Repair Enzyme Poly ADP-ribose Polymerase Research Articles

Sevoflurane Preconditioning Rescues PKMζ Gene Expression from Broad Hypoxia-Induced mRNA Downregulation Correlating with Improved Neuronal Recovery

Hypoxia due to stroke is a major cause of neuronal damage, leading to loss of cognition and other brain functions. Sevoflurane preconditioning improves recovery after hypoxia. Hypoxia interferes with protein expression at the translational level; however, its effect on mRNA levels for neuronal protein kinase and anti-apoptotic genes is unclear. To investigate the link between sevoflurane preconditioning and gene expression, hippocampal slices were treated with 4% sevoflurane for 15 min, a 5 min washout, 10 min of hypoxia, and 60 min of recovery. We used quantitative PCR to measure mRNA levels in the CA1 region of rat hippocampi. The mRNA levels for specific critical proteins were examined, as follows: Protein kinases, PKCγ (0.22), PKCε (0.38), and PKMζ (0.55) mRNAs, and anti-apoptotic, bcl-2 (0.44) and bcl-xl (0.41), were reduced 60 min after hypoxia relative to their expression in tissue not subjected to hypoxia (set to 1.0). Sevoflurane preconditioning prevented the reduction in PKMζ (0.88 vs. 1.0) mRNA levels after hypoxia. Pro-apoptotic BAD mRNA was not significantly changed after hypoxia, even with sevoflurane preconditioning (hypoxia 0.81, sevo hypoxia 0.84 vs. normoxia 1.0). However, BAD mRNA was increased by sevoflurane in non-hypoxic conditions (1.48 vs. 1.0), which may partially explain the deleterious effects of volatile anesthetics under certain conditions. The DNA repair enzyme poly ADP-ribose polymerase 1 (PARP-1) was increased by sevoflurane in tissue not subjected to hypoxia (1.23). PARP-1 mRNA was reduced in untreated tissue after hypoxia (0.21 vs. 1.0); sevoflurane did not improve PARP-1 after hypoxia (0.27). Interestingly, the mRNA level of the cognitive kinase PKMζ, a kinase essential for learning and memory, was the only one protected against hypoxic downregulation by sevoflurane preconditioning. These findings correlate with previous studies that found that sevoflurane-induced improvement of neuronal survival after hypoxia was dependent on PKMζ. Maintaining mRNA levels for critical proteins may provide an important mechanism for preserving neuronal function after stroke.

Effect of ethyl pyruvate on mitochondrial dynamics of lipopolysaccharide-induced human kidney-2 cells

To examine the effects of ethyl pyruvate (EP) on mitochondrial dynamics and cell apoptosis in lipopolysaccharide (LPS)-induced human kidney-2 (HK-2) cells. HK-2 cells were divided into three groups: HK-2 cells were challenged with LPS (800 μg/L) for 24 hours as LPS group, or LPS mixed with EP (0.25 mmol/L) for 24 hours as EP group. Cells were incubated with normal saline for 24 hours as control group. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and intracellular adenosine triphosphate (ATP) were detected by enzyme linked immunosorbent assay (ELISA). JC-1 staining and Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) assays were used to evaluate mitochondrial membrane potential and cell apoptosis, respectively. Western Blot was used to evaluate the protein expressions of mitochondrial dynamics, including death-associated protein kinase 2 (DAPK-2), mitofusin (Mfn-1 and Mfn-2), and apoptotic associated biomarkers, including caspase-3, caspase-9, Bcl-2, Bcl-xL, cytochrome C (Cyt C), and DNA repair enzyme poly ADP-ribose polymerase (PARP). Compared with the NC group, MDA, IL-6, TNF-α of LPS group were significantly increased, the expression of SOD, mitochondrial membrane potential and ATP level were significantly decreased, the expression of mitochondrial fission protein DAPK-2 was significantly increased, and mitochondrial fusion proteins Mfn-1 and Mfn-2 were significantly decreased, cell apoptosis and apoptotic protein caspase-3, caspase-9 and Cyt C were increased, and anti-apoptotic protein Bcl-2, Bcl-xL, PARP were significantly decreased. Compared with the LPS group, the oxidative activities and inflammatory factors above were inhibited in EP group [MDA (μmol/L): 12.35±2.21 vs. 45.95±1.76, SOD (kU/L): 54.68±1.42 vs. 40.73±1.60, IL-6 (ng/L): 67.87±2.61 vs. 338.92±20.91, TNF-α (ng/L): 19.23±1.80 vs. 180.69±6.51], mitochondrial membrane potential and ATP level were significantly increased [mitochondrial membrane potential: (99.43±0.25)% vs. (69.40±0.75)%, ATP (×106 RLU): 0.19±0.01 vs. 0.12±0.05], the expression of mitochondrial fission protein was significantly decreased (DAPK-2/β-actin: 0.03±0.01 vs. 0.61±0.02), mitochondrial fusion proteins were significantly increased (Mfn-1/β-actin: 0.43±0.04 vs. 0.17±0.01, Mfn-2/β-actin: 0.201±0.004 vs. 0.001±0.001), percentage of cell apoptosis was significantly decreased [(5.25±0.17)% vs. (34.42±0.64)%], the expressions of apoptotic proteins were significantly decreased (caspase-3/β-actin: 0.25±0.15 vs. 1.76±0.01, caspase-9/β-actin: 0.09±0.02 vs. 1.52±0.12, Cyt C/β-actin: 0.001±0.001 vs. 0.350±0.030), and the expressions of anti-apoptotic proteins and PARP were significantly increased (Bcl-2/β-actin: 0.500±0.010 vs. 0.009±0.004, Bcl-xL/β-actin: 0.550±0.010 vs. 0.009±0.001, PARP/β-actin: 0.94±0.01 vs. 0.16±0.13), with statistically significant differences (all P < 0.05). There are enhanced mitochondrial fission and diminished mitochondrial fusion in LPS-induced HK-2 cells. EP can protect mitochondria functions by regulate mitochondrial dynamics, and reducethe apoptosis of LPS-induced HK-2 cells.

Abstract 3933: NADPH oxidases 4 inhibition using a hyaluronic acid nanoparticle drug delivery system sensitized therapeutic response to chemo and radiotherapy in drug resistant breast cancer

Abstract Objectives: Resistance to therapy is the major unmet challenge in clinical management of breast cancer. NADPH oxidases 4 (NOX4) is a critical NADPH oxidase subunit because it constitutively reacts with hydrogen peroxide (H2O2), generating ROS that is associated with tumorigenesis, metastasis, and invasion. A high level of NOX4 activity has been associated with drug resistance in human cancers. To improve the therapeutic efficacy, we have developed a hyaluronic acid nanoparticle (HANP) carrying a NOX4 inhibitor and examined the anti-tumor effect in combination with radiotherapy (RT) or chemotherapy. Methods: Biodegradable polymeric HANP was synthesized and encapsulated with GKT831 (HANP/GKT831), a novel inhibitor of NOX4. Targeted delivery of GKT831 with HANP was first studied by optical imaging in an orthotopic human breast cancer patient tissue derived xenograft (PDX) model. Subsequently, the ability of HANP/GKT831 in inhibition of tumor cells and sensitization of tumor cells to chemotherapy and RT was evaluated in breast PDX models following intravenous administration of HANP/GKT831 (containing 5 mg/kg equivalent dose of GKT831) in combination with Doxorubicin (DOX, 5 mg/kg) and RT (2 Gy) for 5 times, once per week. Furthermore, mechanism of NOX4 inhibition on improving therapeutic response in breast cancer was investigated. Results: We have developed HANP/GKT831 with drug loading efficiency of 15% (w/w) determined by high performance liquid chromatography (HPLC). The selective accumulation of HANP/GKT831 in breast PDX tumors following systemic delivery was demonstrated by optical imaging. Following five treatments, nude mice bearing breast PDX tumors that received HANP/GKT831 in combination with DOX or RT had significant stronger tumor growth inhibition compared with either DOX or RT alone (73.71±13.87% and 79.33±19%, respectively) as well as the non-treatment control group (86.33±16.25% and 78.15±14.27%, respectively). Examination of tumor tissues revealed that the level of poly (ADP-ribose) polymerase (PARP) expression was reduced in tumors treated with HANP/GKT831 in combination of RT or DOX, which induces cell death through DNA damage. Conclusions: We have developed a polymeric HANP carrying a NOX4 inhibitor (HANP/GKT831). We found that HANP/GKT831 could be efficiently delivered into tumors following systemic administration and significantly enhanced tumor response to Dox treatment or radiotherapy in two multi-drug resistant breast PDX tumor models. Therefore, HANP/GKT831 is a promising therapy agent for targeted therapy of breast cancer. Currently, mechanism of the effect on chemo- and radiotherapy sensitization is under investigation. One of the possible mechanisms is the downregulation of DNA-damage repairing functions, such as downregulation of DNA damage repair enzyme PARP. Citation Format: Lei Zhu, Yi Zhao, Wei Ping Qian, Dazhi Wang, Bing-Hua Jiang, Lily Yang. NADPH oxidases 4 inhibition using a hyaluronic acid nanoparticle drug delivery system sensitized therapeutic response to chemo and radiotherapy in drug resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3933.

Abstract B16: Cells containing mutations in the cohesin component, STAG2, are sensitive to PARP inhibition

Abstract Synthetic lethality (SL) has been proposed as a strategy to specifically target cells harboring cancer mutations while leaving unmutated, non-cancer cells unaffected, thus minimizing drug side effects. One genetic vulnerability common to many types of cancer is chromosome instability (or CIN). CIN can lead to increased genetic mutation and aneuploidy in cancer cells thereby distinguishing them from their normal neighbors and making it a possible candidate phenotype to target by SL. Here, we focus on the multifunctional cohesin complex that is critical for accurate chromosome segregation and genome stability because it tethers sister chromatids together after DNA replication and maintains cohesion until their separation in anaphase. Although several subunits of cohesin have been shown to be mutated in tumors, recent data identifies a cohesin component, STAG2 (Stromal Antigen 2 or SA-2) as a highly mutated gene in several types of cancer including Ewing's sarcoma, melanoma and glioblastomas (GBMs). As a previous study in our lab had shown that knockdown of any one of the core cohesion subunits SMC1A, SMC3 or RAD21 can render cells sensitive to the poly ADP-ribose polymerase (PARP) inhibitor olaparib, we sought to determine if cell lines harboring cancer-specific STAG2 mutations showed similar sensitivity. Using paired GBM cell lines containing either a truncated STAG2 or wild-type STAG2 knock-in, we found that STAG2 mutation status was, indeed, associated with significantly decreased proliferation in the presence of PARP inhibition. Interestingly, olaparib treatment led to an accumulation of cells in the G2 phase of the cell cycle and an increase in DNA damage markers. Furthermore, in the presence of increasing concentrations of olaparib, a higher percentage of STAG2-mutated cells had micronuclei and fragmented nuclei compared to their wild-type STAG2 knock-in counterparts. Taken together, these data suggest that the DNA repair enzyme PARP may be a potential therapeutic SL target for tumors harboring mutations in STAG2. Citation Format: Melanie L. Bailey, Nigel J. O'Neil, Derek M. van Pel, Phil Hieter. Cells containing mutations in the cohesin component, STAG2, are sensitive to PARP inhibition. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B16.

Elevated levels of DNA repair enzymes and antioxidative enzymes by (+)-catechin in murine microglia cells after oxidative stress

(+)-Catechin possesses a broad range of pharmacological properties, including antioxidative effect. However, little is reported on the mechanism by which (+)-catechin protects microglia cells from DNA damage by oxidative stress. In this study, TUNEL assay and DNA electrophorysis indicated that (+)-catechin markedly blocked DNA fragmentation and apoptosis of microglia cells by tBHP exposure. A potent antioxidative effect of (+)-catechin was confirmed by comparison with a putative antioxidant agent, N-acetylcysteine at the lower doses. Furthermore, the increased intracellular ROS by tBHP exposure were scavenged by elevated activities of catalase (CAT) and superoxide dismutase (SOD) after (+)-catechin treament. (+)-Catechin partially inhibited the activation of caspase-3, thereby both cleavage of poly (ADP-ribose) polymerase (PARP) and degradation of inhibitor of caspase-activated DNase (ICAD) were effectively abolished. In addition, the expression of PARP for repair of impaired DNA was significantly increased by (+)-catechin treatment. Taken together, these data suggest that protective effects of (+)-catechin against oxidative DNA damage of microglia cells is exerted by the increased expression of DNA repair enzyme PARP and antioxidant enzyme activities.

Delta 9-tetrahydrocannabinol induces the apoptotic pathway in cultured cortical neurones via activation of the CB1 receptor.

Delta 9-tetrahydrocannabinol, the principal psychoactive component of marijuana, exerts a variety of effects on the CNS, including impaired cognitive function and neurobehavioural deficits. The mechanisms underlying these neuronal responses to tetrahydrocannabinol are unclear but may involve alterations in neuronal viability. Tetrahydrocannabinol has been shown to influence neuronal survival but the role of the cannabinoid receptors in the regulation of neuronal viability has not been fully clarified. In this study we demonstrate that tetrahydrocannabinol promotes the release of cytochrome c, activates caspase-3, promotes cleavage of the DNA repair enzyme poly-ADP ribose polymerase and induces DNA fragmentation in cultured cortical neurones. These effects of tetrahydrocannabinol were completely abrogated by the CB(1) receptor antagonist AM-251. The findings of this study demonstrate that tetrahydrocannabinol induces apoptosis in cortical neurones in a manner involving the CB1 subtype of cannabinoid receptor.

Effects of free radicals on ciliary movement in the human nasal epithelial cells

Objective: There have been few reports on the effects of free oxygen radicals on ciliary mobility of nasal respiratory epithelial cells. The aim of this study was to determine the effects of free radicals and antioxidants on human nasal epithelial cells (HNECs) using video-computerized analysis. Methods: Human nasal epithelial cells were obtained from the nasal cavity of normal volunteers. Ciliary beat frequency (CBF) was calculated as the mean value of ten randomly selected cells. The proportion of the area with normal CBF (above 8 Hz) was calculated from 10 randomly selected sites per specimen. Free radicals were produced by xanthine–xanthine oxidase enzymatic system. The generation of free radicals was confirmed by chemoilluminometer. CBF and the proportion of the area with normal CBF were measured at every 5 min for 30 min after the addition of enzyme. For the evaluation of the antioxidant effects on free radical-mediated ciliary slowing in HNECs, cells were incubated in superoxide dismutase solution (300 unit/ml) for 30 min and 3-aminobenzamide (5 mM). Results: Superoxide produced by 0.4 mM xanthine and 400 miliunit/ml xanthine oxidase decreased CBF (7.71±1.91 Hz). A total of 2 min later, ciliary slowing was evident (3.87±1.10 Hz). Regarding the changes in proportion of epithelial area that showed normal CBF experimental group showed a significant decrease in percentage of epithelial area with normal CBF over time. Superoxide dismutase prevented ciliary slowing (8.76±0.99 Hz). Moreover, 3-aminobenzamide, an inhibitor of the DNA repair enzyme poly-ADP ribose polymerase, prevented inhibition of CBF (8.32±0.61 Hz). Conclusions: These results suggest that oxygen-mediated damage to DNA may be the mechanism of the deterioration effects of oxygen radicals on the ciliated respiratory nasal epithelium.