Deoxypodophyllotoxin Induces PARP-1-dependent Cell Death (parthanatos) In Glioma Cells

Malignant glioma is the most common type of primary malignant brain tumor and is associated with high morbidity[20]. They are difficult to be removed out radically by surgery and resistant to radiotherapy and chemotherapy. Resistance to apoptosis is found to make glioma cells survive currently used medicines or radiotherapy[21]. Therefore, it is needed to develop new chemicals to induce glioma cell death not via apoptotic pathways.PARP-1, known as poly(ADP-ribose) synthetase 1 or poly(ADP-ribose) transferase 1, is mainly located at eukaryotic nucleus and involved in multiple facets of DNA repair[22]. In malignant gliomas, the basal level of PARP-1 are markedly higher than that in neurons, and its level is found to be positively associated with gliomas’ malignancy and poor survival of the patients[23]. PARP-1 could be activated by chromosomal DNA strand nicks and breaks due to the attack of reactive oxygen species(ROS) or genotoxic alkylating agents[24]. Mild activation of PARP-1 is responsible for repairing damaged DNA, which is an importance factor contributing to the resistance of glioma cells to current chemotherapy and radiotherapy. Thus, PARP-1 inhibitors are used to restore or enhance the sensitivity of glioma cells to these treatments[10]. Paradoxically, genetic evidences show PARP-1 cooperates with patched protein(Ptc1) to suppress the development of medulloblastoma, a subtype of malignant glioma[25]. Furthermore, hyper-activation of PARP-1 has been found recently to be a new pathway accounting for cancer cell death induced by chemical compounds[4, 5]. Therefore, previous studies reveal that PARP-1 is a protein with dual functions in modulating cancer cell survival or death, and previous similar studies suggest that PARP-1 dependent pathway might be a potential target for future treatment of glioma.Deoxypodophyllotoxin(DPT, Fig.1) is an active component and major lignan of the traditional plant Anthriscus sylvestris, which is a common wild plant in northwest Europe, in parts of North America, Africa, Asia and New Zealand[11]. Although DPT is found to have multiple biological functions such as anti-virus, anti-inflammation and anti-allergy[12], accumulating evidences have shown that DPT has potent anti-tumor activity as well. Animal study showed that DPT inhibited the growth of gastric tumor and lung cancer[13,14], and in vitro data demonstrated that DPT could induce death in various types of cell lines from glioma, lung cancer, gastric cancer, breast cancer, prostate cancer, and cervical carcinoma[13-17]. The anti-tumor effect of DPT is found to be associated with destabilization of microtubule, induction of mitochondria-related apoptosis, inhibition of angiogenesis and G2/M cell cycle arrest [11, 18]. Despite it was reported that DPT could induce cell death in U87 and SF126 glioma cells via mitochondria-related apoptosis pathway[12], it is still unclear whether PARP-1 contributes to DPT induced death in cancer cells. Clarifying this issue would help us to understand the mechanism underlying the anti-glioma effects of DPT.Objectives:in this study, we use rat C6 glioma cells and Human SHG-44 glioma cells to investigate whether DPT induces glioma cell death via PARP-1-dependent pathway.Methods:1.MTT assay was used to assess the viability of glioma cells treated with or without DPT.Lactate dehydrogenase cytotoxicity assay kit was used to assay cellular death rate. colony formation assay was used to assess cellular death under the lower dose of DPT colony formation assay was used access the long-term effect of DPT on the proliferation of glioma cells.2.Western Blotting was used to access the protein level of PARP-1 and AIF in cytoplasm and nucleus in a DPT dose and incubation time- dependent manner3.we used 3AB to investigate the role of PARP-1 in DPT-induced glioma cell death and the protein level of PAR,PARP-1 and AIF in cytoplasm and nucleus inhibited by 3AB.4.PARP-1 was genetically knocked down by using small interfering RNA(Si RNA) to clarify the function of PARP-1 in glioma cell death caused by DPT.5.We investigated the DPT-induced changes in mitochondria by using JC-1 staining and flow cytometry analysis.6.Single cell gel electrophoresis(SCGE) and DNA agarose gel electrophoresis are used to detecting strand damages in the DNA of a cell with applications of genotoxic agents7.The level of reactive oxygen species(ROS) was assessed by using redox-sensitive dye DCFH-DA.Results:1.DPT inhibits the viability of C6 and SHG-44 cells in a dose-and time-dependent manner.and DPT at 5nmol/L could effectively inhibit colony formation in both C6 and SHG-44 glioma cells at 6 days,LDH release assay demonstrated that DPT could kill glioma cells2.In C6 or SHG-44 glioma cells, cytoplasmic accumulation of PAR polymer increased with elevation of DPT concentration and incubation time extension. Also, the protein level of PARP-1 and AIF in cytoplasm and nucleus were both increased in a DPT dose- and incubation time- dependent manner3.Pretreatment with 3AB made the cell death ratio reduce markedly in C6 and SHG-44 cell, Western blotting analysis showed that cytoplasmic level of PAR polymer, the level of PARP-1 in cytoplasm and nucleus and nuclear translocation of AIF were all inhibited by 3AB.4.Compared with that in the scramble Si RNA group, cytoplasmic accumulation of PAR polymer and nuclear translocation of AIF were both markedly attenuated by PARP-1 Si RNA.and cell death ratio assay showed that knockdown of PARP-1 mitigated significantly DPT-induced cell death in C6 and SHG-44 glioma cells.5. When C6 and SHG-44 cells were treated respectively with 180nmol/L and 450nmol/L DPT, the red fluorescence decreased, but the green fluorescence increased concomitantly at 12 h and 24 h. Further investigation by flow cytometry proved as well that longer incubation with DPT resulted in more decline in mitochondrial membrane potentials. By contrast, PARP-1 inhibitor 3AB blocked the DPT-induced dissipation in mitochondrial membrane potentials.6.The average length of the comet tail was positively associated with DPT concentration and treatment time and smear bands on agarose gel were observed in the C6 and SHG-44 cells treated 12 h and 24 h with DPT,Further more,pretreatment with 3AB inhibited DPT-induced DNA fragmentation.7.DPT-induced production of ROS in glioma cells increased not only with the elevation in its concentration, but also with the extension of incubation time.DPT-induced excessive ROS production was obviously inhibited by NAC at each indicated time point and DPT concentration.Further more western blotting analysis showed that the production of PAR polymer, the up-regulated expression of PARP-1 within nucleus and cytoplasm, and AIF translocation to nucleus were all markedly mitigated by NAC.Conclusions:1.Glioma cells could be induced to death via PARP-1 dependent parthanatos,and DPT is a parthanatos inducer for glioma cells2.DNA damage and ROS played an important role in DPT-induced parthanatos in glioma cells...