The Effects And Mechanisms Of RIP1 And RIP3 On Shikonin Induced DNA Double-Strand Breaks In Glioma Cells

Background:Malignant glioma is the most common type of primary malignant brain tumor with an average survival not longer than one year,even though the patients accepted surgery in combination with postoperative chemotherapy and radiotherapy[4].The insensitivity of glioma cell to currently-used chemotherapeutic agents and radiotherapeutic regimes is attributed to their resistance to apoptosis[6].However,induction of necroptosis is found to be an effective strategy to kill the cancer cells resistant to apoptosis[7].Different with apoptosis,necroptosis is a type of caspase-independent programmed cell death with morphological similarities to necrosis and is primarily regulated by receptor interacting serine-threonine protein kinases1(RIP1)and 3(RIP3).Upon the induction of necroptosis,the activated RIP1 interacts with its downstream signal RIP3 via their RIP homotypic interaction motifs to form a protein complex named necrosome,in which RIP3 is activated by RIP1 or via auto-phosphorylation[8].Sequential activation of RIP1 and RIP3 were found to lead to energy depletion and cellular membrane disruption which are regard as critical biochemical events causing necroptotic cell death[9,10].However the roles of RIP1 and RIP3 remain unclear in regulation of DNA double strand breaks(DSBs),another hallmark of programmed necrosis[11].DNA double-strand breaks(DSBs)may arise spontaneously during DNA replication or following exposure to ionizing radiation(IR),chemotherapeutic drugs or oxidative stress[12].Moreover,DNA DSBs represent the most dangerous DNA damage,because a single DSB can be lethal when not be repaired or misrepaired[13].Notably,recent studies reveal that chromatinolysis is closely associated with DNA DSBs[11].Upon DSB generation,ataxia telangiectasia mutated(ATM)or DNA-dependent protein kinase catalytic subunit(DNA-PKcs)are activated,and then phosphorylate histone variant H2 AX at serine139 to produce ?H2AX[14].?H2AX specifically accumulates at the sites of DSBs that occur either in vitro or in vivo,and is thus regarded as a sensitive molecular maker of DNA DSBs[15,16].Although ?H2AX serves as a platform for ordered recruitment of the proteins involved in DNA repair,it recruits the AIF translocated from mitochondria to form DNA-degrading complex during the process of necroptosis[11].Thus,generation of DNA DSBs is a crucial step leading to chromatinolysis in the cells undergoing necroptosis.Shikonin is a natural naphthoquinone isolated from Lithospermum Erythrorhizon and has been demonstrated to induce necroptosis not only in glioma cells[17],but also in other types of cancer cells such as breast cancer,multiple myeloma and osteosarcoma[18-20].Additionally,shikonin could also cause DNA damage and inhibit DNA synthesis[21,22].However,it remains unclear whether shikonin-induced DNA damage is regulated by the RIP1 and RIP3.Therefore,we used rat and human glioma cell lines and mice model of xenograft glioma in this study to investigate the roles of RIP1 and RIP3 in shikonin-induced DNA DSBs in glioma cells and the underlying mechanism.Objectives:we used rat and human glioma cell lines and mice model of xenograft glioma in this study to investigate the roles of RIP1 and RIP3 in shikonin-induced DNA DSBs in glioma cells and the underlying mechanism.Methods:1.MTT assay was used to examine the viability of cells.2.Flow cytometry(Annexin V-FITC and PI double staining)was used to investigate the cell death models.3.Single cell gel electrophoresis(comet assay)was used to detect the DNA damage and DNA double strand breaks(DSB)of glioma cells.4.DCFH-DA probe was used to evaluate the level of intracellular reactive oxygen species(ROS).Mito SOX red probe was used to evaluate the level of mitochondrial superoxide.5.The DTNB-GSSH reductase recycling assay kit was used to evaluate the level of intracellular total GSH.6.Knockdown of RIP1 or RIP3 with small interfering RNA was used to investigate the roles of RIP1 or RIP3 in shikonin-induced necroptosis of glioma cells.7.The mice model of C6 xenograft glioma was used to investigate the effect of shikonin on glioma cells in vivo.8.Western-blotting was used to analyze the expressional level of related proteins in glioma cell and xenograft glioma tissue.9.ELISA was used to examine the level of the TNF-? in glioma cells and its culture medium.10.Immunohistochemical staining was used to detect the macrophage infiltration of glioma tissue.11.Immunofluorescence staining was used to detect the expression of ?H2AX in glioma cells through laser scanning confocal microscope.12.Co-immunoprecipitation was used to investigate the interaction of RIP1 and RIP3 in glioma cells and xenograft glioma tissue.Results:1.MTT assay showed that shikonin inhibited the viability of glioma cells in a range of concentration but had limited inhibitory effect of normal cells.Flow cytometry assay showed that shikonin induced necrosis but not apoptosis.2.RIP1 and RIP3 were upregulated in a time-dependent manner in glioma cells exposed to shikonin.Co-immunoprecipitation of RIP1 or RIP3 showed that shikonin promoted their assembly into necrosome.MTT assay showed that RIP1 inhibitor NEC-1,RIP3 inhibitor GSK872 or knockdown of RIP1 and RIP3 with small interfering RNA could prevent shikonin-induced reduction in the viabilities of glioma cells.The upregulated expression of RIP1 caused by shikonin was inhibited when glioma cells was pretreated with NEC-1.The upregulated expression of RIP3 caused by shikonin was inhibited when glioma cells was pretreated with GSK872.CYPD was upregulated in a time-dependent manner in glioma cells treated with shikonin.By contrast,caspase8 was down-regulated with the extension of incubation time.However,caspase3,activated caspase3 and activated caspase8 did not change significantly.ELISA results showed that shikonin inhibited the release of TNF-? from glioma cells.3.Single cell gel electrophoresis(neutral and alkaline comet assay)showed that shikonin elevated the glioma cells with comet tail and the DNA content within the tail,which indicated shikonin induced DNA damage and DNA double strand breaks(DSBs).However,these effect of shikonin could be prevented by NEC-1 and GSK872.As revealed by confocal microscope with immunocytochemistry staining,?H2AX formed in the nuclei of shikonin-treated glioma cells.?H2AX and p-ATM were upregulated in a time-dependent manner in glioma cells exposed to shikonin,which could be inhibited by NEC-1,GSK872 or knockdown of RIP1 and RIP3 with small interfering RNA.4.The mice model of C6 xenograft glioma revealed that the volume and weight of xenograft gliomas became smaller in the shikonin group than that in the control group.Moreover,RIP1,RIP3,?H2AX and p-ATM were upregulated,and the formation of necrosome consisting of RIP1 and RIP3 also increased in the shikonin group.The expression of HMGB1,HSP70 and calreticulin were upregulated in the shikoni group.Immunohistochemistry staining demonstrated that shikonin induced macrophage infiltration in glioma tissue.5.The ROS detected by DCFH-DA probe was overproduced in shikonin-treated glioma cells,which could be inhibited by NEC-1 and GSK872.The mitochondrial superoxide detected by Mito SOX red probe was overproduced in shikonin-treated glioma cells,which could be inhibited by NEC-1 and GSK872.GSH levels assay revealed that the intracellular total GSH decreased in shikonin-treated glioma cells,which could be reversed by prior administration of NEC-1 or GSK872.6.ROS detected by DCFH-DA probe was overproduced in glioma cells exposed to H2O2.MTT assay showed that the viabilities of glioma cells incubated with H2O2 reduced,which can be blocked by NAC.Comet assay revealed that H2O2 elevated glioma cells with comet tail and the DNA content within the tail,which indicated H2O2 induced DNA damage and DNA DSBs.?H2AX and p-ATM were upregulated in glioma cells exposed to H2O2,which could be prevented by NAC.The overproduction of ROS,reduction in viabilities,DNA damage,DNA DSBs,and upregulated expression of RIP1,RIP3,?H2AX and p-ATM could be blocked by NAC.Moreover,NAC and NEC-1 could inhibit the formation of necrosome consisting of RIP1 and RIP3.Conclusions:1.Shikonin induced glioma cell death in vivo and in vitro.2.Shikonin elevated the level of RIP1 and RIP3,and promoted the interaction of RIP1 and RIP3 in glioma cells.Thus,shikonin induced necroptosis in glioma cells.3.Shikonin induced DNA double strand breaks,in which ROS is the key effector.4.RIP1 and RIP3 promoted the overproduction of ROS via causing excessive generation of mitochondrial superoxide and depletion fo GSH..