Research On The Effects And Mechanisms Of Ivermectin And Moxidectin Induce Autophagy In Glioma Cells

Gliomas are the most common tumors.With the current standard of medical treatment,surgery is the main therapeutic method,followed by adjuvant radiotherapy and chemotherapy.Ivermectin(IVM)and Moxidectin(MOX)belongs to macrolides which are broad-spectrum antiparasite drugs.IVM/MOX has an inhibitory effect on viability of glioma cells in vitro and in vivo.Thus,IVM/MOX has been identified as promising anticancer agent for glioma cell.In this study,IVM/MOX were used to treat U251 cells and C6 cells.Autophagy was measured by transmission electron microscopy(TEM),immunofluorescence,western blot and immunohistochemistry.Cell viability was detected with MTT and colony formation assay.The apoptosis rate was measured by flow cytometry and terminal deoxynucleotidyl transferase d UTP nick end labelling(TUNEL).Western blot and immunohistochemistry analyses were used to detect relevant protein expression.U251-derived xenografts were established for examination of MOX-induced autophagy on glioma in vivo.The main objective of this study was to explore autophagy induced by IVM/MOX in glioma cells.These results will provide theoretical basis for using of IVM and MOX.Results are as follows:(1)IVM/MOX induced autophagyof glioma cells in vivoThe effect of IVM/MOX on the induction cell autophagy in glioma cells was examined by TEM and GFP-LC3 transfection.TEM revealed obviously characteristic autophagosomes in glioma cells treated with IVM/MOX.Abundant LC3 puncta appeared in IVM/MOX-treated cells.The proteins related to cell autophagy were measured by western blot.As shown in results,LC3-II/LC3-I expression was more pronounced,and the level of P62 was decreased in glioma cells with an increased dose of IVM/MOX.(2)The machanisms of IVM/MOM on autophagy of glioma cellsWestern blot analyse was used to detect autophagyt relevant protein expression.As shown in results,p-AKT,P-m TOR were decreased with IVM/MOX treatment in a dose-dependent manner Finaly,the relative ATP levels were down-regulated in glioma cells treated with IVM/MOX.(3)The role of autophagy in IVM/MOX-induced cell apoptosisCells were treated with CQ for 1 h before treatment with IVM/MOX.Cell viability was detected with MTT and colony formation assay.These findings showed co-treatment with either CQ or 3-MA decreased the cell growth ability treated with IVM in glioma cells.But,cell growth ability was increased by inhibited autophagy treated with MOX.Cell apoptosis detected by flow cytometry analysis,the percentage of apoptotic cells by CQ in IVM-treated cells compared with those treated with IVM alone enhanced.These results showed that inhibition of autophagy repressed MOX-induced apoptosis in U251 and C6 cells.(4)IVM/MOX induced autophagy of glioma cells in vivoU251 cells were injected subcutaneously into athymic nude mice.Western b lot and immunohistochemistry analyses were used to detect relevant protein expression.A massive LC3 accumulation was on tumor sections in IVM/MOX-treated xenografts compared with the control group.IVM/MOX significantly induced autophagy of glioma cells in vivo.(5)The role of autophagy in IVM/MOX-induced cell apoptosis in vivoAn internal engraftment model was established and tested.Tumor growth curves in mice treated with IVM+CQ had a relatively slow trend compared with the IVM group.TUNEL staining and immunohistochemistry demonstrated more dead cells and the evident increase in apoptosis proportion in IVM+CQ-treated tumor tissues.Furthermore,compared with MOX group,co-treatment of MOX and CQ was less effective in reducing the tumor size.Immunohistoche mistry and TUNEL assay demonstrated a greater number of dead cells and an evident increase in the apoptotic proportion in MOX treated tumor tissues compared with the MOX+CQ treated tumor tissues.In conclusion,IVM and MOX significantly induced autophagy of glioma cells in vitro and in vivo.IVM/MOX inhibited AKT/m TOR signaling pathway and induced energy damage in glioma cells.IVM-induced inhibited promoted apoptosis in glioma cells,while MOX-induced autophagy autophagy apoptosis.Therefore,regulating autophagy combined with IVM/MOX may be an effective way to treat glioma.