Cleaved Poly Adenosine Diphosphate-ribose Research Articles

Pitavastatin induces caspase-mediated apoptotic death through oxidative stress and DNA damage in combined with cisplatin in human cervical cancer cell line.

Pitavastatin (PITA) is a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor to treat hypercholesterolemia and in recent studies is focused that its potential anti-cancer effect. This study was aimed to elucidate the effect of PITA alone and in combination with cisplatin on cervical cancer cells (HeLa) in vitro. Cytotoxicity of PITA (5-200 μM) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red uptake (NRU) assays for 24, 48, and 72 h. Cell apoptosis and cell cycle analyses were performed in flow cytometry (0.1-100 μM). The evaluation of genotoxic effects and oxidative DNA damage of PITA (2-200 μM) were performed with standard comet assay, formamidopyrimidine glycosylase (fpg)-modified comet assay, and reactive oxygen species (ROS) activation in HeLa cells. PITA alone reduced cell viability in a dose-dependent manner (20-200, 20-200, and 5-200 μM for 24, 48, and 72 h, respectively, in MTT). The combined treatment of PITA with cisplatin resulted in significantly greater inhibition of cell viability. ROS and DNA damage increased significantly at 100 μM for 4 h and 20 μM for 24 h, respectively. PITA-induced apoptosis, an increased proportion of sub G1 cells, was monitored, and also, it increased the expression of active caspase-9 and caspase-3 and upregulated cleaved poly adenosine diphosphate ribose polymerase (PARP) by western blotting and caspase 3/8/9 multiple assay kit. We conclude that PITA can be used to efficiently cervical cancer studies, and promising findings have been obtained for further studies.

Kolaviron offered a novel cardioprotection against ischemic/reperfusion injury by modulation of Akt/protein kinase B (PKB)/p38MAPK/Caspase 3/poly‐ADP‐ribose polymerase signaling pathway

Free radical generation and oxidative stress have been implicated in the pathogenesis and pathophysiology of ischemic/reperfusion injury. Kolaviron is a bioflavonoid with arrays of biological and pharmacological activities. The study was designed to investigate the modulatory role of Kolaviron on ischemic/reperfusion injury in experimental animal models. Twenty male Wistar albino rats were randomly divided into two groups of ten rats per group: Group 1 received corn oil as a vehicle (Control) and rats in Group 2 were pre‐treated with Kolaviron at 200 mg/kg for 4 weeks. After 4 weeks of Kolaviron administration, hearts were excised and mounted on the working heart perfusion system. Western blot analysis for protein expressions was carried out on frozen heart samples. Results revealed significant (p < 0.05) reduction in the activity of cardiac catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)with concomitant reduction in oxygen radical absorbance capacity (ORAC) in ischemic rat heart of Control compared to group pre‐treated with Kolaviron. Similarly, intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) contents were significantly elevated in control compared to Kolaviron pre‐treated rats. Western blot showed that pre‐treatment with Kolaviron significantly increased total Akt/protein kinase B (PKB), phosphorylated Akt/PKB at serine 473 with concomitant significant reduction in the expressions of p38 mitogen‐activated protein kinase (p38MAPK), Caspase 3, and cleaved poly adenosine diphosphate ribose polymerase (PARP). Taken together, Kolaviron offered significant cardioprotection via free radical scavenging activity and upregulation of pro‐survival signaling pathway.

Expression and significance of MAPK/ERK in the specimens and cells of epithelial ovarian cancer

Objective: To detect phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) protein expression in epithelial ovarian cancer and cell lines, and to examine the effects of mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor AZD6244 on cell proliferation, apoptosis as well as cell cycle of ovarian cancer cells. To explore the function and significance of MAPK/extracellular signal-regulated kinase (ERK) signaling pathway in the development of ovarian cancer. Methods: (1) A total of 104 cases of patients with ovarian cancer who accepted the treatment of gynecological surgery and being confirmed by pathological examination in First Affiliated Hospital, Dalian Medical University from January 2004 to December 2013 were selected. The expressions of p-ERK1/2 protein were detected by immunohistochemistry in ovarian cancer specimens, and the relationship between the expressions of p-ERK1/2 and the clinical features of patients was analyzed. (2) p-ERK1/2 and other related proteins were determined by western blot in various ovarian cancer cells, including SKOV3, OV2008, C13, A2780S, A2780CP, OVCAR4, OVCAR5, OVCAR8 and CAOV3 treated with or without MEK inhibitor. The cellular proliferation, apoptosis and cell cycle of ovarian cancer cells after treatment with MEK inhibitor were analyzed by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively. Results: (1) The immunohistochemical method showed that p-ERK1/2 between low grade serous carcinoma and clear cell carcinoma were not significantly higher expressed (P>0.05) . However, a lower level of the p-ERK1/2 expression were observed among high grade serous carcinoma, mucinous carcinoma and endometrioid carcinoma (all P<0.05) . There was no significant correlation between the protein expression of p-ERK1/2 and patients' age, pathological stage of surgery, and preoperative serum CA(125) level (P>0.05). (2) Western blot showed that the protein p-ERK1/2 was widely expressed in various ovarian cancer cell lines such as SKOV3, OV2008, C13, A2780S, A2780CP, OVCAR4, OVCAR5, OVCAR8 and CAOV3. After treatment with AZD6244 (5, 10 μmol/L), the level of p-ERK1/2 in OVCAR5 and OVCAR8 decreased significantly in dose-dependent manner. Additionally, we found a reduction of the expression level of cyclin D1, caspase-3 and appeared cleaved poly adenosine diphosphate ribose polymerase (PARP) in OVCAR5 and OVCAR8, compared with control groups. MTT assays showed that OVCAR5, OVCAR8 and A2780S were differently inhibited in the dose-dependent manner after being treated with different concentrations of AZD6244 (0, 2.5, 5, 10, 25, 50 and 100 μmol/L, all P<0.05). Further tested by flow cytometry, the results showed that AZD6244 (5, 10 μmol/L) was able to induce the apoptosis of OVCAR5, OVCAR8 and A2780S, as well as G(0)/G(1) phase arrest, both in a dose-dependent manner (P<0.05). Conclusions: As the main active and functional unit of MAPK/ERK signaling pathway, p-ERK1/2 protein is expressed in both the tissues and various ovarian cancer cell lines. AZD6244 could down-regulated the expression of p-ERK1/2 in ovarian cancer cells, accompanied by the decreased proliferation and increased cell apoptosis of ovarian cancer cells. In conclusion, MAPK/ERK signaling pathway might play a role in the development and progression of ovarian cancer, and may be provide a novel option for molecular targeted therapies of the disease.

Erythropoietin alleviates spinal neuron apoptosis via c-Jun N-terminal kinase-c-Jun pathway

Objective To study the neural protective mechanism of erythropoietin (EPO) following root avulsion (RA). Methods Sixty-three adult Wistar rats were randomly divided into two groups: control group (avulsion + 1 ml normal saline s. c. on alternate days), EPO-treated group (avulsion + 1 000 U/kg EPO s. c. on alternate days). The rats were sacrificed at 0 (after awake), 1, 2, 4, 7 and 14 days after avulsion for harvesting C5-T1 spinal samples. Immunohistochemistry was used to detect cleaved-poly adenosine diphosphate-ribose polymerase (Cleaved-PARP), c-Jun and phosphorylated c-Jun N-terminal kinase (p-JNK) in spinal neurons. Results (1) The expression of Cleaved-PARP was significantly decreased in EPO-treated group at 2, 4, and 7 days post-avulsion (3 173.3±153.1, 8 943.2±351.5 and 7 017.9±424.7) as compared with control group (10 575.1±594.3, 12 737.1±385.3 and 10 256.9±629.4) (F=4 363.938, 1 587.489, 545.925, P<0.01). (2) The expression of c-Jun in EPO-treated group was significantly lower at 1, 2, 4 and 7 day(s) (1 351.2±253.7, 2 386.8±305.8, 1 898.2±138.0 and 1 547.1±163.9) than in control group (2 165.8±205.7, 3 378.1±272.5, 2 835.2±166.8, 2 579.8±264.8) (F=186.613, 175.719, 562.005, 329.896, P<0.01). (3) The expression of p-JNK in EPO-treated group was significantly lower at 1, 2, 4 days after avulsion (1 407.1±121.7, 1 925.6±126.6 and 4 892.3±330.2) than in control group (3 041.5±139.6, 9 243.8±573.9 and 5 486.6±358.4) (F=2 336.447, 4 651.806, 44.617, P<0.01). The peak of p-JNK was postponed to 4th day in EPO-treated group as compared with 2nd day in control group. Conclusion Down-regulation of p-JNK and c-Jun is the anti-apoptosis mechanism of EPO for spinal neurons. Key words: Root avulsion injury; Erythropoietin; Spinal cord neuron; C-poly adenosine diphosphate-ribose polymerase; C-Jun N-terminal kinase; C-Jun

Study on the neuroprotection and its molecular mechanisms of erythropoietin against brachial plexus root avulsion injury in rats

Objective The neuroprotective and adverse effects, associated with low dosage erythropoietin (EPO) for the spinal cord neuron following nerve avulsion will be researched. Methods 180 Wistar rats were randomly divided into two groups: control group (avulsion + 1 ml normal saline s. c. on alternate days), EPO treated group (avulsion + 1 000 U/kg EPO s. c. on alternate days). The rats were sacrificed at day 0 (before avulsion), 1, 2, 4, 7 and 14 d after avulsion for harvesting C5-T1 spinal samples. We used Nissl, terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL), and Cleaved-poly adenosine diphosphate-ribose polymerase (c-PARP) stainings to analyze the survival and apoptotic neurons. Results (1) The percentage of survivial neurons was significantly decreased, EPO group has more survival neurons compared with C group at 7 d [(78.42±2.64)% and (67.46±3.78)%] and 14 d [(57.32±4.07)% and (46.42±3.14)%]. (2) TUNEL positive (apoptotic) neurons appeared at 2 d, followed by a gradual change until 14 d. The TUNEL in EPO group was significantly decreased at 4 d [(303.1±59.9) and (861.6±85.4)] and 7 d [(419.8±74.2) and (983.2±83.9)] compared to C group. (3) The c-PARP, which is a critical apoptotic factor, was showed a pronounced decrease in EPO group than control ones at 2 d and 4 d. (4)There are no significant differences on blood routine examination in two groups at all time points. Conclusion With administration of 1 000 U/kg EPO, the survival rate of neurons was enhanced and apoptosis was alleviated without significant erythropothesis effect. Key words: Brachial plexus root avulsion injury; Neuron; Apoptosis; Cleaved-poly adenosine diphosphate-ribose polymerase

Autophagy attenuates the oxidative stress-induced apoptosis of Mc3T3-E1 osteoblasts.

The oxidative stress-induced osteoblast apoptosis plays an important role in the pathological process of osteoporosis, but the roles of autophagy in oxidative stress and apoptosis of osteoblasts remain unclear. This study aimed to observe the role of autophagy in oxidative stress injury of osteoblasts and the relationship between autophagy and apoptosis. Mc3T3-E1 cells were stimulated with different concentrations (0.1, 0.5, and 1 mM) of hydrogen peroxide. The cell viability was detected via cell counting kit 8 (CCK8) at different time points (0, 2, 6, 8, and 12 h), the apoptosis was detected via Western blotting and flow cytometry, and the autophagy was detected via macrophage-derived chemokine (MDC) and transmission electron microscope. The changes in expression of autophagy-associated protein, Beclin1, and LC3II/I ratio, were detected via Western blotting. Moreover, the intracellular reactive oxygen species (ROS) level and extracellular superoxide dismutase (SOD) level were observed using the autophagy regulators, rapamycin (Rap) and 3-methyladenine (3-MA), so as to clarify the interaction between autophagy and cellular oxidation. Hydrogen peroxide-induced apoptosis and autophagy of osteoblasts were in dose- and time-dependent manners; the hydrogen peroxide inhibitors could inhibit the autophagy level, and autophagy inhibitor (3-MA) could significantly enhance the hydrogen peroxide-induced ROS level and apoptosis rate in cells. Besides, Western blotting confirmed that the cleaved caspase-3 and cleaved poly adenosine diphosphate ribose polymerase (PARP) proteins were increased. The autophagy inducer (Rap) partially inhibited the hydrogen peroxide-induced oxidative stress and apoptosis. Autophagy inhibits the oxidative stress-mediated apoptosis of osteoblasts, which is a potential target for the osteoporosis treatment.

Kolaviron, a biflavonoid of Garcinia kola seed mitigates ischemic/reperfusion injury by modulation of pro-survival and apoptotic signaling pathways.

The study was designed to investigate the ameliorative effect of Kolaviron (KV) on ischemic/reperfusion injury in experimental animal models. Male Wistar rats were randomly divided into two groups: Group 1 received corn oil as a vehicle and rats in Group 2 were administered KV at 200 mg/kg for 4 weeks. The rats were fed with rat standard chow pellet and water administered ad libitum. After 4 weeks of KV administration, hearts were excised and mounted on the working heart perfusion system. Western blot analysis for protein expression was carried out on frozen heart samples. There was significant (P < 0.05) reduction in the activity of catalase, superoxide dismutase, and glutathione peroxidase with concomitant reduction in oxygen radical absorbance capacity in ischemic rat heart of control compared to group pre-treated with KV, respectively. Similarly, intracellular reactive oxygen species and malondialdehyde were significantly elevated in control compared to KV pre-treated rats. KV significantly increased total Akt/protein kinase B (PKB), phosphorylated Akt/PKB at serine 473 and also caused a significant reduction in p38 mitogen-activated protein kinase, Caspase 3, and cleaved poly adenosine diphosphate ribose polymerase. Taken together, KV offered significant cardioprotection via free radical scavenging activity and upregulation of pro-survival pathway.

Akt inhibitor MK-2206 enhances the effect of cisplatin in gastric cancer cells.

The phosphoinositide 3-kinase/Akt pathway activation commonly occurs in various types of human cancer and has an important role in chemoresistance. Combination of traditional chemotherapy drugs and molecular-targeted agents is a promising strategy for cancer therapy, which has shown enhanced cytotoxicity and lower drug resistance. The present study found that the Akt inhibitor, MK-2206, can increase the effect of cisplatin in the gastric cancer cell line AGS, which has higher Akt phosphorylation, but exhibited a poor combination effect in MKN-45 and MGC-803 cells, which have limited Akt activation. The MTT assay demonstrated that sequential treatment of cisplatin, followed by the Akt inhibitor, MK-2206, caused a synergistic effect of proliferation inhibition, and the apoptosis assay by propidium iodide/fluorescein isothiocyanate staining also showed that combination treatment induced more apoptosis compared to the monotherapy groups. Using western blot analysis, MK-2206 was shown to significantly suppress the phosphorylation of Akt (Ser473), however, the expression of total Akt remained the same, and the combination treatment also increased the expression of cleaved poly adenosine diphosphate ribose polymerase, which contributed to apoptosis.

Novel analogs targeting histone deacetylase suppress aggressive thyroid cancer cell growth and induce re-differentiation.

To develop novel therapies for aggressive thyroid cancers, we have synthesized a collection of histone deacetylase (HDAC) inhibitor analogs named AB1 to AB13, which have different linkers between a metal chelating group and a hydrophobic cap. The purpose of this study was to screen out the most effective compounds and evaluate the therapeutic efficacy. AB2, AB3 and AB10 demonstrated the lowest half-maximal inhibitory concentration (IC50) values in one metastatic follicular and two anaplastic thyroid cancer cell lines. Treatment with each of the three ABs resulted in an increase in apoptosis markers, including cleaved poly adenosine diphosphate ribose polymerase (PARP) and cleaved caspase 3. Additionally, the expression of cell-cycle regulatory proteins p21(WAF1) and p27(Kip1) increased with the treatment of ABs while cyclin D1 decreased. Furthermore, AB2, AB3 and AB10 were able to induce thyrocyte-specific genes in the three thyroid cancer cell lines indicated by increased expression levels of sodium iodide symporter, paired box gene 8, thyroid transcription factor 1 (TTF1), TTF2 and thyroid-stimulating hormone receptors. AB2, AB3 and AB10 suppress thyroid cancer cell growth via cell-cycle arrest and apoptosis. They also induce cell re-differentiation, which could make aggressive cancer cells more susceptible to radioactive iodine therapy.