The Study Of Cyclovirobuxine D Induces Mitochondrial Damage And Apoptosis In Glioblastoma Cells Via MitoSOX/Cofilin Pathway

BackgroundGlioma is a type of primary intracranial tumor,it accounted for 81% of CNS neoplasms.Glioma was grouped into Ⅰ-Ⅳ by WHO,the most fatal is Ⅳ called glioblastoma(GBM).At present,the clinical of standard treatment is surgery combined with radiation therapy,drug adjuvant chemotherapy.However,the mainly problem of treatment is that GBM resistance to the chemotherapeutic easily,which will put clinical treatment in a predicament.Therefore,it is meaningful that find an novel anti-GBM agent to improve the effect of treatment.Increased studies have shown that there is great potential for the development of active monomers of natural products into antitumor drugs.Cyclovirobuxine D(CVBD),is a steroidal alkaloid extracted from Buxus sinica,have been treated for cardiovascular diseases with Huangyangning tablets in clinical at present.Surprisingly,researchers have found that CVBD also has anti-tumor pharmacological activities,which could inhibit tumor cells proliferation of breast cancer,hepatocellular carcinoma,and gastric cancer,but the effect of CVBD in anti-glioma is rarely explored.Our previous study found that CVBD have the anti-proliferation effect in T98G、U251、U87 of GBM cells,and could lead to the mitochondrial damage and cell apoptosis.The aim of this paper is to deeply explore the molecular mechanism of CVBD induces the mitochondrial damage and apoptosis in GBM cells.ObjectiveTo clarify the mechanism of CVBD induces mitochondrial damage and apoptosis in GBM cells and provide a theoretical basis for the development of CVBD as a newly anti-GBM agent.MethodsWe used CCK-8 method to detected Cell viability;Detection of mitochondrial superoxide(MitoSOX)and cell apoptosis by flow cytometry;Western blot was used to detect the protein expression;Generation ROS and colocalization of cofilin with mitochondrial were determined by immunofluorescence assay.Results1.CVBD inhibits the cell proliferation of GBM cellsTreatment of T98 G,U251,and U87 cells with gradient concentrations of CVBD,the results shown that cell clone forming ability was clearly decreased in concentration dependently(P<0.05).The cell viability was also decreased in concentration dependently2.CVBD induces caspase 3-mediated apoptosis of GBM cellsCell apoptotic rate was apparently elevated by CVBD induction.The cleaved-PARP and cleaved-Caspase 3 levels were upregulated obviously(P<0.05).Z-VAD-FMK(inhibitor of caspase)was used to pretreat cells,could inhibit PARP degradation induced by CVBD and the cleaved-PARP,cleaved-Caspase 3 could significantly downregulation(P<0.05).These results shown that CVBD induced apoptosis is caspase 3 dependently.3.CVBD induces mitochondrial damage in GBM cellsThe mitochondrial membrane potential was decreased after cells treated with CVBD,it means CVBD may cause mitochondrial damage(P<0.05).We further observed by staining cells with Mito Tracker,the mitochondria were fission and become from filamentous to punctate.TEM reveled that the mitochondrial morphology was shrunken from rodlike to spheres and the mitochondrial cristae were indistinct.Furthermore,Western blot results shown that the expression level of cytochrome C(Cyto C)in cytoplasm was increased(P<0.05).These results shown that CVBD get rise to mitochondrial damage.4.CVBD induces mitochondrial damage through promoting mitochondrial translocation of cofilinWestern blot results shown that cells treated with CVBD could active dephosphorylation of cofilin and translocate to the mitochondria.Observation with confocal revealed that cofilin with mitochondrial could colocalization induced by CVBD.Knockdown of cofilin with sh RNA significantly attenuated mitochondrial damage and apoptosis induced by CVBD(P<0.05),which is manifested by: the mitochondrial fragmentation was significantly decreased;the cell apoptosis rate was apparently decreased;the degradation of apoptosis related protein PARP was inhibited,and the expression of C-PARP,C-Caspase 3 was downregulation apparently;the expression of Cyto C in cytoplasm was decreased.These results indicated that CVBD induces mitochondrial damage though promoting mitochondrial translocation of cofilin.5.CVBD promotes mitochondrial translocation of cofilin through inducing mitochondrial superoxide generationIntracellular ROS and mitochondrial superoxide(MitoSOX)levels increased obviously in a dose-dependent manner after cells treated with CVBD(P<0.05).Pretreated cells with NAC(ROS inhibitor)and Mito Q(MitoSOX inhibitor)respectively.Inhibition of cell viability induced by CVBD was clearly decreased.Cell apoptotic rate and the expression level of apoptotic-related protein was decreased(P<0.05).Furthermore,inhibition of mitochondrial superoxide could significantly downregulate expression of cofilin in mitochondrial and attenuate colocalization of cofilin with mitochondria(P<0.05).These results indicated that CVBD promotes mitochondrial translocation of cofilin by inducing generation of mitochondrial superoxide.ConclusionCVBD increases production of mitochondrial superoxide(MitoSOX)by promoting oxidant stress in GBM cells and thereby causing mitochondrial translocation of cofilin,leading to mitochondrial damage,and inducing apoptosis.