Cyclophilin A Contributes To Shikonin-induced Glioma Cell Necroptosis Via Promotion Of Chromatinolysis

Background:Glioma is one of the most common primary malignant tumors of the nervous system with a high incidence rate.The treatment of glioma in the clinic primarily involves surgery with adjuvant radiotherapy and chemotherapy.However,following systematic treatment,the average survival of most patients is less than one year.It may be that glioma cells are anti-apoptotic,making them insensitive to radiotherapy and chemotherapy.Therefore,an effective treatment involves inducing necroptosis in the glioma cells.Different from apoptosis,necroptosis is a caspase-independent mode of programmed cell death that involves a mechanism similar to that of apoptosis,but with morphological features similar to that of necrosis.In the process of necroptosis,receptor interacting serine-threonine protein kinases1(RIP1)is activated first.Then the activated RIP1 interacts with its downstream signal RIP3 through their RIP homotypic interaction motifs to form a protein complex called necrosome.In addition,RIP3 can also be activated by self-phosphorylation.The process of necroptosis is usually accompanied by cell swelling,cellular membrane disruption,DNA damage and energy depletion.DNA damages such as DNA single strand break,DNA double strand breaks(DSBs)and many other forms occur.The most serious form of DNA damage is DNA DSBs,which can cause chromatinolysis and cell death.When DNA replication is affected by chemotherapeutic drugs,ionizing radiation,or oxidative stress,it will lead to DNA damage and DNA DSBs.The role of necroptosis in DNA DSBs remains elusive.In the process of DNA DSBs,DNA-dependent protein kinase catalytic subunit(DNA-PKcs)and ataxia telangiectasia mutated(ATM)will be activated to promote the phosphorylation of histone variant H2 AX to ?-H2 AX.Thus,?-H2 AX is generally considered as a marker of DNA DSBs.Meanwhile,?-H2 AX can also recruit AIF in the mitochondria and Cyclophilin A(Cyp A)in the cell to the nucleus,forming a DNA degradation complex and promoting chomatinolysis.DNA DSBs is the key factor resulting chomatinolysis.Shikonin is a kind of naphthpquinone that is extracted from Lithospermum erythrorhizon.It has been used for more than 2000 years for the treatment of infections,inflammation,and bleeding diseases.Moreover,Shikonin has been demonstrated to induce necroptosis in multiple myeloma and osteosarcoma.In our previous studies,we have demonstrated that Shikonin can induce necroptosis in glioma cells,activate its downstream signaling factor MLKL by activating RIP1 and RIP3,and promote chromatinolysis by causing nuclear translocation of AIF and the formation of ?-H2 AX.At the same time,it has been reported that Cyp A is also involved in the nuclear translocation of AIF during apoptosis and can positively regulate the nuclear translocation of AIF,and promote chromatinolysis by combining with AIF and ?-H2 AX to form the DNA degradation complex.However,it is unclear whether Cycp A is involved in Shikonin-induced necroptosis and whether it is regulated by RIP1 and RIP3.Therefore,in this study,we use human and rat glioma cell lines and mice model of xenograft glioma to investigate the role of Cyp A in Shikonin-induced chromatinolysis and the underlying mechanism.Objectives:In this study,we use human and rat glioma cell lines and mice model of xenograft glioma to investigate the role of Cyclophilin A in Shikonin-induced chromatinolysis and the underlying mechanism.Methods:1.MTT assay was used to examine the viability of glioma cells after being treated with Shikonin.2.LDH release assay was used to examine the death rate of glioma cells after being treated with Shikonin.3.The morphological changes of the glioma cells were observed by laser scanning confocal microscope combine with Hoechst 33258 staining.4.Mitochondrial membrane potential was determined by JC-1 via analyzed with flow cytometry.5.DNA agarose gel electrophoresis was used to detect the DNA damage of glioma cells.6.Comet assay was used to detect the DNA damage of glioma cells.7.DCFH-DA was used to evaluate the level of intracellular ROS and Mito SOX was used to assay the mitochondrial superoxide.The stained cells were observed via fluorescence microscope,the fluorescence density was measured by microplate reader.8.Knockdown of Cyclophilin A,AIF,RIP1 or RIP3 with si RNA was used to investigate their roles in shikonin-induced necropotosis.9.The effect of shikonin on glioma cells in vivo was investigated by C6 cell lines xenograft glioma mice model.10.Western Blot was used to investigated the expressional level of related proteins.11.The expression of Cyclophilin A,AIF and ?-H2 AX in glioma cells were observed by laser scanning confocal microscope and immunofluorescence staining.Results:1.MTT and LDH release assay showed that shikonin could induce glioma cell necroptosis and kill glioma cells in a time-dependent manner.2.Cyclophilin A was upregulated in a time-dependent manner in glioma cells after treated with shikonin.Cyclophilin A inhibitor Cs A or knockdown of Cyclophilin A and AIF with si RNA could prevent shikonin-induced glioma cells death.3.DNA agarose gel electrophoresis and comet assay showed shikonin could elevate glioma cells DNA damage and DNA double strand breaks.However,these effects could be prevented by Cs A or si RNA Cyclophilin A and si RNA AIF.Shikonin could induce the expression of ?-H2 AX,p-ATM and p-DNAPKcs,while the overexpression of?-H2 AX,p-ATM and p-DNAPKcs could be inhibited by Cs A and si RNA Cyclophilin A.4.The morphology of glioma cells after treated with shikonin was chatacterized by necroptosis,such as cell swelling,cellular membrane disruption and chromatinolysis.Laser confocal microscopy combined with immunofluorescence staining was used to observe glioma cells.Cyclophilin A expression in glioma nucleus increased after the effect of shikonin.Meanwhile,AIF expression was decreased in mitochondria and increased in nucleus.?-H2 AX is formed in the nucleus.5.The intracellular ROS and the mitochondrial superoxide were increased after treated with shikonin.However,these effects could be prevented by Cs A or si RNA Cyclophilin A.6.C6 cell lines xenograft glioma mice model showed that compared with the control group,the size and volume of tumor decreased significantly in shikonin group.Meanwhile,DNA double strand breaks and related proteins were increased.Conclusions:1.Shikonin induces necroptosis in glioma cells and promotes the expression of Cyclophilin A.2.In the process of shikonin-induced necroptosis of glioma cells,activated Cyclophilin A can target mitochondria and trigger overproduction of superoxide in the mitochondria,resulting in mitochondrial depolarization and nuclear translocation of AIF.Meanwhile,excessive mitochondrial superoxide can increase intracellular ROS and accelerate the formation of ?-H2 AX.3.In the process of shikonin-induced necroptosis of glioma cells,Cyclophilin A promotes nuclear translocation of AIF and can form DNA degradation complex with AIF and ?-H2 AX,resulting in chromatinolysis.