Yinan Luo

Shikonin Kills Glioma Cells through Necroptosis Mediated by RIP-1

Background and Purpose Shikonin was reported to induce necroptosis in leukemia cells, but apoptosis in glioma cell lines. Thus, it is needed to clarify whether shikonin could cause necroptosis in glioma cells and investigate its underlying mechanisms. Methods Shikonin and rat C6 glioma cell line and Human U87 glioma cell line were used in this study. The cellular viability was assayed by MTT. Flow cytometry with annexin V-FITC and PI double staining was used to analyze cellular death modes. Morphological alterations in C6 glioma cells treated with shikoinin were evaluated by electronic transmission microscopy and fluorescence microscopy with Hoechst 33342 and PI double staining. The level of reactive oxygen species was assessed by using redox-sensitive dye DCFH-DA. The expressional level of necroptosis associated protein RIP-1 was analyzed by western blotting. Results Shikonin induced cell death in C6 and U87 glioma cells in a dose and time dependent manner. The cell death in C6 and U87 glioma cells could be inhibited by necroptosis inhibitor necrotatin-1, not by pan-caspase inhibitor z-VAD-fmk. Shikonin treated C6 glioma cells presented electron-lucent cytoplasm, loss of plasma membrane integrity and intact nuclear membrane in morphology. The increased ROS level caused by shikonin was attenuated by necrostatin-1 and blocking ROS by anti-oxidant NAC rescued shikonin-induced cell death in both C6 and U87 glioma cells. Moreover, the expressional level of RIP-1 was up-regulated by shikonin in a dose and time dependent manner as well, but NAC suppressed RIP-1 expression. Conclusions We demonstrated that the cell death caused by shikonin in C6 and U87 glioma cells was mainly via necroptosis. Moreover, not only RIP-1 pathway, but also oxidative stress participated in the activation of shikonin induced necroptosis.

Inhibition of autophagy induced by proteasome inhibition increases cell death in human SHG-44 glioma cells

AbstractAim:The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Recent studies suggest that proteasome inhibitors may reduce tumor growth and activate autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the effect of autophagy on the destiny of glioma cells remains unclear. In this study, we sought to investigate whether inhibition of the proteasome can induce autophagy and the effects of autophagy on the fate of human SHG-44 glioma cells.Methods:The proteasome inhibitor MG-132 was used to induce autophagy in SHG-44 glioma cells, and the effect of autophagy on the survival of SHG-44 glioma cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by MTT assay. Apoptosis and cell cycle were detected by flow cytometry. The expression of autophagy related proteins was determined by Western blot.Results:MG-132 inhibited cell proliferation, induced cell death and cell cycle arrest at G2/M phase, and activated autophagy in SHG-44 glioma cells. The expression of autophagy-related Beclin-1 and LC3-I was significantly up-regulated and part of LC3-I was converted into LC3-II. However, when SHG-44 glioma cells were co-treated with MG-132 and 3-MA, the cells became less viable, but cell death and cell numbers at G2/M phase increased. Moreover, the accumulation of acidic vesicular organelles was decreased, the expression of Beclin-1 and LC3 was significantly down-regulated and the conversion of LC3-II from LC3-I was also inhibited.Conclusion:Inhibition of the proteasome can induce autophagy in human SHG-44 glioma cells, and inhibition of autophagy increases cell death. This discovery may shed new light on the effect of autophagy on modulating the fate of SHG-44 glioma cells.

Celastrol causes apoptosis and cell cycle arrest in rat glioma cells.

Abstract Glioma still remains a major health problem in the world. Celastrol has been proved to be an effective natural proteasome inhibitor and was used for treatment of autoimmune disease, chronic inflammation and neurodegenerative disease. However, its effect on glioma is unclear. In this study, we investigated the therapeutic effects of celastrol on C6 glioma cells. The results demonstrated that celastrol inhibited cell proliferation in a time- and dose-dependent manner, suppressed proteasome chymotrypsin-like activity and induced apoptosis and cell cycle arrest at G2/M phase in C6 cells. Proapoptosis proteins bax and caspase-3 were up-regulated, as well as cell cycle G2/M-related proteins cyclin B1, p21 and p27. Conversely, anti-apoptosis proteins bcl-2 and XIAP and cell cycle regulator cyclin-dependent kinase 2 were down-regulated. Taken together, our data suggest that celastrol can suppress proteasome activity and induce apoptosis and cell cycle arrest in C6 glioma cells, which make it be a potential drug for glioma.

Gross Total Resection of Glioma with the Intraoperative Fluorescence-guidance of Fluorescein Sodium

Objective. High dose fluorescein sodium has been utilized for fluorescence-guided tumor resection with conflicting reports on the efficacy of this procedure. The aim of this study was to reevaluate the utility and clinical limitations of using fluorescein sodium for the treatment and resection of glioma brain tumors. Methods. Patients diagnosed with glioma were divided into two groups with a total of 22 patients enrolled in the study: 1) the study group (n=10), patients that received intravenous injection of fluorescein sodium and 2) the control group (n=12), patients that did not receive injections during surgical resection. Quality of life was evaluated according to Karnofsky Performance Scale (KPS) score and neurological status. Fluorescein sodium was intravenously injected at a dose of 15-20mg/kg of body weight. Glioma resection was evaluated preoperative and postoperatively with enhanced Magnetic Resonance Imaging (MRI). Results. Significant differences in the gross total resection (GTR) rates were observed between the two patient groups (Fishers Exact Test p=0.047). Progressive free survival was significantly longer in the study group (Students T-Test p=0.033) as well as in the GTR group (Students T-Test p=0.0001) compared to the control and non-GTR groups, respectively. Three patients in the study group and four patients in the control group had transient neurological deterioration. One patient in the control group had permanent hemiplegia. Conclusion. The intraoperative utility of using fluorescein sodium can significantly increase the GTR rate without obvious deterioration. In addition, we find that it is better to apply the fluorescein sodium in the cases with BBB (blood-brain barrier) disruption, which had been enhanced in preoperative MRI.

Deoxypodophyllotoxin triggers parthanatos in glioma cells via induction of excessive ROS

Parthanatos is a new form of programmed cell death that is regulated by hyper-activated PARP-1, and is emerging as a new strategy to kill cancer cells. Deoxypodophyllotoxin (DPT) is a natural chemical that is found to induce cancer cell death, in which the role of parthanatos is unknown. Thus, we investigated this issue in this study by using glioma cell lines and mice model of xenograft glioma. We found that DPT induced glioma cell death in vitro and inhibited the growth of xenograft glioma in vivo, which was accompanied with parthanatos-related biochemical events including expressional upregulation of PARP-1, cytoplasmic accumulation of PAR polymer, and nuclear translocation of AIF. In vitro study revealed that genetic knockdown of PARP-1 with small interfering RNA attenuated DPT-induced elevation in the cytoplasmic PAR-polymer and the nuclear AIF, as well as protected glioma cells against the toxicity of DPT. Further, antioxidant NAC, as well as PARP-1 inhibitor 3AB, not only alleviated the overproduction of ROS caused by DPT, but also reversed the above-mentioned biochemical events, maintained mitochondrial membrane potential and rescued glioma cells death. Therefore, we demonstrated that deoxypodophyllotoxin triggered parthanatos in glioma cells via induction of excessive ROS.

Protection of ischemic postconditioning against neuronal apoptosis induced by transient focal ischemia is associated with attenuation of NF-κB/p65 activation.

Background and Purpose Accumulating evidences have demonstrated that nuclear factor κB/p65 plays a protective role in the protection of ischemic preconditioning and detrimental role in lethal ischemia-induced programmed cell death including apoptosis and autophagic death. However, its role in the protection of ischemic postconditioning is still unclear. Methods Rat MCAO model was used to produce transient focal ischemia. The procedure of ischemic postconditioning consisted of three cycles of 30 seconds reperfusion/reocclusion of MCA. The volume of cerebral infarction was measured by TTC staining and neuronal apoptosis was detected by TUNEL staining. Western blotting was used to analyze the changes in protein levels of Caspase-3, NF-κB/p65, phosphor- NF-κB/p65, IκBα, phosphor- IκBα, Noxa, Bim and Bax between rats treated with and without ischemic postconditioning. Laser scanning confocal microscopy was used to examine the distribution of NF-κB/p65 and Noxa. Results Ischemic postconditioning made transient focal ischemia-induced infarct volume decrease obviously from 38.6%±5.8% to 23.5%±4.3%, and apoptosis rate reduce significantly from 46.5%±6.2 to 29.6%±5.3% at reperfusion 24 h following 2 h focal cerebral ischemia. Western blotting analysis showed that ischemic postconditioning suppressed markedly the reduction of NF-κB/p65 in cytoplasm, but elevated its content in nucleus either at reperfusion 6 h or 24 h. Moreover, the decrease of IκBα and the increase of phosphorylated IκBα and phosphorylated NF-κB/p65 at indicated reperfusion time were reversed by ischemic postconditioning. Correspondingly, proapoptotic proteins Caspase-3, cleaved Caspase-3, Noxa, Bim and Bax were all mitigated significantly by ischemic postconditioning. Confocal microscopy revealed that ischemic postconditioning not only attenuated ischemia-induced translocation of NF-κB/p65 from neuronal cytoplasm to nucleus, but also inhibited the abnormal expression of proapoptotic protein Noxa within neurons. Conclusions We demonstrated in this study that the protection of ischemic postconditioning on neuronal apoptosis caused by transient focal ischemia is associated with attenuation of the activation of NF-κB/p65 in neurons.

Ischaemic Postconditioning Rescues Brain Injury Caused by Focal Ischaemia/Reperfusion via Attenuation of Protein Oxidization

OBJECTIVE: To investigate the effects of ischaemic postconditioning on brain injury and protein oxidization in focal ischaemia/reperfusion. METHODS: Adult male Wistar rats (n = 30) were randomly divided into sham-operated, ischaemia, and ischaemic postconditioning groups. Ischaemia was produced by middle cerebral artery occlusion and ischaemic postconditioning was performed using three cycles of 30-s/30-s reperfusion/reocclusion after 2 h of ischaemia. Brain infarction size, hydrogen peroxide concentration, superoxide dismutase (SOD), catalase (CAT) and proteasome activities, protein carbonyl derivatives and advanced oxidized protein products (AOPPs) were evaluated. RESULTS: The size of brain infarction after ischaemic postconditioning was significantly smaller compared with the ischaemia group, and was concomitant with significant reduction in protein carbonyl derivatives and AOPPs. The activities of SOD, CAT and proteasomes were elevated by ischaemic postconditioning compared with the ischaemia group. CONCLUSIONS: Ischaemic post-conditioning is an effective way of reducing the size and effects of brain infarction caused by focal ischaemia/reperfusion, possibly due to a decrease in oxidized protein levels. Decreasing protein oxidization may, therefore, be a useful target for preventing cerebral injury.

Proteasome inhibitor MG-132 induces C6 glioma cell apoptosis via oxidative stress.

Aim:Proteasome inhibitors have been found to suppress glioma cell proliferation and induce apoptosis, but the mechanisms are not fully elucidated. In this study we investigated the mechanisms underlying the apoptosis induced by the proteasome inhibitor MG-132 in glioma cells.Methods:C6 glioma cells were used. MTT assay was used to analyze cell proliferation. Proteasome activity was assayed using Succinyl-LLVY-AMC, and intracellular ROS level was evaluated with the redox-sensitive dye DCFH-DA. Apoptosis was detected using fluorescence and transmission electron microscopy as well as flow cytometry. The expression of apoptosis-related proteins was investigated using Western blot analysis.Results:MG-132 inhibited C6 glioma cell proliferation in a time- and dose-dependent manner (the IC50 value at 24 h was 18.5 μmol/L). MG-132 (18.5 μmol/L) suppressed the proteasome activity by about 70% at 3 h. It induced apoptosis via down-regulation of antiapoptotic proteins Bcl-2 and XIAP, up-regulation of pro-apoptotic protein Bax and caspase-3, and production of cleaved C-terminal 85 kDa PARP). It also caused a more than 5-fold increase of reactive oxygen species. Tiron (1 mmol/L) effectively blocked oxidative stress induced by MG-132 (18.5 μmol/L), attenuated proliferation inhibition and apoptosis in C6 glioma cells, and reversed the expression pattern of apoptosis-related proteins.Conclusion:MG-132 induced apoptosis of C6 glioma cells via the oxidative stress.

JNK Activation Contributes to Oxidative Stress-Induced Parthanatos in Glioma Cells via Increase of Intracellular ROS Production

Parthanatos is a form of PARP-1-dependent programmed cell death. The induction of parthanatos is emerging as a new strategy to kill gliomas which are the most common type of primary malignant brain tumor. Oxidative stress is thought to be a critical factor triggering parthanatos, but its underlying mechanism is poorly understood. In this study, we used glioma cell lines and H2O2 to investigate the role of JNK in glioma cell parthanatos induced by oxidative stress. We found that exposure to H2O2 not only induced intracellular accumulation of ROS but also resulted in glioma cell death in a concentration- and incubation time-dependent manner, which was accompanied with cytoplasmic formation of PAR polymer, expressional upregulation of PARP-1, mitochondrial depolarization, and AIF translocation to nucleus. Pharmacological inhibition of PARP-1 with 3AB or genetic knockdown of its level with siRNA rescued glioma cell death, as well as suppressed cytoplasmic accumulation of PAR polymer and nuclear translocation of AIF, which were consistent with the definition of parthanatos. Moreover, the phosphorylated level of JNK increased markedly with the extension of H2O2 exposure time. Either attenuation of intracellular ROS with antioxidant NAC or inhibition of JNK phosphorylation with SP600125 or JNK siRNA could significantly prevent H2O2-induced parthanatos in glioma cells. Additionally, inhibition of JNK with SP600125 alleviated intracellular accumulation of ROS and attenuated mitochondrial generation of superoxide. Thus, we demonstrated that JNK activation contributes to glioma cell parthanatos caused by oxidative stress via increase of intracellular ROS generation.

Inhalation of hydrogen gas attenuates cognitive impairment in transient cerebral ischemia via inhibition of oxidative stress

Abstract Objective: To investigate the effects of inhalation of hydrogen gas on cognitive impairment induced by transient cerebral ischemia and its potential mechanism. Methods: Two-vessel occlusion rat model was used to produce 10-minute transient global cerebral ischemia. One hundred and twenty male Wistar rats were randomly divided into sham, sham+H2, ischemia, and ischemia+H2 groups (n = 30 each group). Inhalation of 2% hydrogen gas was performed immediately at the end of operation and lasted for 3 hours. Cognitive function of rats was evaluated via Morris water maze. Neuronal damage in the CA1 region was quantified according to their morphological changes revealed by hematoxylin–eosin staining. The levels of oxidative stress products malondialdehyde (MDA) and 8-iso-prostaglandin F2alpha, and the activities of anti-oxidative enzymes catalase and superoxide dismutase were measured to investigate the effects of inhalation of hydrogen gas on oxidative stress. Results: Inhalation of hydrogen gas decreased significantly the average latency of the ischemic rats in finding hidden platform and elongated markedly their retention in the target quadrant. The neuronal density 3·3±2·1 cells/mm in CA1 region of the ischemic rats increased to 21·7±2·6 cells/mm after they were treated with hydrogen gas. Moreover, hydrogen gas made higher levels of MDA and 8-iso-PGF2α in the ischemic rats attenuate to 3·2±0·2, 3·5±0·5, 3·4±0·3 and 26·4±2·3, 28·2±2·6, 26·8±2·1 at reperfusion 4, 24, and 72 hours, respectively (P<0·01 versus ischemia group at each indicated time). By contrast, the activities of superoxide dismutase and catalase damaged by ischemia/reperfusion recovered to 129·7±14·8, 100·5±12·2 and 11·4±0·8, 9·6±1·1 at reperfusion 24 and 72 hours, respectively (P<0·01 versus ischemia group at each indicated time). Conclusion: Inhalation of hydrogen gas could attenuate cognitive impairment in the ischemic rats. This protection is associated with decreased neuronal death in CA1 region and inhibition of oxidative stress.