JNK Contributes To Glioma Cell Parthanatos During The Oxidative Stress

Malignant glioma is the most common type of primary malignant brain tumor with higher morbidity[25, 26]. Resistance to apoptosis is found to make glioma cells survive currently used chemotherapy or radiotherapy[26]. PARP-1, as a highly expressed nuclear protein that senses DNA-strand breaks and is involved in multiple facets of DNA repair and maintenance of genomic stability in glioma cells[27], was thought to be a factor responsible for the resistance of glioma cells to apoptosis after being mildly activated, and inhibition of PARP-1 activation has been a strategy to recover the sensitivities of glioma cells to chemotherapy or radiotherapy[28-30]. However, recent evidences have shown that hyper-activation of PARP-1 leads to cell death[32,33], which is established as a new modality of programmed cell death and named as “Parthanatos” to distinguish it from caspase dependent apoptosis, necrosis and other modalities of cell death[27].According to a cell death nomenclature committee, parthanatos is defined by two main criteria. One is excessive PAR synthesis should accompany cell death, and the other is the cell death should be completely or partially prevented by PARP-1 deletion or inhibitor treatment[34]. Experimental studies reveal that parthanatos is not only involved in multiple pathological conditions such as diabetes, inflammation, cerebral hypoxia/ischemia and head trauma[34-37], but also contributed to chemicals-induced death in cancer cells such as glioma, esophageal squamous-cell carcinoma and gastric cancer[32,33,38]. As a distinct cell death pathway, parthanatos is characterized by a series of choreographed biochemical events including hyper-activation of PARP-1, cytoplasmic accumulation of PAR polymer, mitochondrial depolarization, and nuclear translocation of AIF. Finally, the nuclear AIF initiates chromatinolysis or condensation and results in cell death [27, 34].Although the activation of PARP-1 is thought to be induced by chromosomal DNA strand nicks and breaks due to the attack of genotoxic agents[27], ROS was also found to participate in regulation of parthanatos under the condition of genotoxic stress. Chiu et al proved that inhibition of ROS attenuated PARP-1-dependent cell death in the mouse embryonic fibroblasts treated with MNNG, an alkylating agent that damages DNA directly[39]. Similarly, ROS was found in our previous study to account for the hyper-activation of PARP-1 in the glioma cells treated with deoxypodophyllotoxin[32]. However, it is still poorly understood which signal pathway is involved in PARP-1 hyper-activation in glioma cells under the condition of oxidative stress, despite MAPK subfamily ERK pathway was reported to participate in the regulation of parthanatos caused by oxidative stress in human lymphocytes[40]. JNK, which is another subfamily of MAPK, could also be activated by ROS and plays an important role in regulation of programmed glioma cell death[41]. We thus speculate that JNK might have a regulatory effect on parthanatos caused by oxidative stress. Given that H₂O₂ is a member of ROS that also include superoxide and hydroxyl radicals[16] and extensively used to induce oxidative stress in various types of cancer cells such as gastric carcinoma, cervical cancer, colorectal cancer, breast cancer and glioma[17-24], we thus used human glioma cell lines and H₂O₂ in this study to investigate the role of JNK in glioma cell parthanatos during oxidative stress.Objectives:we use glioma cell lines and H₂O₂ to investigate how JNK regulates parthanatos in glioma cells during the oxidative stress. Methods:1、MTT assay was used to assess the glioma cells’ viabilities after being treated with H₂O₂.2、Lactate dehydrogenase cytotoxicity assay kit was used to assay cellular death rate after the glioma cells being treated with H₂O₂.3、annexin V-FITC detection kit was used for assessment of cell death modality and the Mitochondrial membrane potential was determined by the retention of the dye Rhodamine 123 via analyzed with flow cytometry。4、The average level of intracellular ROS was evaluated by using redox-sensitive dye DCFH-DA and mitochondrial superoxide was assayed by using Mito SOX red,the stained cells were observed under the fluorescence microscope,the fluorescence density was measured by fluorescence spectrometer。5、After the glioma cells being treated with H₂O₂,incubated with Heochst33342,then the cells were visualized under fluorescence microscope.6、Knocked down PARP-1 or JNK with small interfering RNA and examined H₂O₂-induced parthanatos in glioma cells.7、Expression changes in cytoplasmic PAR 、PARP-1、JNK、p-JNK、AIF were examined by western blotting after the glioma cells being treated with H₂O₂. Results:1、MTT assay proved that H₂O₂ induced glioma SHG-44, U251 and U87 glioma cells death,LDH release assay indicated that H₂O₂ triggered SHG-44、U251 and U87 glioma cell death in a concentration-dependent manner. Pretreatment with 3AB inhibited PARP-1、knocked down PARP-1 with small interfering RNA、pretreatment with NAC inhibited Intracellular ROS or pretreatment with SP600125 inhibited JNK phosphorylation significantly improved the glioma cells’ viabilities after being treated with H₂O₂.2、Annexin V-FITC detection kit via analyzed with flow cytometry proved that pretreatment with 3AB inhibited PARP-1 improved the percentage of living cells after being treated with H₂O₂. Rhodamine 123 via analyzed with flow cytometry proved that pretreatment with 3AB inhibited PARP-1 prevented the declined mitochondrial depolarization after being treated with H₂O₂. Annexin V-FITC detection kit via analyzed with flow cytometry proved that knocked down PARP-1 with small interfering RNA or pretreatment with NAC inhibited Intracellular ROS or pretreatment with SP600125 inhibited JNK phosphorylation reversed the reduction of cellular viabilities and cell death caused by H₂O₂.3、The redox-sensitive dye DCFH-DA and using Mito SOX red stained the cells observed under the fluorescence microscope revealed that the glioma cells treated with H₂O₂ had much brighter green and red fluorescence than the cells in control group,the fluorescence density measured by fluorescence spectrometer indicated that H₂O₂ induced accumulation of intracellular ROS and over-generation of mitochondrial superoxide、pretreatment with NAC inhibited Intracellular ROS or pretreatment with SP600125 inhibited JNK phosphorylation counteracted the increase of intracellular ROS、pretreatment with SP600125 inhibited JNK phosphorylation counteracted the over-generation of mitochondrial superoxide.4、Incubated with Heochst33342,the cells were visualized under fluorescence microscope after the glioma cells being treated with H₂O₂, AIF accumulated obviously with nuclei.5、The western blotting examined the expression changes in cytoplasmic PAR 、PARP-1、JNK、p-JNK、AIF after the glioma cells being treated with H₂O₂:PARP-1 up-regulated in both cytoplasm and nuclei,cytoplasmic PAR polymer significantly up-regulated and AIF accumulated obviously with nuclei after the glioma cells treated by H₂O₂ in a concentration and incubation time dependent manner. Pretreatment with 3AB inhibited PARP-1、knocked down PARP-1 with small interfering RNA or pretreatment with NAC inhibited Intracellular ROS, inhibited the up-regulated expression of PARP-1, suppressed the increase of cytoplasmic PAR polymer and AIF translocation into nucleus after the glioma cells treated by H₂O₂。Exposure to H₂O₂ triggered concentration-dependent upregulation of phosphorylated JNK.Pretreatment with SP600125 inhibited JNK phosphorylation or knocked down JNK with small interfering RNA,inhibited JNK phosphorylation,also inhibited the up-regulated expression of PARP-1, suppressed the increase of cytoplasmic PAR polymer and AIF translocation into nucleus after the glioma cells treated by H₂O₂。 Conclusions:1、ROS induced glioma cell parthanatos.2、JNK activation contributes to glioma cell parthanatos.3、JNK regulate intracellular ROS level contributes to glioma cell parthanatos...