Transferase-mediated Deoxyuridine Triphosphate Nick End Research Articles

Sperm DNA and detection of DNA fragmentations in sperm.

The questionable effectiveness of routine sperm parameters in determining male factor infertility problems and increasing the success rates of assisted reproductive techniques have led to the investigation of more detailed sperm parameters that could affect the male fertility and reproduction. Thus, the effects of different sperm parameters such as sperm DNA integrity was started to be investigated thanks to the previously described methods such as single cell gel electrophoresis (COMET) assay, sperm chromatin structure assay (SCSA), acridine orange test (AOT), terminal deoxynucleotidyl transferase-mediated deoxyuridine (TdT) triphosphate (dUTP) nick end labeling (TUNEL) assay and sperm chromatin dispersion (SCD) test. However, studying on sperm DNA might be very complex because the sperm DNA differs from the somatic cell DNA with its unique structure. Also, the sperm DNA undergoes many changes during spermatogenesis and it is condensed by being packaged tightly with different types and numbers of protamines in different species. Despite all these difficulties, these methods provide important information about the reasons and consequences of DNA damages in sperm and the effects of these damages on reproduction.

Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick End Labeling (TUNEL) Assay to Characterize Histopathologic Changes Following Thermal Injury.

BackgroundDespite the wide application of lasers and radiofrequency (RF) surgery in dermatology, it is difficult to find studies showing the extent of damage dependent on cell death.ObjectiveWe evaluated histopathologic changes following in vivo thermal damage generated by CO2 laser, 1,444 nm long-pulsed neodymium:yttrium-aluminum-garnet (LP Nd:YAG) laser and RF emitting electrosurgical unit.MethodsThermal damage was induced by the above instruments on ventral skin of rat. Specimens were stained with hematoxylin and eosin, along with a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay, to highlight the degree of irreversible cellular injury.ResultsThe volume of vaporization was largest with the CO2 laser. Area of cell death area identified by TUNEL assay, when arranged from widest to narrowest, was 1,444 nm LP Nd:YAG laser, CO2 laser, and RF emitting electrosurgical unit.ConclusionThis histopathologic evaluation of the acute characterization of injury across devices may be advantageous for attaining better treatment outcomes.

Alpha1-adrenergic receptor antagonist tamsulosin ameliorates aging-induced memory impairment by enhancing neurogenesis and suppressing apoptosis in the hippocampus of old-aged rats

ABSTRACTAge-related memory decline is closely associated with decreased neurogenesis and increased apoptosis in the hippocampus. Noradrenaline exerts its effect by selectively binding to and activating adrenergic receptors (ARs). Tamsulosin, α1-AR antagonist, is reported to have access to the brain and interact with α1-AR. In this study, the effects of tamsulosin on short-term and spatial learning memory in terms of neurogenesis and apoptosis were investigated using rats. Step-down avoidance test for short-term memory and radial 8-arm maze test for spatial learning memory were conducted. Neurogenesis was detected by 5-bromo-2’-deoxyuridine (BrdU) immunohistochemistry and apoptosis was evaluated by caspase-3 immunohistochemisty and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNE) staining. Western blot for protein kinase C (PKC), cAMP-responsive element-binding protein (CREB), brain-derived neurotrophic factor (BDNF), tyrosine kinase B (TrkB), phosphatidylinositol 3-kinase (PI 3-kinase), Akt, Bcl-2, and Bax was conducted. In the aged rats, short-term and spatial learning memory was declined. Hippocampal nerogenesis was suppressed and hippocampal apoptosis was enhanced in the aged rats. In addition, phosphorylation of PKCα, CREB, PI-3 kinase, and Akt was decreased in the hippocampus of old-aged rats. Tamsulosin activated PKC/CREB and PI-3 kinase/Akt pathways. With these pathways, BDNF-TrkB signaling enhanced hippocampal neurogenesis and suppressed apoptosis in the old-aged rats. As the results, tamsulosin improved performance of short-term and spatial learning memory in the aged rats.

Ginkgetin inhibits proliferation of human leukemia cells via the TNF-α signaling pathway.

Ginkgetin is known to be an anticancer agent in many studies. However, its effectiveness in treating chronic myeloid leukemia [corrected] remains unknown. The present study aimed to evaluate the effects of ginkgetin on the growth of the K562 cell line. The MTT assay was employed to examine the proliferation of K562, and a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining was conducted to detect the apoptotic rates. Furthermore, changes of tumor necrosis factor-α (TNF-α) were detected by Western blot analysis. Ginkgetin inhibited the proliferation of K562 cells in a dose- and time-dependent manner. Concentrations of ginkgetin required to induce 50% death of K562 at 24, 48 and 72 h were 38.9, 31.3 and 19.2 μM, respectively. Moreover, treatment of ginkgetin increased K562 apoptosis in vitro along with increased levels of TNF-α. Interestingly, anti-TNF-α antibody prevented ginkgetin-induced K562 cell apoptosis and growth inhibition via deactivation of caspase-8, caspase-9 and caspase-3. Concomitantly, downregulation of TNF-α by etanercept in vivo attenuated ginkgetin-induced inhibitory effects on the tumor growth in an xenograft mouse model. Our results indicate that ginkgetin effectively inhibits K562 cell proliferation, and TNF-α plays a key role in ginkgetin-induced cell apoptosis.

Prevention of doxorubicin-induced cardiomyopathy using targeted MaFGF mediated by nanoparticles combined with ultrasound-targeted MB destruction.

The present study seeks to observe the preventive effects of doxorubicin-induced cardiomyopathy (DOX-CM) in rats using targeted non-mitogenic acidic fibroblast growth factor (MaFGF) mediated by nanoparticles (NP) combined with ultrasound-targeted MB destruction (UTMD). DOX-CM rats were induced by intraperitoneally injected doxorubicin. Six weeks after intervention, the indices from the transthoracic echocardiography and velocity vector imaging showed that the left ventricular function in the MaFGF-loaded NP (MaFGF-NP) + UTMD group was significantly improved compared with the DOX-CM group. The increased malondialdehyde and decreased superoxide dismutase were observed in the DOX-CM group, while a significant increase in superoxide dismutase and a decrease in malondialdehyde were detected in the groups treated with MaFGF-NP + UTMD. From the Masson staining, the MaFGF-NP + UTMD group showed a significant difference from the DOX-CM group. The cardiac collagen volume fraction and the ratio of the perivascular collagen area to the luminal area number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling positive cells in the MaFGF-NP + UTMD group decreased to 8.9%, 0.55-fold, compared with the DOX-CM group (26.5%, 1.7-fold). From terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling staining, the results showed the strongest inhibition of apoptosis progress in MaFGF-NP + UTMD group. The immunohistochemical staining of the TGF-β1 in MaFGF-NP + UTMD group reached 3.6%, which was much lower than that of the DOX-CM group (12.6%). These results confirmed that the abnormalities, including left ventricular dysfunction, myocardial fibrosis, cardiomyocytes apoptosis and oxidative stress, could be suppressed by twice weekly MaFGF treatments for 6 consecutive weeks (free MaFGF or MaFGF-NP+/UTMD), with the strongest improvements observed in the MaFGF-NP + UTMD group. Western blot analyses of the heart tissue further revealed the highest pAkt levels, highest anti-apoptosis protein (Bcl-2) levels and strongest reduction in proapoptosis protein (Bax) levels in the MaFGF-NP + UTMD group. This study confirmed the preventive effects of DOX-CM in the rats with MaFGF-NP and UTMD by retarding myocardial fibrosis, inhibiting oxidative stress, and decreasing cardiomyocyte apoptosis.

The Differentiation-Inducing Effect of a Retinoic Acid Derivative on a Pulmonary Carcinoma Cell Line

Pulmonary carcinoma is a main cause of cancer death internationally. Chemotherapeutic regimens in most solid tumors have limited effect. And in the targeted inhibition of carcinogenic driver mutations with molecular therapies, in a large number of cases, patients acquire resistance to drugs. Therefore, the development of new therapeutic approaches is highly desirable. In the present study, we evaluated the applicability of 4[(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl- 2-naphthalenyl)carbamoyl]benzoic acid (Am80), a synthetic retinoid, as a differentiation inducer in pulmonary carcinoma and examined its effect on the viability and differentiation of Calu-6 cells (a human nonsmall cell lung cancer cell line), with the final goal of establishing a new approach to pulmonary carcinoma therapy. The cell viability of Calu-6 cells after Am80 treatment was assessed by the CellTiter-Glo Luminescent Cell Viability Assay. The early apoptosis of cells was detected by Terminal deoxynucleotidyl Transferase-mediated deoxyuridine triphosphate Nick End Labeling (TUNEL) staining. The differentiation-inducing effect of Am80 on Calu-6 cells was investigated by immunostaining. Am80 induced the differentiation of Calu-6 to alveolar type I and II cells. However, Am80 reduced the viability of Calu-6 cells without inducing apoptosis. In addition, in order to investigate whether Am80 inhibits tumor growth in vivo, we conducted an in vivo experiment to determine he effect of the intratumoral administration of Am80 in mice harboring sc tumors composed of Calu-6 cells. Am80 significantly influenced the suppression of tumor growth.

Simvastatin attenuates renal ischemia/reperfusion injury from oxidative stress via targeting Nrf2/HO-1 pathway.

Ischemia-reperfusion (I/R) injury of the kidneys is commonly encountered in the clinic. The present study assessed the efficacy of simvastatin in preventing I/R-induced renal injury in a rat model and investigated the corresponding molecular mechanisms. Rats were divided into 3 groups, including a sham, I/R and I/R + simvastatin group. The results revealed that in the I/R group, the levels of blood urea nitrogen, serum creatinine and lactate dehydrogenase were significantly higher than those in the sham group, which was significantly inhibited by simvastatin pre-treatment. I/R significantly decreased superoxide dismutase activity compared with that in the sham group, which was largely rescued by simvastatin. Furthermore, I/R significantly increased the malondialdehyde content compared with that in the sham group, which was reduced by simvastatin. Hematoxylin-eosin staining revealed no obvious morphological abnormalities in the sham group, while I/R led to notable tubular cell swelling, vacuolization, cast formation and tubular necrosis, which was rescued by simvastatin. A terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay demonstrated that I/R significantly increased the number of apoptotic cells compared with that in the sham group, which was significantly inhibited by simvastatin. Western blot analysis demonstrated that simvastatin upregulated I/R-induced increases of nuclear factor erythroid-2-related factor 2 (Nrf2) and anti-oxidant enzyme heme oxygenase-1 (HO-1). Reverse-transcription quantitative PCR indicated that changes in the mRNA levels of Nrf2 and HO-1 were consistent with the western blot results. It was concluded that simvastatin treatment led to upregulation of HO-1 protein levels through activating the Nrf2 signaling pathway to ultimately protect the kidneys from I/R-associated oxidative damage.

Melatonin Inhibits Neural Cell Apoptosis and Promotes Locomotor Recovery via Activation of the Wnt/β-Catenin Signaling Pathway After Spinal Cord Injury.

The spinal cord is highly sensitive to spinal cord injury (SCI) by external mechanical damage, resulting in irreversible neurological damage. Activation of the Wnt/β-catenin signaling pathway can effectively reduce apoptosis and protect against SCI. Melatonin, an indoleamine originally isolated from bovine pineal tissue, exerts neuroprotective effects after SCI through activation of the Wnt/β-catenin signaling pathway. In this study, we demonstrated that melatonin exhibited neuroprotective effects on neuronal apoptosis and supported functional recovery in a rat SCI model by activating the Wnt/β-catenin signaling pathway. We found that melatonin administration after SCI significantly upregulated the expression of low-density lipoprotein receptor related protein 6 phosphorylation (p-LRP-6), lymphoid enhancer factor-1 (LEF-1) and β-catenin protein in the spinal cord. Melatonin enhanced motor neuronal survival in the spinal cord ventral horn and improved the locomotor functions of rats after SCI. Melatonin administration after SCI also reduced the expression levels of Bax and cleaved caspase-3 in the spinal cord and the proportion of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) positive cells, but increased the expression level of Bcl-2. These results suggest that melatonin attenuated SCI by activating the Wnt/β-catenin signaling pathway.

The role of inversely operating glutamate transporter in the paradoxical analgesia produced by glutamate transporter inhibitors

Controlling extracellular glutamate level in a physiological range is important to maintain normal sensory transmission. Here, we investigated the paradoxical action of glutamate transporters in the rat formalin test to elucidate a possible role of inversely operating transporters in its analgesic mechanism. The effects of glutamate transporter inhibitor on formalin-induced pain behavior were examined. Then we performed a microdialysis study to clarify the differential change in extracellular glutamate concentration by intrathecal administration of transportable and non-transportable blockers. And we further investigated the mechanism pharmacologically via pretreatment with antagonists of various receptors and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining. Intrathecally-injected glutamate transporter inhibitors, non-transportable DL-threo-β-benzyloxyaspartat (TBOA) and transportable trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC), produced paradoxical antinociception in the formalin test. In normal rats, inhibition of the glutamate transporter increased extracellular glutamate. In the formalin model rats, TBOA suppressed while t-PDC enhanced glutamate release. When tPDC was pretreated 30min prior to formalin injection, glutamate release was blocked. Blocking α-2 adrenergic receptors reversed the tPDC analgesia. Increased apoptosis was not apparent in the spinal dorsal horn of tPDC-treated rats compared to the control group. These data suggest that glutamate transporters in a formalin-induced pain state work in a reverse mode and can be blocked from releasing glutamate by TBOA and preloaded tPDC. The analgesic mechanism of TBOA may be related to the blockade of inversely operating transporter, and that of tPDC may be associated with the activation of noradrenergic neurotransmission but not with dorsal horn neurotoxicity.

Exploration of Bcl-2 family and caspases-dependent apoptotic signaling pathway in Zearalenone-treated mouse endometrial stromal cells

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin found in several food commodities worldwide. Although the toxicity of ZEA have been widely studied in a number of cell types, the mechanistic role of ZEA on apoptosis of endometrial stromal cells (ESCs) remains poorly understood. The objective of this study was to determine the effects of ZEA on apoptosis of mouse ESCs and explore the signaling pathway underlying the cytotoxicity of ZEA. The results showed that ZEA treatment caused obvious apoptosis in ESCs as determined by the flow cytometry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Immunoblotting and real-time quantitative polymerase chain reaction (RT-qPCR) revealed that ZEA treatment increased the ratio of Bax/Bcl-2. The enzymatic activity assays revealed that caspases-3 and caspase-9 were activated by ZEA treatment in a dose-dependent manner. In addition, flow cytometry show that the apoptotic percentages of cells pretreated with Z-VAD-FMK and Z-LEHD-FMK were markedly reduced compared to the ZEA-treated cells. Overall, the results suggested that ZEA induced obvious apoptosis in ESCs via a Bcl-2 family and caspases-dependent signaling pathway.

Cell cycle arrest induced by inhibitors of epigenetic modifications in maize (Zea mays) seedling leaves: characterization of the process and possible mechanisms involved.

Epigenetic modifications play crucial roles in the regulation of chromatin architecture and are involved in cell cycle progression, including mitosis and meiosis. To explore the relationship between epigenetic modifications and the cell cycle, we treated maize (Zea mays) seedlings with six different epigenetic modification-related inhibitors and identified the postsynthetic phase (G2 ) arrest via flow cytometry analysis. Total H4K5ac levels were significantly increased and the distribution of H3S10ph signalling was obviously changed in mitosis under various treatments. Further statistics of the cells in different periods of mitosis confirmed that the cell cycle was arrested at preprophase. Concentrations of hydrogen peroxide were relatively higher in the treated plants and the antioxidant thiourea could negate the influence of the inhibitors. Moreover, all of the treated plants displayed negative results in the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) and γ-H2AX immunostaining assays after exposure for 3d. Additionally, the expression level of topoisomerase genes in the treated plants was relatively lower than that in the untreated plants. These results suggest that these inhibitors of epigenetic modifications could cause preprophase arrest via reactive oxygen species formation inhibiting the expression of DNA topoisomerase genes, accompanied by changes in the H4K5ac and H3S10ph histone modifications.

Targeted inhibition of survivin with YM155 promotes apoptosis of hypoxic human pulmonary arterial smooth muscle cells via the upregulation of voltage-dependent K⁺ channels.

Hypoxic pulmonary hypertension (PH) is a common disease characterized by a disturbance to the balance of apoptosis and cell proliferation in pulmonary artery smooth muscle cells (PASMCs). The anti-apoptotic protein, survivin, has been observed to be upregulated in pulmonary arteries (PAs) of chronic hypoxia-induced PH rats. The present study aimed to investigate the therapeutic potential of sepantronium bromide (YM155), a selective survivin inhibitor, on hypoxic human PASMCs and examine the potential underlying mechanisms. Cultured human PASMCs (HPASMCs) were randomly divided into the following groups: i) Normoxia (N); ii) normoxia + 100 nmol/l YM155 (NY100); iii) hypoxia (H); iv) hypoxia + 1 nmol/l YM155 (HY1); v) hypoxia + 10 nmol/l YM155 (HY10); and hypoxia + 100 nmol/l YM155 (HY100) groups. The cells were exposed to the different conditions for 24 h, according to the group. Cell viability was then determined using a Cell Counting Kit-8 assay, and apoptosis was detected using a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. The expression levels of survivin were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunocytochemistry and Western blot analyses. The expression levels of the voltage-dependent K+ (Kv) channels, Kv1.5 and Kv2.1, were measured using RT-qPCR and Western blotting. Cell proliferation in the hypoxic PASMCs was significantly increased by hypoxia, however, apoptosis of the HPASMCs was suppressed, the expression of survivin were upregulated and the expression levels of Kv1.5 and Kv2.1 were downregulated. YM155 treatment ameliorated the hypoxia-induced increase in cell proliferation and expression of survivin in a concentration-dependent manner, increased apoptosis, and increased the expression levels of Kv1.5 and Kv2.1 (P<0.05). By contrast, YM155 treatment in normoxic HPASMCs had no significant effects on proliferation, apop-tosis, or the expression levels of survivin and Kv channels in the PASMCs. The present study is the first, to the best of our knowledge, to demonstrate that YM155, a selective survivin inhibitor, has a beneficial therapeutic effect on hypoxic HPASMCs, and that YM155 induces a pro-apoptotic effect by downregulating the apoptosis inhibitor, survivin, possibly through a Kv channel-mediated mechanism.

Role of p97/Valosin-containing protein (VCP) and Jab1/CSN5 in testicular ischaemia-reperfusion injury.

The most significant complication of testicular ischaemia is loss of the testis, which may lead to infertility. Testicular ischaemia damages protein degradation pathways which, together with the overproduction of damaged proteins and consequent upregulation of ubiquitin-conjugated protein aggregates. Despite recent advances, the factors leading to impairment of spermatogenesis owing to testicular ischaemia remain poorly understood. This study was undertaken to gain insight into the cellular and molecular mechanism underlying torsion induced germ cell apoptosis. Male rats were subjected to 2 h torsion, and testes were examined at 2, 4, 12 and 24 h after torsion repair (reperfusion). Ischaemia-reperfusion (IR) of the testes resulted in apoptosis which was revealed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) technique. At 12 h after torsion repair germ cell loss reached peak, then decreased at 24 h repair. Western blotting showed that apoptotic proteins (active caspase 3, caspase 9 and Bax) gradually was upregulated at 12 h reperfusion, however anti-apoptotic protein (Bcl2) was downregulated in the relevant IR treatment. Furthermore, Jab1/CSN5 expression was gradually upregulated and p97/VCP expression was downregulated in IR injury according to western blotting and immunohistochemistry. To test further whether polyubiquitination was also involved in IR injury, the expression of polyubiquitinated proteins was examined, which showed that polyubiquitinated proteins were significantly increased in IR injury. These finding suggest that p97/VCP and Jab1/CSN5 provide a novel signaling pathway for testicular ischaemia and may play an important role in IR injury induced cell death in rat testis.

Effects of Rapamycin and Rapamycin-loaded Poly(lactic-co-glycolic)Acid Nanoparticles on Apoptosis and Expression of bcl-2 and p27(kip1) Proteins of Human Umbilical Arterial Vascular Smooth Muscle Cell

To investgate the effects of rapamycin(RPM)and RPM-loaded poly(lactic-co-glycolic)acid(PLGA)nanoparticles(NPs)on the apoptosis of human umbilical arterial vascular smooth muscle cells(HUASMCs)in vitro and expression of bcl-2 and p27(kip1) protein. HUASMCs were cultured in vitro and divided to RPM and RPM-PLGA-NPs groups treated at 3 different concentration by 12 and 24 hours,with M231-smooth muscle growth supplements medium and null-PLGA-NPs treated groups as controlled. The apoptosis of HUASMCs was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining and flow cytometry. The expressions of bcl-2 and p27(kip1) were detected by streptacidin/peroxidase immunohistochemical method. The effect on cellular proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidecolorimetry. The proliferation of HUASMCs was inhibited by RPM and RPM-PLGA-NPs in a dose-dependent manner. DNA electrophoresis showed DNA ladder in RPM and RPM-PLGA-NPs groups and classical scalar strips in control groups. The apoptotic indexes of RPM 100 ng/ml group and RPM-PLGA-NPs 500 ng/ml group detected by flow cytometry were(45.45<2.36)% and(35.04<5.64)%,respectively,which were significantly higher than that of M231-smooth muscle growth supplements control group [(2.60<0.95)%,all P<0.01]. The apoptotic indexes of groups incubated with RPM and RPM-PLGA-NPs for 24 hours were significantly higher than those of groups which incubated for 12 hours(P<0.05,P<0.01). The positive expression indexes(PEI)of p27(kip1) and bcl-2 protein were higher in RPM and RPM-PLGA-NPs groups than that of control groups. The Spearman's rank correlation coefficient test showed that there was no significant correlation between the PEI of p27(kip1) and the apoptotic indexes in the RPM group and RPM-PLGA-NPs group(P>0.05). Rapamycin-loaded PLGA nanoparticles and rapamycin have similar effects in inhibiting proliferation and inducing apoptosis;meanwhile,they upregulate the expression of p27(kip1) protein without downregulating the expression of bcl-2 protein in HUASMCs in vitro. RPM-PLGA-NPs has more potent pro-apoptotic effect than equivalent dose of RPM but is not linearly correlated with the p27(kip1) expression level.

Abstract 3337: Titanium peroxide nanoparticles enhance antitumor efficacy through reactive oxygen species in pancreatic cancer radiation therapy

Abstract Purpose: Pancreatic cancer is a highly lethal disease and notoriously resistant to many types of cytotoxic chemotherapy and radiotherapy. A novel strategy should be explored for these radioresistant tumors. We originally synthesized a titanium peroxide nanoparticle (TiOxNP) which had a distinct ability to produce reactive oxygen species (ROS) upon X-ray irradiation (IR). Here, the characterization and efficacy of TiOxNP as radiosensitizer for pancreatic cancer therapy were investigated. Methods: TiOxNPs were synthesized from anatase titanium oxide nanoparticles (TiO2NPs) by H2O2 processing and coated by polyacrylic acid. The ability of TiOxNPs to enhance ROS generation upon X-ray irradiation (IR) was tested by using 3′-(p-Aminophenyl) fluorescein and several antioxidants. A xenograft mouse model using the human pancreatic cancer cell line MIAPaCa-2 was used to evaluate the cytotoxic effects of a combination of TiOxNPs and IR in vivo. MIAPaCa-2 cells were injected subcutaneously into the hind legs of immunodeficient BALB/c mice. Seven days after injection, the tumor was treated with a TiOxNP suspension injected directly into the tumor and application of 5 Gy of IR. Tumor size and health of the mice were monitored for 43 days after the treatment. Induction of apoptosis was evaluated by immunohistochemistry using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining. We also characterized TiOxNPs by ESR and XAFS analyses. Results: ROS generation was enhanced dramatically by TiOxNPs in a concentration- and radiation dose- dependent manner compared to enhancement by TiO2NPs. At the highest concentration of TiOxNPs, ROS generation upon an irradiation dose of 30 Gy was enhanced by a factor of more than 10. Also, ROS generated by TiOxNPs upon IR were scavenged by vitamin C or glutathione. Growth inhibition of the tumor in the group receiving both TiOxNP and IR was significantly greater than single treatment subgroups (P &amp;lt; 0.05). The number of apoptotic cells was also significantly higher in the combination group (P &amp;lt; 0.05). Histologically, large pools of TiOxNPs around tumor cells and small amounts inside the tumor cells were observed. No mice died within a 43-day observation period or showed any apparent body weight loss. In the ESR spectra, some signals were observed for TiOxNPs. The spectral shapes of Ti K-edge X-ray absorption near edge structure, spectra were the same among the mineral anatase, TiO2NPs and TiOxNPs. Conclusions: TiOxNPs induced remarkable ROS production upon IR. They were safe and effective in a xenograft mouse model using engrafted human pancreatic cancer cells. Further studies would be necessary to elucidate a mechanism of radical generation and long-term toxicity, however our study shows that TiOxNPs are promising radiosensitizers for application in pancreatic cancer therapy. Citation Format: Masao Nakayama, Ryohei Sasaki, Toru Mukohara, Chiaki Ogino, Kenta Morita, Mitsuo Umetsu, Satoshi Ohara, Kazuyoshi Sato, Chiya Numako, Seiichi Takami, Akihiko Kondo. Titanium peroxide nanoparticles enhance antitumor efficacy through reactive oxygen species in pancreatic cancer radiation therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3337. doi:10.1158/1538-7445.AM2015-3337

Effects of pioglitazone pre-treating on acinar cells apoptosis induced by caerulein in acute pancreatitis

Objective To investigate the effects of pioglitazone pre-treating on pancreatic acinar cell (AR42J cells) apoptosis induced by caerulein. Methods AR42J cells were divided into blank control group (with normal culture), pioglitazone group (40 μmol/L), caerulein control group (1×10-8 mol/L), pioglitazone+ caerulein group (40 μmol/L pioglitazone+ 1×10-8 mol/L caerulein) and pioglitazone+ GW9662+ caerulein group (40 μmol/L pioglitazone+ 5 μmol/L GW9662+ 1×10-8 mol/L caerulein). Pioglitazone and GW9662 were added 30 minutes earlier than caerulein. Cell proliferation rate of each group was determined by MTT assay at three, six, 12 and 24 hour. The cell apoptosis rate was detected by flow cytometry with Annexin Ⅴ/PI staining and terminal dexynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining. The activity of Caspase 3, 8 and 9 of each group was measured. Mitochondrial membrane potential (MMP) was detected by flow cytometry with JC-1 staining. Single factor analysis of variance and LSD test were performed for data analysis. Results At six, 12 and 24 hour, the cell proliferation rate of pioglitazone group and pioglitazone+ caerulein group was 0.19±0.02, 0.22±0.02, 0.36±0.02 and 0.20±0.04, 0.23±0.02, 0.38±0.02, respectively, which were significantly lower than those of blank control group (0.25±0.04, 0.28±0.03 and 0.46±0.02) and caerulein group (0.23±0.02, 0.29±0.01 and 0.46±0.05, tcontrol group=-3.16, -4.61 and -6.25, tcaerulein group=-1.58, -4.61 and -6.15, all P 0.05), but cell apoptosis of these two groups were higher than those of control group ((5.52±0.64)%, (5.30±0.97)% and (5.47±0.88)%) and caerulein group ((5.98±1.21)%, (7.47±0.58)% and (8.11±1.32)%) respectively, and the differences were statistically significant (tcontrol group=9.81, 4.45 and 10.74, tcaerulein group=4.38, 7.62 and 6.98, all P <0.05). There was no significant difference in apoptosis rate between pioglitazone+ GW9662+ caerulein group ((5.82±0.26)%, (6.05±0.83)% and (9.23±0.90)%) and caerulein group; while significantly higher when compared with those of pioglitazone+ caerulein group (t=-4.63, -10.07 and -5.70, all P<0.05). At 12 hour, the apoptosis rate detected by TUNEL staining of pioglitazone group ((3.93±0.40)%) was significantly higher than that of control group ((2.73±0.68)%), the apoptosis rate of pioglitazone+ caerulein group ((8.43±1.65)%) was significantly higher than that of caerulein group ((2.80±0.56)%), the apoptosis rate of pioglitazone+ GW9662+ caerulein group ((3.87±0.35)%) was lower than that of pioglitazone+ caerulein group (t=7.93, 8.92, -5.35, all P<0.05). At 24 hour, the activity of Caspase 3, 8 and 9 of pioglitazone+ caerulein group (1.28±0.05, 1.38±0.04 and 1.53±0.09) significantly increased compared with those of caerulein group (1.12±0.88, 1.22±0.02 and 0.53±0.07, t=3.20, 8.62 and 1.29, all P<0.05). After treated with GW9662, part of activity of Caspase enzymes recovered. The number of cells with potential change of mitochondrial membrane in pioglitazone group and pioglitazone+ caerulein group was more than that of caerulein group (28.50±0.91)% and (28.20±2.56)% vs (15.00±3.67)%) and part of membrane potential recovered after GW9662 added ((20.67±2.20)%). Conclusions Pioglitazone might promote AR42J cell apoptosis through the activation of caspases enzymes and changing membrane potential. And the antagonist GW9662 would partially inhibit the apoptosis induced by pioglitazone. Key words: Pioglitazone; Caerulein; AR42J cells; Acute pancreatitis

Autophagy and Apoptosis Associated with Abortive Pollen Development in the Cytoplasmic Male-sterile Line GP1BC1-12 of Platycodon grandiflorum

Platycodon grandiflorum (balloon flower) is widely cultivated for medicinal, edible, and ornamental purposes. The cytoplasmic male-sterile line GP1BC1-12 of P. grandiflorum has been used to produce hybrids, but its mechanism of sterility has not been studied. In this work, the mechanism was evaluated by a combination of transmission electron microscopy (TEM) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. Observations showed that autophagy and apoptosis were simultaneously present in the abortive anthers. Autophagy was indicated by the autophagic vesicles, abnormal arrangement of endoplasmic reticulum, and the vacuole’s invagination. Apoptosis was characterized by chromatin aggregation and DNA cleavage. It was concluded that programmed cell death is one of the direct reasons for cytoplasmic male sterility in P. grandiflorum. This study first noted the simultaneous presence of the features of apoptosis, microautophagy, and macroautophagy in the abortive anthers of P. grandiflorum.

Detection of cell apoptosis in pelvic lymph nodes of patients with cervical cancer after neoadjuvant chemotherapy

To investigate if the administration of neoadjuvant chemotherapy (NACT) reduces pelvic lymph node metastasis by inducing tumour cell apoptosis in patients with cervical cancer. This study enrolled patients with stage Ib2-IIb cervical cancer who underwent surgery with (NACT group) or without (control group) prior cisplatin-based chemotherapy. Immunohistochemical staining of caspase-3 and an in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling assay were used to measure the levels of apoptosis in primary tumours and pelvic lymph nodes. A total of 185 patients participated in the study: 102 in the NACT group and 83 in the control group. Treatment was considered to be clinically effective in 69.6% (71/102) of the NACT group. The rate of metastasis in the NACT group (20.6%; 21/102) was significantly lower than the control group (42.2%; 35/83). The level of caspase-3 immunostaining and the rate of apoptosis in primary tumours and pelvic lymph nodes in the NACT group were significantly higher than in the control group. NACT appeared to limit pelvic node metastasis by inducing tumour cell apoptosis in patients with cervical cancer.

Inhibitors of the p53-Mdm2 interaction increase programmed cell death and produce abnormal phenotypes in the placozoon Trichoplax adhaerens (F.E. Schulze)

Recent identification of genes homologous to human p53 and Mdm2 in the basal phylum Placozoa raised the question whether the network undertakes the same functions in the most primitive metazoan organism as it does in more derived animals. Here, we describe inhibition experiments on p53/Mdm2 interaction in Trichoplax adhaerens by applying the inhibitors nutlin-3 and roscovitine. Both inhibitors had a strong impact on the animals' survival by significantly increasing programmed cell death (cf. apoptosis, measured via terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay). Treatment with roscovitine decreased cell proliferation (visualized by means of bromodeoxyuridine incorporation), which is likely reducible to its function as cyclin-dependent kinase inhibitor. Obvious phenotypic abnormalities have been observed during long-term application of both inhibitors, and either treatment is highly lethal in T. adhaerens. The findings of this study suggest a conserved role of the p53/Mdm2 network for programmed cell death since the origin of the Metazoa and advocate the deployment of Placozoa as a model for p53, apoptosis, and possibly cancer research.

Protective effects of fluvoxamine against ischemia/reperfusion injury in isolated, perfused guinea-pig hearts.

Serotonin (5-hydroxytryptamine; 5-HT) is known to be activated during ischemia-reperfusion and triggers contractile dysfunction and pathological apoptosis. Here, the beneficial effects of the selective serotonin reuptake inhibitor (SSRI) fluvoxamine was demonstrated on ischemia-reperfusion injury in guinea-pig hearts perfused using the Langendorff technique. The recovery (%) of left ventricular developed pressure (LVDP) by fluvoxamine (5×10(-8) M) was 95.4% (control: 32%), which was consistent with the inhibition of mitochondrial Ca(2+)([Ca(2+)]m) uptake induced by changes in the Ca(2+) content and acidification of the perfusate, and similar to reperfusion following global ischemia in Langendorff-perfused hearts. Fluvoxamine inhibited the increase in [Ca(2+)]m induced by changes in the Ca(2+) content of the perfusate in perfused preparations of mitochondria, which was similar to the results obtained with the mitochondrial permeability transition pore (MPTP) opener atractyroside. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells were significantly less in fluvoxamine-treated hearts than in control hearts, with decreases in caspase-3 activity. These results suggest that SSRI inhibits opening of the MPTP by preventing [Ca(2+)]m overload-induced apoptosis related to the endogenous accumulation of 5-HT in ischemia-reperfusion hearts.