The Effects And Mechanisms Of JNK On Pristimerin Induced Necroptosis In Glioma Cells

Background:Malignant glioma is the most common type of primary malignant brain tumor with higher rates of morbidity and recurrence [1]. Glioma cells could survive current chemotherapy and radiotherapy due to their resistance to apoptosis[2], so that alternative strategy is needed to kill glioma cells not via induction of apoptosis.Necrosis was regarded previously as a passive form of cell death, but mounting evidences indicate that it is also regulated by complex signal transduction pathways and execution mechanisms[3]. As a counterpart to uncontrolled necrosis, the term“programmed necrosis” was introduced to define this controlled necrosis[3-4].Programmed necrosis is found to be involved in either physiological scenarios such as embryonic development or pathological settings such as ischemic disorders [5].Moreover, induction of necroptosis by natural or synthesized chemicals such as shikonin, β-Lapachone and sorafenib is demonstrated to be an effective treatment strategy for cancer cells which are resistance to current treatments[6-8]. However, our understanding remains poor on how to induce programmed necrosis in cancer cells.Pristimerin is a natural quinonemethide triterpenoid compound(Figure 1)isolated from various plant species in the Celastraceae and Hippocrateaceae families[9]. It was found that pristimerin induced caspase-dependent apoptosis via causing mitochondrial dysfunction in various types of cancer cell lines such as ovarian carcinoma, hepatocellular carcinoma, cervical cancer, breast cancer and glioma[9-13].Moreover, previous studies have revealed that mitochondria play a crucial role not only in regulation of apoptosis, but also in modulation of programmed necrosis [14-16].Thus, we speculated that pristimerinmight induce programmed necrosis in glioma cells. In this study, we used glioma cell lines and mice model of xenograft glioma to examine whether pristimerin induces glioma cell death via necroptosis pathway and investigate the underlying mechanism.Objectives:In this study, we used glioma cell lines and mice model of xenograft glioma to examine whether pristimerin induces glioma cell death via necroptosis pathway and investigate the underlying mechanism.Methods:1.MTT assay was used to examine the toxic effect of pristimerin on glioma cells.2.We used flow cytometry with Annexin V-FITC and PI dual staining to examine the cell death form. Hoechst 33342/PI dual staining was used to determine the nuclear morphology and membrane integrity. DNA band on agarose gel electrophoresis which was used to discriminate necrosis from apoptosis. Morphological changes in C6 and U251 glioma cells induced by pristimerin is observed through TEM(Transmission Electron Microscopy).3. Mitochondrial membrane potential was determined by the retention of the dye Rhodamine 123. We investigated the protein levels of Bax, Bcl-2, nuclear AIF and caspase 3 by western blotting.4. In order to clarify the role of AIF in pristimerin-induced necrosis in glioma cells, the level of AIF was knocked down by using small interfering RNA.5. We investigated the role of JNK activation in pristimerininduced glioma cell death by using its specific inhibitor SP600125 and JNK Si RNA.6.The anti-glioma effect of pristimerin in vivo was investigated by using nude mouse with subcutaneous xenograft glioma.7. The average level of intracellular ROS was evaluated by using redoxsensitive dye DCFH-DA. Mitochondrial superoxide was assayed by using Mito SOX red as described by manufacture’s instruction. Intracellular total GSH was measured byusing the DTNB-GSSH reductase recycling assay kit as described by the manufacture.Results:1. The viabilities of C6, U251 and U87 glioma cells decreased significantly with the increase of pristimerin doses and the extension of incubation time.2. The necrosis(Annexin V-/Pl+) rates of glioma cells treated with pristimerin increased markedly with the extension of incubation time in C6, U251 and U87 cells.The nuclei of the cells treated with pristimerin could be also stained with PI(in red color) and had no signs of nuclear condensation or fragmentation. The glioma cells treated with pristimerin presented smearing DNA band on agarose gel electrophoresis.pristimerin-treated cells presented chromatinolysis, electron-lucent cytoplasm, loss of plasma membrane integrity, and intact nuclear membrane3. Pristimerin-induced changes in mitochondrial membrane potential and found that it decreased at 1.5 h and further decreased at 6 h. pristimerin induced mitochondrial depolarization via up-regulating the ratio of Bax/Bcl-2 and caused AIF accumulation within the nuclei of glioma cells.4. MTT assay and flow cytometry analysis showed that the reduced viability and the increased necrosis caused by pristimerin at 6 hours were both partially reversed when AIF was knocked down by Si RNA5. Western blotting analysis showed that the levels of JNK and phosphor-JNK(the active form of JNK) were both up-regulated significantly at 1.5 h and peaked at 6h, indicating JNK was insistently activated by pristimerin in glioma cells. As well as inhibition of JNK phosphorylation, SP600125 reversed pristimerin-induced mitochondrial membrane potential decline, abnormal expression of Bax and Bcl-2 and nuclear translocation of AIF. The up-regulated nuclear AIF, the increased Baxl/Bcl-2and the declined mitochondrial membrane potential caused by pristimerin were all prevented in the cells transfected with JNK Si RNA.6. Treatment with pristimerin at the dosage of 1 mg/kg and 3 mg/kg inhibited the growth of xenograft glioma at day 5 and day 4, respectively. Moreover, this inhibitory effect was more significantly in the 3 mg/kg group. At day 8 when the mice were sacrificed, both the average volume and the average weight of the glioma in 1mg/kg group were significantly bigger than that in the 3 mg/kg group.7. The intracellular ROS was overproduced in the cells treated with pristimerin because the green fluorescence density increased to 2.57 ± 0.47 times in C6 cells andto 3.16 ± 0.52 times in U251 cells when compared with that in control cells. we also found that GSH, an intracellular ROS scavenger, decreased at 1.5 h of incubation with pristimerin, and reduced further at 3.0 h when compared with that in control group.Western blotting analysis revealed that inhibition of ROS with NAC attenuated pristimerin-induced upregulation and phosphorylation of JNK.Conclusions:1. Pristimerin induced programmed necrosis in glioma cells was AIF-dependent.2. Pristimerin triggers AIF-dependent programmed necroptosis in glioma cells via activation of JNK.3. Pristimerin triggers necroptosis in glioma cells via activation of JNK by ROS overproduced in mitochondria..