial lncRNAs and their potential roles in myocardial ischaemia-reperfusion injury (IRI)., Methods: EdgeR bioconductor package was used to screen differentially expressed lncRNAs in myocardial IRI, and lncRNA AK12348 was selected. The mRNA levels of lncRNA AK12348 in normal and anoxia/reoxygenation (A/R) cardiomyocytes were determined by qRT-PCR. After transfection with siRNA-lncRNA, AK12348, LDH release and cell apoptotic rates in normal and A/R cardiomyocytes were determined. The protein expression values of PARP and Caspase-3 were also determined by western blotting., Results: The relative level of lncRNA AK12348, LDH release and cell apoptotic rate in A/R cardiomyocytes was significantly higher than that in normal cardiomyocytes. After transfection with siRNA-lncRNA AK12348, LDH release and cell apoptotic rates in A/R cardiomyocytes were reduced, while the values in normal cardiomyocytes had almost no change. The protein expression values of PARP and Caspase-3 in A/R cardiomyocytes were much higher than the Control. After knockdown of lncRNA AK12348, the values decreased., Conclusion: Long non-coding RNAs AK12348 could be potential therapeutic targets for the treatment of myocardial IRI., (Copyright © 2017 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). Published by Elsevier B.V. All rights reserved.)

The present study aimed to examine the effects of sodium selenite on the SW982 human synovial sarcoma cell line in relation to cell viability, apoptosis and autophagy. The results indicated that sodium selenite reduced cell viability and induced apoptosis by activating caspase‑3 and members of the poly (ADP‑ribose) polymerase and Bcl‑2 protein families in SW982 cells. Furthermore, autophagy was also suppressed by sodium selenite treatment in SW982 cells, and apoptosis was upregulated in cells co‑treated with sodium selenite and the autophagy inhibitor 3‑methyladenine. By contrast, apoptosis was downregulated when sodium selenite was combined with rapamycin, an inducer of autophagy. The results indicated that autophagy may protect cells from the cytotoxicity of sodium selenite. The present study results demonstrated that sodium selenite induced apoptosis and inhibited autophagy and autophagy‑protected cells from death by antagonizing sodium selenite‑induced apoptosis in SW982 cells in vitro.

Objectives: The purpose of this study was to investigate the effects of 5-aminolaevulinic acid mediated photodynamic therapy (ALA-PDT) on the survival activity and apoptosis of human melanoma cell line A375 and non-melanoma skin carcinoma cell line A431 cells. The mechanism for cellular apoptosis was explored., Methods: The cell survival activity was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and the proportion of apoptotic cells was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The expression levels of Bcl-2, Bax, caspase-3, caspase-8 and caspase-9 protein were assessed by western blot. The subcellular localization of cytochrome c was comparatively investigated by immunohistochemistry between pre-ALA-PDT and post- ALA-PDT., Results: ALA-PDT significantly inhibited the survival activity of A375 cells and A431 cells in a dose- and time-dependent manner. The optimum inhibition efficiencies for A375 cells and A431 cells were obtained at 0.6 mM ALA at 4 h and 8 h after ALA-PDT, respectively. The phenomena of apoptosis were observed in ALA-PDT treated cells by TUNEL assay. The apoptotic rates of A375 cells and A431 cells were 90.0% and 61.5% at 6 h after ALA-PDT, respectively. Apoptosis induced by ALA-PDT involved in down-regulation of Bcl-2 protein, up-regulation of Bax protein and cleaved-PARP protein. It was observed that the expression of cleaved- caspase-3, caspase-8 and caspase-9 proteins in A375 cells and A431 cells gradually increased in 2 h and 4 h but decreased at 4-6 h and 6-8 h after ALA-PDT, respectively. In apoptosis cells immunohistochemical localization show that cytochrome C diffused from the mitochondria into the cytosol., Conclusion: ALA-PDT could significantly inhibit the survival activity of A375 and A431 cells. The apoptosis induced by ALA-PDT in A375 and A431 cells was related to the caspase-dependent death-receptor pathway and Cytochrome c-dependent mitochondrial pathway., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We previously demonstrated that arsenic, cadmium, and lead mixture at environmentally relevant doses induces astrocyte apoptosis in the developing brain. Here, we investigated the mechanism and contribution of each metal in inducing the apoptosis. We hypothesized participation of transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), reported to affect astrocyte survival. We treated cultured rat astrocytes with single metals and their combinations and performed apoptosis assay and measured PPARγ expression levels. We found that cadmium demonstrated maximum increase in PPARγ as well as apoptosis, followed by arsenic and then lead. Interestingly, we observed that the metals mimicked PPARγ agonist, troglitazone, and enhanced PPARγ-transcriptional activity. Co-treatment with PPARγ-siRNA or PPARγ-antagonist, GW9662, suppressed the astrocyte apoptosis, suggesting a prominent participation of PPARγ in metal(s)-induced astrocyte loss. We explored PPARγ-transcriptional activity and identify its target gene in apoptosis, performed in silico screening. We spotted PPARγ-response elements (PPREs) within poly(ADP-ribose) polymerase (PARP) gene, and through gel-shift assay verified metal(s)-mediated increased PPARγ binding to PARP-PPREs. Chromatin-immunoprecipitation and luciferase-reporter assays followed by real-time PCR and Western blotting proved PPRE-mediated PARP expression, where cadmium contributed most and lead least, and the effects of metal mixture were comparable with troglitazone. Eventually, dose-dependent increased cleaved-PARP/PARP ratio confirmed astrocyte apoptosis. Additionally, we found that PPARγ and PARP expressions were c-Jun N-terminal kinases and cyclin-dependent kinase5-dependent. In vivo treatment of developing rats with the metals corroborated enhanced PPARγ-dependent PARP and astrocyte apoptosis, where yet again cadmium contributed most. Overall, our study enlightens a novel PPARγ-dependent mechanism of As-, Cd-, and Pb-induced astrocyte apoptosis.

Peroxynitrite is a powerful oxidant, formed from the reaction of nitric oxide and superoxide. It is known to interact and modify different biological molecules such as DNA, lipids and proteins leading to alterations in their structure and functions. These events elicit various cellular responses, including cell signaling, causing oxidative damage and committing cells to apoptosis or necrosis. This review discusses nitrosative stress-induced modification in the DNA molecule that results in the formation of 8-nitroguanine and 8-oxoguanine, and its role in disease conditions. Different approaches of cell death, such as necrosis and apoptosis, are modulated by cellular high-energy species, such as ATP and NAD + . High concentrations of peroxynitrite are known to cause necrosis, whereas low concentrations lead to apoptosis. Any damage to DNA activates cellular DNA repair machinery, like poly(ADP-ribose) polymerase (PARP). PARP-1, an isoform of PARP, is a DNA nick-sensing enzyme that becomes activated upon sensing DNA breakage and triggers the cleavage of NAD + into nicotinamide and ADP-ribose and polymerizes the latter on nuclear acceptor proteins. Peroxynitrite-induced hyperactivation of PARP causes depletion of NAD + and ATP culminating cell dysfunction, necrosis or apoptosis. This mechanistic pathway is implicated in the pathogenesis of a variety of diseases, including circulatory shock (which is characterized by cellular hypoxia triggered by systemic altered perfusion and tissue oxygen utilization leading end-organ dysfunction), sepsis and inflammation, injuries of the lung and the intestine. The cytotoxic effects of peroxynitrite centering on the participation of PARP-1 and ADP-ribose in previously stated diseases have also been discussed in this review.

Irinotecan is a natural alkaloid agent widely used in cancer therapy. High-mobility group protein B1 as a non-histone chromosomal protein plays a fundamental role in gene expression and inflammation. In this study, the effect of irinotecan on high-mobility group protein B1 and MMP9 content, gene expression, cell cycle, and cell growth in human breast cancer cells (MCF-7) was investigated. The cells were exposed to various concentrations of irinotecan and the viability determined by trypan blue exclusion and 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyltetrazolium bromide assays. High-mobility group B proteins were extracted from the control and drug-treated cells and analyzed by immunoblot. High-mobility group protein B1 and MMP9 messenger RNA expression was studied by reverse transcription polymerase chain reaction. The results demonstrated reduction of cell viability upon increasing irinotecan concentration, up-regulated high-mobility group protein B1 gene expression, and down-regulated MMP9 mRNA. Although the content of high-mobility group protein B1 was decreased in chromatin extract upon drug action, no high-mobility group protein B1 release to extracellular space was detected by immunoblot analysis. Irinotecan decreased H3K9 acetylation and increased poly ADP-ribose polymerase fragmentation to 89 kDa and anion superoxide production suggesting induction of apoptosis in these cells. Propidium iodide staining of the cells 24 h after the drug treatment revealed arrest of the cells in S-phase. From the results, it is concluded that overexpression of high-mobility group protein B1 in the presence of irinotecan precedes breast cancer cells into apoptosis and in this response the binding of irinotecan to chromatin or high-mobility group protein B1 may condense/aggregate chromatin, preventing high-mobility group protein B1 release from chromatin.

BRCA1/2 mutation status in epithelial ovarian cancer (EOC) presently relies on genetic testing which is resource consuming. Immunohistochemistry is cheap, fairly reproducible, and may identify gene product alterations due to both germline and somatic mutations and other defects along the BRCA gene pathway (BRCAness phenomenon), which is important when treatment with poly (adenosine-diphosphate-ribose) polymerase (PARP) inhibitors is considered. The aim of this study was to investigate immunohistochemical detection of BRCA1 and PARP expression in EOC and their possible prognostic relevance. Tumor tissue from 170 patients with EOC was stained immunohistochemically with BRCA1 and PARP antibodies. Semiquantitative analyses were performed to determine loss of, equivocal, and retained BRCA1 and high versus low PARP protein expression. These parameters were analyzed for relation with patient and clinicopathologic characteristics and overall survival. BRCA1 expression was reduced in 21.2 % of the tumors and 36.5% showed high PARP expression. No correlation between the 2 parameters or between PARP and clinicopathologic features was found. Overall survival was significantly increased in the BRCA1-reduced and equivocal groups [median survival 2.4 y (95% CI, 1.6-6.6) and 4.9 y (95 % CI, 2.3-6.7) vs. 1.5 y (95% CI, 1.3-1.9); P=0.0002]. Multivariate analysis confirmed these findings; hazard ratio=0.53 (95% CI, 0.34-0.81; P=0.0037; loss of BRCA1 expression). In conclusion, immunohistochemical BRCA1 expression in EOC holds considerable prognostic information, whereas PARP expression did not influence the outcome. The results call for validation in prospective trials.

Triple-negative breast cancers have unfavorable outcomes due to their inherent aggressive behavior and lack of targeted therapies. Breast cancers occurring in BRCA1 mutation carriers are mostly triple-negative and harbor homologous recombination deficiency, sensitizing them to inhibition of a second DNA damage repair pathway by, e.g., PARP inhibitors. Unfortunately, resistance against PARP inhibitors in BRCA1-deficient cancers is common and sensitivity is limited in BRCA1-proficient breast cancers. RK-33, an inhibitor of the RNA helicase DDX3, was previously demonstrated to impede non-homologous end-joining repair of DNA breaks. Consequently, we evaluated DDX3 as a therapeutic target in BRCA pro- and deficient breast cancers and assessed whether DDX3 inhibition could sensitize cells to PARP inhibition. High DDX3 expression was identified by immunohistochemistry in breast cancer samples of 24% of BRCA1 (p = 0.337) and 21% of BRCA2 mutation carriers (p = 0.624), as compared to 30% of sporadic breast cancer samples. The sensitivity to the DDX3 inhibitor RK-33 was similar in BRCA1 pro- and deficient breast cancer cell lines, with IC50 values in the low micromolar range (2.8-6.6 μM). A synergistic interaction was observed for combination treatment with RK-33 and the PARP inhibitor olaparib in BRCA1-proficient breast cancer, with the mean combination index ranging from 0.59 to 0.62. Overall, we conclude that BRCA pro- and deficient breast cancers have a similar dependency upon DDX3. DDX3 inhibition by RK-33 synergizes with PARP inhibitor treatment, especially in breast cancers with a BRCA1-proficient background.

Despite advances in diagnosis and treatment options, breast cancer is one of the main causes of cancer related death among women worldwide. Present study is aimed to preliminarily evaluate our hypothesis that the combination of resveratrol (RSV), a natural antioxidant, and lower dose of sorafenib (SF), a multi-kinase inhibitor and a component of ERK1/2 (extracellular signal-regulated kinase 1/2) pathway, would augment apoptosis in human breast cancer MCF7 cells. MCF7 cellexpressions s were treated with RSV, SF and their combination. MTT (3-[4,5-dimethylthiazol-2-yl] -2, 5-diphenyl-tetrazolium bromide) assay, DNA fragmentation assay, Hoechst33342, H 2 DCFDA (2', 7'-Dichlorodihydrofluorescein diacetate), Rhodamine123 staining, and Western Blot to detect different signaling protein expressions, were conducted to test the hypothesis. Combination of RSV and SF showed higher cytotoxicity on MCF7 cells than their individual treatment. Results from morphology change, Hoechst33342 staining, and DNA fragmentation suggested higher apoptosis data in the combinational treatment. Intracellular ROS (reactive oxygen species) levels, p53 and Bax/Bcl2 expressions, and decrease in mitochondrial membrane potential were also higher in the combinational treatment. Up-regulation of apaf-1, cl. caspase 9, cl. caspase 3 and cl. PARP (poly (ADP-Ribose) polymerase) were also noticed, while the expressions of cyclinD1 and cyclinB1 were decreased in the combinational group. The increase in apoptosis and signaling protein expressions with RSV and SF combinational treatment were increased over time. The combination of RSV and lower dose of SF at 6μM showed enhanced apoptotic activity than SF alone. Therefore, RSV can be considered as a neo-adjuvant to improve SF efficacy in breast cancer treatment., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

This study aims to clarify the molecular mechanism of fluorine exposure that leads to nerve injury. PC12 cells were treated with fluorine at different concentrations (0.5, 1.0, 1.5, and 2.0 mM). Cytoactivity was detected at different time points (2, 4, 6, 8, 12, 24, and 48 h). After 2 h, DCF was used to detect and mark the level of reactive oxygen species (ROS) within cells. After 24 h, cellular metamorphosis was observed using an inverted microscope. After 2 h, Hoechst-33342 was used to detect apoptosis. After 24 h, Western blot analysis was performed to detect apoptosis-related poly (ADP-ribose) polymerase (PARP) protein, p-elF, and expression of the endoplasmic reticulum stress-related X-box binding protein 1 (XBP-1). The results showed that Fluorine exposure resulted in a reduction of cell viability, which was negatively correlated with fluorine dose. Within certain fluorine exposure duration, the ROS level within the cell and the apoptotic level are linearly related to fluorine exposure level. XBP-1 and PARP protein are sensitive to variations in fluorine concentration, which indicates that oxidative stress from fluorine exposure can lead to apoptosis. XBP-1 and PARP may be the key proteins during the entire process. These results provide a valid basis for fluorine-induced free radical injury theory.

The aim of this study was to explore the correlation between hepatocyte cell adhesion molecule (hepaCAM) and SMAD family member 2/3 (SMAD2/3) in bladder carcinoma, and the involvement of the SMAD2/3 pathway in hepaCAM-induced tumor apoptosis. Immunohistochemistry was used to measure hepaCAM and p-SMAD2/3 protein levels in bladder cancer tissues. Flow cytometry and Hoechst staining were used to study the effect of hepaCAM on cellular apoptosis. Western blot was employed to determine the expression of hepaCAM and SMAD2/3/caspase pathway molecules using a hepaCAM overexpression adenovirus, a caspase inhibitor (Z-VAD-FMK), and a SMAD2/3 activator (transforming growth factor (TGF)-β1), respectively. Translocation of p-SMAD2/3 was measured by immunofluorescence and western blot. HepaCAM proteins were significantly decreased (P < 0.05), while p-SMAD2/3 proteins were remarkably increased (P < 0.05) in bladder carcinoma compared to adjacent tissues. However, the low hepaCAM and high p-SMAD2/3 were not statistically associated with clinicopathological characteristics of the patients. A negative linear correlation between hepaCAM and p-SMAD2/3 was observed according to Pearson analysis (r = -0.712/-0.724, P = 0.008/0.011). Overexpression of hepaCAM activated caspase 3/8/9 and downregulated poly-ADP ribose polymerase (PARP) and p-SMAD2/3. Treatment of bladder cancer cells with Z-VAD-FMK + hepaCAM significantly downregulated procaspase 3/8/9 and PARP and induced cellular apoptosis, compared with that using Z-VAD-FMK alone. Similarly, combined treatment of TGF-β1 + hepaCAM significantly downregulated p-SMAD2/3, procaspase 3/8/9, and PARP and induced apoptosis of bladder cancer cells, compared with TGF-β1 alone. Overexpression of hepaCAM prevented the p-SMAD2/3 translocation from the cytoplasm to the nucleus in bladder cancer cells BIU-87 and T24. Our findings uncover that the p-SMAD2/3 pathway is critical for hepaCAM-induced cancer cell apoptosis and provide valuable insights for current and future Ad-hepaCAM and p-SMAD2/3 clinical trials.

APR-246 (PRIMA-1(Met)) is able to bind mutant p53 and restore its normal conformation and function. The compound has also been shown to increase intracellular ROS levels. Importantly, the poly-[ADP-ribose] polymerase-1 (PARP-1) enzyme plays an important role in the repair of ROS-induced DNA damage. We hypothesize that by blocking this repair with the PARP-inhibitor AZD2281 (olaparib), DNA damage would accumulate in the cell leading to massive apoptosis. We observed that APR-246 synergistically enhanced the cytotoxic response of olaparib in TP53 mutant non-small cell lung cancer cell lines, resulting in a strong apoptotic response. In the presence of wild type p53 a G2/M cell cycle block was predominantly observed. NOXA expression levels were significantly increased in a TP53 mutant background, and remained unchanged in the wild type cell line. The combined treatment of APR-246 and olaparib induced cell death that was associated with increased ROS production, accumulation of DNA damage and translocation of p53 to the mitochondria. Out data suggest a promising targeted combination strategy in which the response to olaparib is synergistically enhanced by the addition of APR-246, especially in a TP53 mutant background., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Tumorigenesis results from various types of dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation, apoptosis and senescence. The transcription factor Myc-interacting zinc finger protein 1 (Miz-1) can either activate or repress gene expression in concert with binding partners including the Myc oncoprotein in several types of tumors. Known target genes of these complexes encode the cyclin‑dependent kinase inhibitors such as cdkn2b (p15) and cdkn1a (p21). In the present study, we showed that the silencing of Miz-1 expression, through shRNA in a lentiviral vector, influenced various biological processes in two types of esophageal carcinoma cell lines. Silencing of the expression of Miz-1 inhibited cell proliferation and promoted apoptosis in vitro. Loss of Miz-1 reduced the migration ability in esophageal carcinoma cells. High expression of p21 and downregulation of cyclin D1 accompanied the knockdown of Miz-1. Our data demonstrated that esophageal cancer has a cell cycle arrest pathway via Miz-1, p21 and cyclin D1.

Objective: Sinuleptolide, a soft corals-derived bioactive norditerpenoid, is a marine natural product with a potent anti-inflammatory effect. We evaluate the potential anti-oral cancer effects of sinuleptolide and investigate the possible mechanisms involved., Designs: Cell viability, cell cycle, apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and DNA damage analyses were performed., Results: In a cell viability assay, we found that sinuleptolide is dose-responsively antiproliferative against oral gingival cancer Ca9-22 cells but less harmful to normal human gingival fibroblast (HGF-1) cells (P<0.001). In cell cycle analysis, sinuleptolide induced subG1 accumulation at a higher dose and led to G2/M arrest of Ca9-22 cells (P<0.005). Apoptosis was significantly increased in sinuleptolide-treated Ca9-22 cells based on annexin V and poly(ADP-ribose) polymerase (PARP) expressions (P<0.05-0.0001). Based on flow cytometer analysis, sinuleptolide also induced the generation of ROS and decreased MMP in a dose-responsive manner (P<0.05-0.0001). DNA damage increased dose-responsively after sinuleptolide treatments (P < 0.001) based on comet and γH2AX assays., Conclusion: Sinuleptolide can induce an antiproliferation of oral cancer Ca9-22 cells involving apoptosis, oxidative stress and DNA damage, suggesting that sinuleptolide represents a potential chemotherapeutic drug for oral cancer treatment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

DNA repair plays a critical role in response to ionizing radiation (IR) and developing of radiotherapy induced normal tissue reactions. In our study, we investigated the association of radiotherapy related acute side effects, with X-ray repair cross complementing group 1 (XRCC1) and Poly (ADP-ribose) polymerase 1 (PARP1) DNA repair gene expression levels, their changes in protein expression and DNA damage levels in breast cancer patients. The study included 40 women with newly diagnosed breast cancer; an experimental case group (n=20) with acute side effects and the control group (n=20) without side effects. For gene and protein expression analysis, lymphocytes were cultured for 72 h and followed by in vitro 2 Gray (Gy) gamma-irradiation. For detection of DNA damage levels, lymphocytes were irradiated with in vitro 2 Gy gamma-rays and followed by incubation for 72 h. XRCC1 mRNA and protein expression levels were significantly higher in controls than in experimental cases (P=0.020). In terms of DNA damage levels, an increased frequency of micronucleus (MN) was observed in experimental cases versus controls, but this association was not significant (P=0.206). We also observed a significant negative correlation between MN frequency and XRCC1 protein levels in experimental (r=-0.469, P=0.037) vs control (r=-0.734, P<0.001). Our results suggested that decreased XRCC1 expression levels might be associated with the increased risk of therapeutic IR-related acute side effects in patients with breast cancer., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background & Objectives: The main goal when treating malignancies with radiation is to deprive tumour cells of their reproductive potential. One approach is to induce tumour cell apoptosis. This study was conducted to evaluate the ability of carbon ions ( [12] C) to induce apoptosis and cell cycle arrest in human HTB140 melanoma cells., Methods: In this in vitro study, human melanoma HTB140 cells were irradiated with the 62 MeV/n carbon ( [12] C) ion beam, having two different linear energy transfer (LET) values: 197 and 382 keV/μm. The dose range was 2 to 16 Gy. Cell viability was estimated by the sulforhodamine B assay seven days after irradiation. The cell cycle and apoptosis were evaluated 48 h after irradiation using flow cytometry. At the same time point, protein and gene expression of apoptotic regulators were estimated using the Western blot and q-PCR methods, respectively., Results: Cell viability experiments indicated strong anti-tumour effects of [12] C ions. The analysis of cell cycle showed that [12] C ions blocked HTB140 cells in G2 phase and induced the dose dependent increase of apoptosis. The maximum value of 21.8 per cent was attained after irradiation with LET of 197 keV/μm at the dose level of 16 Gy. Pro-apoptotic effects of [12] C ions were confirmed by changes of key apoptotic molecules: the p53, Bax, Bcl-2, poly ADP ribose polymerase (PARP) as well as nuclear factor kappa B (NFκB). At the level of protein expression, the results indicated significant increases of p53, NFκB and Bax/Bcl-2 ratio and PARP cleavage. The Bax/Bcl-2 mRNA ratio was also increased, while no change was detected in the level of NFκB mRNA., Interpretation & Conclusions: The present results indicated that anti-tumour effects of [12] C ions in human melanoma HTB140 cells were accomplished through induction of the mitochondrial apoptotic pathway as well as G2 arrest., Competing Interests: None.

Osteosarcoma is composed of tumor osteoblasts and bone-like tissues, with malignant tumors originating from osteogenesis organization. Osteosarcoma is a primary malignant bone tumor. Invasion and metastasis of osteosarcoma affect the prognosis of patients. However, effective therapeutic treatments remain to be identified. The aim of the present study was to investigate the possible inhibitory and apoptotic effects of ginkgetin in osteosarcoma cells. 3.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays were used to determine the effect ginkgetin exerted on the growth of osteosarcoma cells. Flow cytometry was used to determine cell apoptosis. STAT3 protein expression and activation of caspase-3/9 were measured using western blot analysis and the MTT and LDH assays, respectively. The results showed that ginkgetin inhibited cell growth and induced cell cytotoxicity in osteosarcoma cells in a dose-dependent manner. Treatment with ginkgetin significantly activated the apoptosis of osteosarcoma cells in a concentration-dependent manner. The anticancer activity of ginkgetin significantly inhibited STAT3 and promoted caspase-3/9 activation in osteosarcoma cells. The findings demonstrated that ginkgetin exerts growth inhibitory and apoptotic effects on osteosarcoma cells through the inhibition of STAT3 and activation of caspase-3/9.

Poly (ADP) ribose polymerase (PARP) inhibitors, first evaluated nearly a decade ago, are primarily used in malignancies with known defects in DNA repair genes, such as alterations in breast cancer, early onset 1/2 (BRCA1/2). While no specific mutations in BRCA1/2 have been reported in malignant peripheral nerve sheath tumors (MPNSTs), MPNST cells could be effectively targeted with a PARP inhibitor to drive cells to synthetic lethality due to their complex karyotype and high level of inherent genomic instability. In this study, we assessed the expression levels of PARP1 and PARP2 in MPNST patient tumor samples and correlated these findings with overall survival. We also determined the level of PARP activity in MPNST cell lines. In addition, we evaluated the efficacy of the PARP inhibitor AZD2281 (Olaparib) in MPNST cell lines. We observed decreased MPNST cell proliferation and enhanced apoptosis in vitro at doses similar to, or less than, the doses used in cell lines with established defective DNA repair genes. Furthermore, AZD2281 significantly reduced local growth of MPNST xenografts, decreased the development of macroscopic lung metastases, and increased survival of mice with metastatic disease. Our results suggest that AZD2281 could be an effective therapeutic option in MPNST and should be further investigated for its potential clinical use in this malignancy.

Background. Several investigators have reported increased levels of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme which plays an important role in the repair of damaged DNA, in cells exposed to extremely low dose ionizing radiation which does not cause measurable DNA damage. Objective. To examine whether exposure of the cells to nonionizing radiofrequency fields (RF) is capable of increasing messenger RNA of PARP-1 and its protein levels in mouse bone marrow stromal cells (BMSCs). Methods. BMSCs were exposed to 900 MHz RF at 120 μW/cm(2) power intensity for 3 hours/day for 5 days. PARP-1 mRNA and its protein levels were examined at 0, 0.5, 1, 2, 4, 6, 8, and 10 hours after exposure using RT-PCR and Western blot analyses. Sham-exposed (SH) cells and those exposed to ionizing radiation were used as unexposed and positive control cells. Results. BMSCs exposed to RF showed significantly increased expression of PARP-1 mRNA and its protein levels after exposure to RF while such changes were not observed in SH-exposed cells. Conclusion. Nonionizing RF exposure is capable of inducing PARP-1.

BRCA1/2-mutant cells are hypersensitive to inactivation of poly(ADP-ribose) polymerase 1 (PARP-1). We recently showed that inhibition of PARP-1 by NU1025 is strongly cytotoxic for BRCA1-positive BT-20 cells, but not BRCA1-deficient SKBr-3 cells. These results raised the possibility that other PARP-1 inhibitors, particularly those tested in clinical trials, may be more efficacious against BRCA1-deficient SKBr-3 breast cancer cells than NU1025. Thus, in the presented study the cytotoxicity of four PARP inhibitors under clinical evaluation (olaparib, rucaparib, iniparib and AZD2461) was examined and compared to that of NU1025. The sensitivity of breast cancer cells to the PARP-1 inhibition strongly varied. Remarkably, BRCA-1-deficient SKBr-3 cells were almost completely insensitive to NU1025, olaparib and rucaparib, whereas BRCA1-expressing BT-20 cells were strongly affected by NU1025 even at low doses. In contrast, iniparib and AZD2461 were cytotoxic for both BT-20 and SKBr-3 cells. Of the four tested PARP-1 inhibitors only AZD2461 strongly affected cell cycle progression. Interestingly, the anti-proliferative and pro-apoptotic potential of the tested PARP-1 inhibitors clearly correlated with their capacity to damage DNA. Further analyses revealed that proteomic signatures of the two studied breast cancer cell lines strongly differ, and a set of 197 proteins was differentially expressed in NU1025-treated BT-20 cancer cells. These results indicate that BT-20 cells may harbor an unknown defect in DNA repair pathway(s) rendering them sensitive to PARP-1 inhibition. They also imply that therapeutic applicability of PARP-1 inhibitors is not limited to BRCA mutation carriers but can be extended to patients harboring deficiencies in other components of the pathway(s)., (© 2015 Wiley Periodicals, Inc.)

Background: Cisplatin-resistant non-small cell lung cancer (NSCLC) cells are often characterized by alterations in vitamin B-related metabolic processes, including the overexpression and hyperactivation of poly(ADP-ribose) polymerase 1 (PARP1) and the downregulation of pyridoxal kinase (PDXK), correlating with elevated apoptosis resistance. Low PDXK expression is an established negative prognostic factor in NSCLC., Patients and Methods: We determined by immunohistochemistry the expression of PARP1 and the level of its product, poly(ADP-ribose) (PAR), in two independent cohorts of patients with resected NSCLC., Results: Intratumoral high levels (above median) of PAR (but not PARP1 protein levels) had a negative prognostic impact in both the training (92 stage I subjects) and validation (133 stage I and II subjects) cohorts, as determined by univariate and multivariate analyses. The simultaneous assessment of PAR and PDXK protein levels improved risk stratification., Conclusion: NSCLC patients with high intratumoral PARP1 activity (i.e. elevated PAR levels above median) and low PDXK expression (below median) had a dismal prognosis, while patients with low PARP1 activity and high PDXK expression had a favorable outcome. Altogether, these results underscore the clinical potential and possible therapeutic relevance of these biomarkers., (© The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Ewing's sarcoma is a malignant pediatric bone tumor with a poor prognosis for patients with metastatic or recurrent disease. Ewing's sarcoma cells are acutely hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibition and this is being evaluated in clinical trials, although the mechanism of hypersensitivity has not been directly addressed. PARP inhibitors have efficacy in tumors with BRCA1/2 mutations, which confer deficiency in DNA double-strand break (DSB) repair by homologous recombination (HR). This drives dependence on PARP1/2 due to their function in DNA single-strand break (SSB) repair. PARP inhibitors are also cytotoxic through inhibiting PARP1/2 auto-PARylation, blocking PARP1/2 release from substrate DNA. Here, we show that PARP inhibitor sensitivity in Ewing's sarcoma cells is not through an apparent defect in DNA repair by HR, but through hypersensitivity to trapped PARP1-DNA complexes. This drives accumulation of DNA damage during replication, ultimately leading to apoptosis. We also show that the activity of PARP inhibitors is potentiated by temozolomide in Ewing's sarcoma cells and is associated with enhanced trapping of PARP1-DNA complexes. Furthermore, through mining of large-scale drug sensitivity datasets, we identify a subset of glioma, neuroblastoma and melanoma cell lines as hypersensitive to the combination of temozolomide and PARP inhibition, potentially identifying new avenues for therapeutic intervention. These data provide insights into the anti-cancer activity of PARP inhibitors with implications for the design of treatment for Ewing's sarcoma patients with PARP inhibitors.

PARP1 and poly(ADP-ribosyl)ation (PARylation) have been shown to be essential for the initial steps of cellular reprogramming. However, the mechanism underlying PARP1/PARylation-regulated activation of pluripotency loci remains undetermined. Here, we demonstrate that CHD1L, a DNA helicase, possesses chromatin remodeling activity and interacts with PARP1/PARylation in regulating pluripotency during reprogramming. We found that this interaction is mediated through the interplay of the CHD1L macro-domain and the PAR moiety of PARylated-PARP1. Chromatin immunoprecipitation assays demonstrated the co-occupancy of CHD1L and PARP1 at Pou5f1, Nanog, and Esrrb pluripotency loci. Knockdown of CHD1L significantly blocked the binding activity of PARP1 at pluripotency loci and inhibited the efficiency of PARP1-driven reprogramming. Notably, we found that CHD1L-promoted reprogramming requires both a PARP1-interacting domain and DNA helicase activity, partly contributing to the chromatin-remodeling states of pluripotency loci. Taken together, these results identify CHD1L as a key chromatin remodeler involved in PARP1/PARylation-regulated early-stage reprogramming and pluripotency in stem cells., (© 2015 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Myosin VI (MYO6) is a unique member of the myosin superfamily, and almost no experimental studies link MYO6 to tumorigenesis of breast cancer. However, previous microarray data demonstrated that MYO6 was frequently overexpressed in breast cancer tissues. In this study, to further develop its role in breast cancer, endogenous expression of MYO6 was significantly inhibited in breast cancer ZR-75-30 and MDA-MB-231 cells using lentivirus-mediated RNA interference. Quantitative polymerase chain reaction and western blot were applied to detect the expression level of MYO6. Cell viability of both cell lines was measured by methylthiazol tetrazolium and colony formation assays. Besides, cell cycle assay was utilized to acquire the distribution information of cell phase. The results demonstrated that knockdown of MYO6 markedly reduced cell viability and colony formation, as well as suppressed cell cycle progression in breast cancer cells. The results suggested that MYO6 played a vital role in breast cancer cells and might provide useful information for diagnosis and therapy of human breast cancer in future.

Prostate cancer (PCa) displays infrequent point mutations, whereas genomic rearrangements are highly prevalent. In eukaryotes, the genome is compartmentalized into chromatin loop domains by the attachment to the nuclear matrix (NM), and it has been demonstrated that several recombination hot spots are situated at the base of loops. Here, we have characterized the binding between NM proteins and matrix attachment regions (MARs) in PCa. Nontumor and 44 PCa tissues were analyzed. More aggressive tumors were characterized by an increase in the complexity of the NM protein patterns that was synchronous with a decrease in the number of proteins binding the MAR sequences. PARP-1 was the protein that showed the most evident changes. The expression of the PARP-1 associated with NM increased and it was dependent on tumor aggressiveness. Immunohistochemical analysis showed that the protein was significantly overexpressed in tumor cells. To explore the role of PARP-1 in PCa progression, PCa cells were treated with the PARP inhibitor, ABT-888. In androgen-independent PC3 cells, PARP inhibition significantly decreased cell viability, migration, invasion, chromatin loop dimensions and histone acetylation. Collectively, our study provides evidence that MAR-binding proteins are involved in the development and progression of PCa. PARP could play a key role in the compartmentalization of chromatin and in the development of the more aggressive phenotype. Thus, PARP can no longer be viewed only as an enzyme involved in DNA repair, but that its role in chromatin modulation could provide the basis for a new therapeutic approach to the treatment of PCa., (© 2015 UICC.)

Condensed-bicyclic triazolo-thiadiazoles were synthesized via an efficient "green" catalyst strategy and identified as effective inhibitors of PTP1B in vitro. The lead compound, 6-(2-benzylphenyl)-3-phenyl-[1,2,4]triazolo[3][1,3,4]thiadiazole (BPTT) was most effective against human hepatoma cells, inhibits cell invasion, and decreases neovasculature in HUVEC and also tumor volume in EAT mouse models. This report describes an experimentally unidentified class of condensed-bicyclic triazolo-thiadiazoles targeting PTP1B and its analogs could be the therapeutic drug-seeds.

Background: PARP inhibitors have shown promising clinical results in cancer patients carrying BRCA1/2 mutations. Their clinical efficacy could logically be influenced by PARP1 protein levels in patient tumors., Methods: We screened three cohorts of patients with ovarian cancer, totaling 313 samples, and evaluated PARP1 protein expression by immunohistochemistry with further validation by western blotting., Results: We observed that up to 60 % of tumors showed little PARP1 protein expression. In serous ovarian tumors, comparing intratumoral PARP1 expression between chemo-naïve and post-chemotherapy patients revealed a decrease in intratumoral PARP1 following chemotherapy in all three cohorts (immunohistochemistry: p < 0.001, n = 239; western blot: p = 0.012, n = 74). The findings were further confirmed in a selection of matched samples from the same patients before and after chemotherapy., Conclusion: Our data suggest that patients should be screened for PARP1 expression prior to therapy with PARP inhibitors. Further, the observed reduction of intratumoral PARP1 post-chemotherapy suggests that treating chemo-naïve patients with PARP inhibitors prior to the administration of chemotherapy, or concurrently, might increase the responsiveness to PARP1 inhibition. Thus, a change in the timing of PARP inhibitor administration may be warranted for future clinical trials.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear protein that impacts DNA repair and apoptosis. Both experimental and ongoing clinical studies indicate that PARP-1 inhibitors are potent and promising anticancer agents. However, the outcome of treatment with PARP-1 inhibitors depends on the expression of PARP-1 protein in the tumor cells. This study aimed to assess PARP-1 expression in peripheral blood CD34+ leukemic cells before and after 12 hours of prednisone administration as well as the relation between PARP-1 expression and early treatment response to initial therapy and other prognostic factors (immunophenotype, age, initial peripheral blood white blood count [WBC], and risk factor group). The study comprised 43 children with de novo ALL. Cytospins of peripheral blood were stained with mouse anti-CD34-FITC and anti-PARP-1 antibody followed by goat anti-mouse APC-conjugated antibody. DNA was counterstained with PI (propidium iodide). Cellular fluorescence was measured by a laser scanning cytometer. Statistically significant differences in baseline PARP-1 expression with respect to early treatment response (good vs. poor), ALL immunophenotype (ALL B vs. ALL T), age (children < 1 years and > 6 years vs. children 1-6 years), initial WBC (< 20 000/µl vs. ≥ 20 000/µl), and risk factor group (SR vs. IR vs. HR) were not found. PARP-1 expression was increased 12 hours after treatment in poor early treatment responders, whereas it remained statistically unchanged with respect to ALL immunophenotype, age, initial WBC, risk factor group and early treatment response. The overexpression of PARP-1 in poor early treatment responders suggests that it may contribute to treatment failure in this group of children with ALL. Our observation - if confirmed by other studies - may form the rationale for administration of PARP inhibitors in selected subsets of ALL children.

Unlabelled: In Kaposi's sarcoma-associated herpesvirus (KSHV), poly(ADP-ribose) polymerase 1 (PARP-1) acts as an inhibitor of lytic replication. Here, we demonstrate that KSHV downregulated PARP-1 upon reactivation. The viral processivity factor of KSHV (PF-8) interacted with PARP-1 and was sufficient to degrade PARP-1 in a proteasome-dependent manner; this effect was conserved in murine gammaherpesvirus 68. PF-8 knockdown in KSHV-infected cells resulted in reduced lytic replication upon reactivation with increased levels of PARP-1, compared to those in control cells. PF-8 overexpression reduced the levels of the poly(ADP-ribosyl)ated (PARylated) replication and transcription activator (RTA) and further enhanced RTA-mediated transactivation. These results suggest a novel viral mechanism for overcoming the inhibitory effect of a host factor, PARP-1, thereby promoting the lytic replication of gammaherpesvirus., Importance: Gammaherpesviruses are important human pathogens, as they are associated with various kinds of tumors and establish latency mainly in host B lymphocytes. Replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is a central molecular switch for lytic replication, and its expression is tightly regulated by many host and viral factors. In this study, we investigated a viral strategy to overcome the inhibitory effect of poly(ADP-ribose) polymerase 1 (PARP-1) on RTA's activity. PARP-1, an abundant multifunctional nuclear protein, was downregulated during KSHV reactivation. The viral processivity factor of KSHV (PF-8) directly interacted with PARP-1 and was sufficient and necessary to degrade PARP-1 protein in a proteasome-dependent manner. PF-8 reduced the levels of PARylated RTA and further promoted RTA-mediated transactivation. As this was also conserved in another gammaherpesvirus, murine gammaherpesvirus 68, our results suggest a conserved viral modulation of a host inhibitory factor to facilitate its lytic replication., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Toll-like receptors (TLRs) are signaling receptors in the innate immune system that is specific immunologic response to systemic bacterial infection and injury. TLRs contribute to the initial induction of neuroinflammation in the CNS. In spinal cord injury (SCI) intricate immune cell interactions are triggered, typically consisting of a staggered multiphasic immune cell response, which can become deregulated. The present study aims to evaluate the role of TLR4 signaling pathway in the development of secondary damage in a mouse model of SCI using TLR4-deficient (TLR4-KO) mice such as C57BL/10ScNJ and C3H/HeJ mice. We evaluated behavioral changes, histological, immunohistochemistry and molecular assessment in TLR4-KO after SCI. SCI was performed on TLR4-KO and wild-type (WT) mice by the application of vascular clips (force of 24g) to the dura via a four-level T5-T8 laminectomy. Mice were sacrificed at 24h after SCI to evaluate the various parameters. SCI TLR4 KO mice developed severer hind limb motor dysfunction and neuronal death by histological evaluation, myeloid differentiation primary response 88 (Myd88) expression as well as an increase in nuclear factor NF-κB activity, tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels, glial fibrillary acidic protein (GFAP), microglia marker (CD11β), inducible nitric oxide synthases (iNOS), poly-ADP-ribose polymerase (PARP) and nitrotyrosine expression compared to WT mice. Moreover, the absence of TLR4 also caused a decrease in phosphorylated interferon regulatory transcription factor (p-IRF3) and interferon (IFN-β) release. In addition, SCI TLR4 KO mice showed in spinal cord tissues a more pronounced up-regulation of Bax and a down-regulation of Bcl-2 compared to SCI WT mice. Finally, we clearly demonstrated that TLR4 is important for coordinating post-injury sequel and in regulating inflammation after SCI., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Unlabelled: In this study, 3-nitropropionic acid (3-NPA) was separated and purified from endophytic fungi belonging to Phomopsis sp. and its cytotoxicity was determined by MTT assay. Treatment with 3-NPA for 24 h resulted in a dose-dependent apoptosis in MCF-7 cells. Through quantitative detection of the genes that are closely related to the Bcl-2 signalling pathway, there was an increased expression of p53 and Bax and a decreased expression of Bcl-2, which indicated apoptosis in these cells. Meanwhile, the overexpression of PARA (poly ADP-ribose polymerase) and apoptosis inducing factor (AIF) also suggested that 3-NPA induced cellular apoptosis through a caspase-3-independent pathway in caspase-3-deficient MCF-7 cells. The fermentation condition was also improved to produce more 3-NPA: glucose as a carbon source and yeast extract as a nitrogen source, fermentation for 8 days at 32°C and a solution environment of pH 5·0. Under these conditions, the yield of 3-NPA was increased to 529 mg l(-1) compared with 410 mg l(-1) under traditional fermentation conditions., Significance and Impact of the Study: 3-Nitropropionic acid is a mitochondrial inhibitor and has some useful bioactivities such as antibacterial activity. In this paper we found that 3-NPA also has obvious cytotoxicity, so we studied its antitumour activity and tried to determine the antitumour molecular mechanism, opening a new perspective for potential antitumour prodrug development. As 3-NPA is often obtained from natural products with a low yield, in order to overcome the disadvantage of an endophytic fungi source of 3-NPA, we optimized the fermentation conditions for 3-NPA in Phomopsis sp. to obtain the maximum production of 3-NPA., (© 2015 The Society for Applied Microbiology.)

Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at "difficult-to-replicate" sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3'-5' DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.

In this issue of Biochemical Journal, Chen and colleagues characterize an interaction between ACBD3 (acyl-CoA-binding domain-containing 3) protein and PARP [poly(ADP-ribose) polymerase]-1 through the activation of ERKs (extracellular-signal-regulated kinases). This study envisages a pathway through which ABCD3 translates enhanced fatty acid levels to ERK and consequently PARP-1 activation. The consequences of PARP-1 activation lead to cellular and tissue damage, implying that the ACBD3/PARP-1 pathway is an important pathway in lipotoxicity events., (© 2015 Authors; published by Portland Press Limited.)

NAD(+) plays essential roles in cellular energy homoeostasis and redox state, functioning as a cofactor along the glycolysis and citric acid cycle pathways. Recent discoveries indicated that, through the NAD(+)-consuming enzymes, this molecule may also be involved in many other cellular and biological outcomes such as chromatin remodelling, gene transcription, genomic integrity, cell division, calcium signalling, circadian clock and pluripotency. Poly(ADP-ribose) polymerase 1 (PARP1) is such an enzyme and dysfunctional PARP1 has been linked with the onset and development of various human diseases, including cancer, aging, traumatic brain injury, atherosclerosis, diabetes and inflammation. In the present study, we showed that overexpressed acyl-CoA-binding domain containing 3 (ACBD3), a Golgi-bound protein, significantly reduced cellular NAD(+) content via enhancing PARP1's polymerase activity and enhancing auto-modification of the enzyme in a DNA damage-independent manner. We identified that extracellular signal-regulated kinase (ERK)1/2 as well as de novo fatty acid biosynthesis pathways are involved in ACBD3-mediated activation of PARP1. Importantly, oxidative stress-induced PARP1 activation is greatly attenuated by knocking down the ACBD3 gene. Taken together, these findings suggest that ACBD3 has prominent impacts on cellular NAD(+) metabolism via regulating PARP1 activation-dependent auto-modification and thus cell metabolism and function., (© 2015 Authors; published by Portland Press Limited.)

Sulfiredoxin 1 (Srxn1), an endogenous antioxidant protein, plays an important neuroprotective role in cerebral ischemia. However, the exact mechanisms of action of Srxn1 in cerebral ischemia have not yet been fully elucidated. Therefore, in the present study, rat primary cortical astrocytes transfected with a lentiviral vector encoding short hairpin RNA (shRNA) were exposed to oxygen-glucose deprivation (OGD) for 4 h or to 100 µM hydrogen peroxide (H2O2) for 6 h, in order to construct an in vitro model of cerebral ischemia-induced damage. We found that following exposure to OGD or H2O2, the knockdown of Srxn1 resulted in a decrease in cell viability, as shown by MTS assay, an increase in cell damage, as shown by lactate dehydrogenase assay and an increase in cellular apoptosis, as shown by Hoechst 33342 staining and flow cytometry. Furthermore, we found that following exposure to OGD or H2O2, the knockdown of Srxn1 resulted in a decrease in mitochondrial transmembrane potential (Δψm) as indicated by JC-1 staining, an increase in the cytoplasmic expression of cytochrome c (Cyt.C), caspase-3, caspase-9, poly(ADP-ribose) polymerase (PARP) and Bax protein at the protein level, but a decrease in the expression of the anti-apoptotic Bcl-2 protein; these effects were tightly associated with the mitochondrial apoptotic pathway. However, we found that there was no obvious change in the intracellular calcium ([Ca2+]i) levels and caspase-12 expression following the knockdown of Srxn1. Taken together, the results from the present study demonstrate that Srxn1 protects primary rat cortical astrocytes from OGD- or H2O2-induced apoptosis and that involves the activation of the mitochondrial apoptotic pathway, which suggests that Srxn1 may be a potential target in the treatment of cerebral ischemia.

Activation of nuclear factor kappa-B (NF-κB) is implicated in drug resistant of lung cancer cells. Our previous data showed that thiacremonone inhibited activation of NF-κB. In the present study, we investigated whether thiacremonone enhanced susceptibility of lung cancer cells to a common anti-cancer drug paclitaxel by further inhibition of NF-κB. Thus, we used the threefold lower doses of IC50 values (50 μg/ml thiacremonone and 2.5 nM paclitaxel). We found that combination treatment with thiacremonone and paclitaxel was more susceptible (combination index; 0.40 in NCI-H460 cells and 0.46 in A549 cells) in cell growth inhibition of two types of lung cancer cell lines compared to a single agent treatment. Consistent with the combination effect on cancer cell growth inhibition, the combination treatment further induced apoptotic cell death and arrested the cancer cells in G2/M phase accompanied with a much lower expression of cdc2 and cyclin B1, and inhibited colony formation. Much more inactivation of NF-κB and greater expression of NF-κB target apoptosis regulated genes such as caspase-8 and PARPs were found by the combination treatment. Molecular model and pull down assay as well as MALDI-TOF analysis demonstrated that thiacremonone directly binds to p50. These data indicated that thiacremonone leads to increased apoptotic cell death in lung cancer cell lines through greater inhibition of NF-κB by the combination treatment with paclitaxel.

Poly (ADP-ribose) polymerase-1 (PARP-1) functions at the center of cellular stress and sways the immune system at several key points, thus modulates inflammatory diseases. The antiinflammatory properties of PARP-1 inhibitors have been demonstrated ameliorating effect in various neuroinflammatory disorders. It has been reported that there is a close relationship between the inflammatory processes and major depressive disorder. In the present study, we have elucidated the role of oxidative-nitrosative stress-PARP-1 pathway in lipopolysaccharide (LPS)-induced neurobehavioral and neurochemical alterations in mice. 3-Aminobenzamide (10 and 30mg/kg) and imipramine (10 and 30mg/kg) were administered once daily for 14days. Mice were challenged with LPS (1mg/kg, i.p.) 30min after drug administration on the 14th day. The mRNA expression level of PARP-1 (12h after LPS injection) in the hippocampus was measured through quantitative real-time PCR. All the behavioral and biochemical parameters were assessed at 24h after LPS injection. The expression level of PARP-1mRNA was found significantly up-regulated in the hippocampus at 12h after LPS administration. Results showed that the LPS-challenged mice exhibited an increase in immobility time seen in forced swimming test and tail suspension test. LPS increased the levels of proinflammatory cytokines and oxido-nitrosative stress parameters in the hippocampus. However, pretreatment with 3-aminobenzamide (30mg/kg) significantly reversed the LPS-induced alterations in behavioral parameters, proinflammatory cytokines, oxidative-nitrosative stress and PARP-1 mRNA levels. Imipramine failed to prevent the up-regulation of PARP-1 induced by LPS administration. Our results emphasized that oxidative-nitrosative stress-PARP-1 cascade can play a key role in LPS-induced neurobehavioral and neurochemical anomalies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: The purpose of this study was to investigate the incidence of DNA damage during postnatal development of the retina and the relationship between DNA damage and cell death., Methods: DNA damage in the developing postnatal retina of C57BL/6 mice was assessed by determining the amounts of 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is indicative of DNA oxidation and related to the formation of DNA single-strand breaks (SSBs), and phosphorylated histone H2AX (γ-H2AX), a marker of DNA double-strand breaks (DSBs). Poly(ADP-ribose) polymerase (PARP) activation was measured by ELISA and Western blotting. The location of γ-H2AX-positive and dying cells was determined by immunofluorescence and TUNEL assays., Results: Oxidative DNA damage was maintained at low levels during high PARP activation between postnatal days 0 (P0) and P7. Phosphorylated histone H2AX gradually increased between P0 and P14 and decreased thereafter. Phosphorylated histone H2AX-positive cells with cell death morphology or TUNEL positivity were more abundant at P7 than at P14., Conclusions: Oxidative DNA damage in postnatal retina increases during development. It is low during the first postnatal week when PARP-1 activity is high but increases thereafter. The rise in DSBs when PARP activity is downregulated may be attributable to accumulated oxidative damage and SSBs. At P7 and P14, γ-H2AX-positive cells are repairing naturally occurring DNA damage, but some are dying (mostly at P7), probably due to an accumulation of irreparable DNA damage., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)

Background: Eicosapentaenoic acid (EPA) suppresses the proliferation of cell lines derived from colon, pancreatic, breast and other cancers. Few reports have described the effect of EPA on esophageal cancer cell lines., Materials and Methods: We investigated the effect of EPA on the proliferation of the esophageal squamous cell carcinoma cell lines TE11 and KYSE180 with a WST-1 assay. Apoptosis was evaluated with a DNA fragmentation assay. Levels of apoptosis-related proteins (caspase-3, -7, -9 and poly (ADP-ribose) polymerase (PARP)) and cleaved caspase-3, -7, -9 and PARP were evaluated by western blot analysis., Results: After exposure to EPA for 24 h, KYSE180 and TE11 cell proliferation was suppressed in a dose-dependent manner (p<0.05). In addition, caspase -3, -7, -9 and PARP were activated. EPA (0.1 μM, 1 μM, 10 μM) induced apoptosis in a dose-dependent manner, as detected by the DNA fragmentation assay., Conclusion: EPA shows potential as a new treatment for esophageal cancer., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Emodin, an extracted natural compound from the root and rhizome of Rheum palmatum L, has been shown to have multiple biological activities including anticancer functions in previous studies. In this study, we investigated the anti-leukemic activity of emodin alone or emodin in the presence all-trans retinoic acid (ATRA) in acute myeloid leukemia (AML) cells and the potential signaling pathway involved. We demonstrated that emodin could significantly enhance the sensitivity to ATRA and present additive differentiation-inducing effects in AML cell line NB4 cells and, especially, in NB4-derived ATRA-resistant MR2 cells. Further study showed that increasing dose of emodin could effectively induce growth inhibition and apoptotic effects in both cell lines as well as in primary leukemic cells from AML patients. Moreover, the apoptotic induction in AML cells was associated with the activation of caspase cascades involving caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP) cleavage. In addition, leukemic cell response to emodin stimuli in vitro was observed through the decreased expression levels of Bcl-2 and retinoic acid receptor α (RARα). Importantly, emodin was demonstrated as a new inhibitor of PI3K/Akt in AML cells, even in primary AML cells. It inhibited Akt phosphoration (p-Akt) at Ser473 as efficiently as mTOR at Ser2448. Consistently, it exerted suppression effects on the phosphoration of mTOR downstream targets, 4E-BP1 and p70S6K. Taken together, these findings indicate that emodin might be developed as a promising anti-leukemic agent to improve the patient outcome in AML.

GMX1778 and its prodrug GMX1777 represent a new class of cancer drugs that targets nicotinamide phosphoribosyltransferase (NAMPT) as a new strategy to interfere with biosynthesis of the key enzymatic cofactor NAD, which is critical for a number of cell functions, including DNA repair. Using a genome-wide synthetic lethal siRNA screen, we identified the folate pathway-related genes, deoxyuridine triphosphatase and dihydrofolate reductase, the silencing of which sensitized non-small cell lung carcinoma (NSCLC) cells to the cytotoxic effects of GMX. Pemetrexed is an inhibitor of dihydrofolate reductase currently used to treat patients with nonsquamous NSCLC. We found that combining pemetrexed with GMX1777 produced a synergistic therapeutic benefit in A549 and H1299 NSCLC cells in vitro and in a mouse A549 xenograft model of lung cancer. Pemetrexed is known to activate PARPs, thereby accelerating NAD consumption. Genetic or pharmacologic blockade of PARP activity inhibited this effect, impairing cell death by pemetrexed either alone or in combination with GMX1777. Conversely, inhibiting the base excision repair pathway accentuated NAD decline in response to GMX and the cytotoxicity of both agents either alone or in combination. These findings provide a mechanistic rationale for combining GMX1777 with pemetrexed as an effective new therapeutic strategy to treat nonsquamous NSCLC., (©2014 American Association for Cancer Research.)

Long-term memory (LTM) formation requires new protein synthesis and new gene expression. Based on our work in Aplysia, we hypothesized that the rRNA genes, stimulation-dependent targets of the enzyme Poly(ADP-ribose) polymerase-1 (PARP-1), are primary effectors of the activity-dependent changes in synaptic function that maintain synaptic plasticity and memory. Using electrophysiology, immunohistochemistry, pharmacology and molecular biology techniques, we show here, for the first time, that the maintenance of forskolin-induced late-phase long-term potentiation (L-LTP) in mouse hippocampal slices requires nucleolar integrity and the expression of new rRNAs. The activity-dependent upregulation of rRNA, as well as L-LTP expression, are poly(ADP-ribosyl)ation (PAR) dependent and accompanied by an increase in nuclear PARP-1 and Poly(ADP) ribose molecules (pADPr) after forskolin stimulation. The upregulation of PARP-1 and pADPr is regulated by Protein kinase A (PKA) and extracellular signal-regulated kinase (ERK)--two kinases strongly associated with long-term plasticity and learning and memory. Selective inhibition of RNA Polymerase I (Pol I), responsible for the synthesis of precursor rRNA, results in the segmentation of nucleoli, the exclusion of PARP-1 from functional nucleolar compartments and disrupted L-LTP maintenance. Taken as a whole, these results suggest that new rRNAs (28S, 18S, and 5.8S ribosomal components)--hence, new ribosomes and nucleoli integrity--are required for the maintenance of long-term synaptic plasticity. This provides a mechanistic link between stimulation-dependent gene expression and the new protein synthesis known to be required for memory consolidation.

Background and Aims: Aberrant expression of PARP-1 has been reported in various human malignancies and was involved in the progression and metastasis of cancers. However, little is known about PARP-1 expression in prostate cancer (PCa). This study aimed to investigate the expression of PARP-1 and its active polymer poly(ADP-ribose) (PAR) in PCa and benign prostatic hyperplasia (BPH) tissues from Chinese patients., Methods: The expression of PARP-1 and PAR in PCa and benign prostate hyperplasia tissues was assessed by immunohistochemistry in 78 PCa patients and 49 BPH patients. The relationship between the expression of PARP-1 or PAR and clinicopathological parameters in PCa patients was also analyzed., Results: Both the positive and strong positive expression rates of PARP-1 in PCa tissues were significantly higher than those in BPH tissues. Although spearman correlations analysis showed the over-expression of PARP-1 and PAR in PCa tissues was not correlated with age, serum PSA level and Gleason scores (GS), an increasing trend was observed between over-expression of PARP-1 or PAR and the PSA levels (TPSA >20 vs TPSA ≤20) or GS grade (GS ≥8 vs GS ≤6)., Conclusion: PARP-1 and PAR expression is markedly elevated in PCa than that in BPH tissues, which may implicate that PARP-1 and PAR are involved in the development of PCa, and the possible expansion in the use of poly(ADP-ribose) polymerase inhibitors for targeting therapy of PCa in select patients alone or combined with chemotherapy or radiation.

We previously demonstrated that the deletion of the poly(ADP-ribose)polymerase (Parp)-1 gene in mice enhances oxidative metabolism, thereby protecting against diet-induced obesity. However, the therapeutic use of PARP inhibitors to enhance mitochondrial function remains to be explored. Here, we show tight negative correlation between Parp-1 expression and energy expenditure in heterogeneous mouse populations, indicating that variations in PARP-1 activity have an impact on metabolic homeostasis. Notably, these genetic correlations can be translated into pharmacological applications. Long-term treatment with PARP inhibitors enhances fitness in mice by increasing the abundance of mitochondrial respiratory complexes and boosting mitochondrial respiratory capacity. Furthermore, PARP inhibitors reverse mitochondrial defects in primary myotubes of obese humans and attenuate genetic defects of mitochondrial metabolism in human fibroblasts and C. elegans. Overall, our work validates in worm, mouse, and human models that PARP inhibition may be used to treat both genetic and acquired muscle dysfunction linked to defective mitochondrial function., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The aim of the present study was to investigate and compare the effects of diferuloylmethane (curcumin) and diphenyldifluoroketone (EF-24) on cell growth and apoptosis induction in human osteogenic sarcoma cells. This was examined by MTT assay, nuclear DAPI staining, caspase-activation assay, flow cytometry analysis and immunoblotting in Saos2 human osteogenic sarcoma cells. Curcumin and EF-24 inhibited the growth of Saos2 cells in a dose-dependent manner. The apparent potency of EF-24 was more than 3-fold higher that of curcumin. Treatment with curcumin or EF-24 resulted in nuclear condensation and fragmentation in the cells. The caspase-3/-7 activities were detected in living cells treated with curcumin or EF-24. Flow cytometry showed that the rate of apoptosis was increased by curcumin and EF-24 compared to the control. Curcumin and EF-24 promoted the proteolytic cleavages of procaspase-3/-7/-8/-9 with increases in the amount of cleaved caspase-3/-7/-8/-9. The curcumin- or EF-24-induced apoptosis in the Saos2 cells was mediated by the expression of Fas and activation of caspase-8, caspase-3 and poly(ADP-ribose) polymerase. Immunoblotting revealed the Bid and Bcl-2 proteins to be downregulated, and truncated-Bid, Bax and p53 proteins to be upregulated by curcumin and EF-24. Curcumin and EF-24 increased the Bax/Bcl-2 ratio significantly. These results suggest that the curcumin and EF-24 inhibit cell proliferation and induce apoptotic cell death in Saos2 human osteogenic sarcoma cells via both the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway, and may have potential properties for anti-osteosarcoma drug discovery.

PARP-1 plays an important role in DNA damage repair and maintaining genome integrity by repairing DNA single-strand breaks (SSBs) by base excision repair (BER). The aim of the present study was to examine the expression of PARP-1 in breast cancer (BC) patients and to assess the relationship between the subcellular localization of this protein and clinicopathological characteristics. The reactivity of PARP-1 was analyzed by immunohistochemistry in a homogeneous group of 83 stage II ductal BC patients with a 15-year follow-up. Immunostaining of PARP-1 was also evaluated in 4 human BC cell lines and resistance prediction profile for 11 anticancer agents was performed using 3 models of drug-resistant cell lines. Nuclear-cytoplasmic expression (NCE) was associated with shorter overall survival, which was not statistically significant during the 10-year follow-up but became statistically significant after 10 years of observation, during the 15-year follow-up (P=0.015). Analysis performed in subgroups of patients with (N+) and without (N-) nodal metastases showed that NCE was associated with poor clinical outcome in N- patients (P=0.017). Multivariate analysis confirmed a significant impact of NCE on unfavorable prognosis in N- early BC. The presence of PARP-1 NCE may be a new potential unfavorable prognostic factor in lymph node- negative early BC.