Research On The Effects And Mechanisms Of Doramectin Inducing Apoptosis And Autophagy In Glioma Cells

Glioma is an aggressive primary central nervous system tumor that kills a large number of patients each year.Because of invasiveness and drug resistance,traditional treatment methods cannot completely remove glioma,and the prognosis is poor,so it is of great significance to develop new treatment methods and chemotherapy drugs for glioma patients.As a derivative from avermectin,doramectin belongs to macrolide class antiparasitic drug,which can widely inhibit parasite activity in animals in vivo and in vitro,and is widely used in livestock industry.Compared with other Macrolides are antiparasitic drugs,doramectin is absorbed more quickly,has a longer lasting effect and plasma half-life in animals.The previous studies have shown that ivermectin and avermectin inhibited the growth of glioma cells inducing apoptosis in vitro and in vivo,so they are considered as new type of potential anticancer drugs.However,the anticancer effect of doramectin has still not yet been documented.Our study explore d the role of doramectin in glioma cells.The proliferation effects of doramectin on glioma cells were detected by MTT and colony formation assay;the migration inhibition of doramectin on glioma cells were observed by scratch test;the contents of cell apoptosis rate and cell cycle arrest were detected by using flow cytometry;TEM and GFP-LC3 plasmid transfection showed that doramectin can induce autophagy;the expression levels of cell cycle,apoptosis and autophagy-related proteins were detected by Western blot analysis;High-throughput sequencing to explore changes in glioma cells at the transcriptional level.In animal xenografted model,HE staining,TUNEL staining and immunostaining were used to detect the relevant indicators of glioma cell viability,apoptosis and autophagy.In this way,the role of doramectin in the biological behavior of glioma cells was revealed,which provided data support and theoretical basis for the clinical treatment of glioma with doramectin.Results are as follows:（1）Doramectin inhibited the proliferation activity and migration activity of glioma cells.According to the MTT and colony-formation assay results,doramectin inhibited the cell viability of U87 and C6 cells in a time and dose-dependent manner,and had a lesser effect on SVGP12.Secondly,the scratch test showed that the migration ability of U87 and C6 cells was decreased after 24 h treatment with doramectin.（2）Doramectin induced cell cycle arrest at G₀/G₁ phaseFlow cytometry and Western blot showed that doramectin significantly increased the amount of DNA in U87 and C6 cells during G0/G1 phase and decreased the related cyclin expression in a dose-dependent manner（P<0.001）.（3）Doramectin induced apoptosis in glioma cellsDAPI staining results showed that a large number of nuclear condensations in doramectin treated U87 and C6 cells.Secondly,flow cytometry detection results showed that the apoptosis rate increased with the increase of doramectin concentration（P<0.001）.Finally,Western blot results showed that doramectin induced apoptosis of U87 and C6 cells by mitochondrial pathway（P<0.001）.（4）Doramectin induced autophagy in glioma cellsTransmission electron microscopy showed that there were aut ophagosomes in U87 and C6cells treated with doramectin for 48 h.The results of GFP-LC3 transient transfection and Western blot also demonstrated that doramectin could increase autophagy flow and autophagy protein expression in U87 and C6 cells（P<0.001）.（5）Autophagy can significantly suppressed the growth and promoted apoptosisAfter chloroquine was used to inhibit autophagy,MTT absorbance and clonal formation were increased（P<0.01）.Secondly,DAPI and flow cytometry results showed that when autophagy was inhibited,the chromatin condensation degree in the nucleus and the apoptosis rate were also decreased（P<0.01）（6）Doramectin induced apoptosis,autophagy and cell cycle changes in glioma cells at the transcriptional level.As we know from KEGG and GO enrichment analyses,the differentially expressed genes of doramectin were significantly enriched in cell cycle,apoptosis,and autophagy.In addition,signal pathway maps were shown that doramectin induces G0/G1 phase arrest in C6 cells.Secondly,doramectin induced apoptosis through the mitochondrial pathway.Finally,doramectin induced autophagy through the PI3K/AKT/m TOR pathway.（7）In animal xenografted model,doramectin inhibited glioma cell growthIn animal xenografted model,the tumours treated with doramectin were smaller and showed lower side effects in animals（P<0.01）.Moreover,HE and ki67 results also demonstrated that after treatment with doramectin,the cellular activity of glioma cells was reduced（P<0.01）.（8）In animal xenografted model,doramectin induced apoptosis and autophagy in glioma cells.TUNEL results showed that the number of positive cells was significantly increased af ter doramectin treatment.Furthermore,immunohistochemistry and western blot results showed that the number of positive cells and protein expression of apoptotic protein Cleaved caspase-3 and Cleaved caspase-9 were significantly increased,while the number of positive cells and expression of autophagic protein LC3 were significantly increased,while the number of positive cells and expression of p62 were significantly decreased（P<0.01）.（9）In animal xenografted model,doramectin induced apoptosis and autophagy in glioma cells.It can be seen from the tumor volume that after inhibiting autophagy,the tumor volume becomes larger.According to the results of ki67,the number of ki67 positive cells increased after autophagy inhibition.Secondly,TUNEL results showed that TUNEL positive results decreased after autophagy was inhibited.In conclusion,we found that doramectin significantly inhibited the activity of glioma cells in vivo and in vitro,and cell migration was also inhibited.Meanwhile,doramectin induced G₀/G₁ arrest and induced apoptosis and autophagy in glioma cells.In addition,autophagy can play a promoted role inhibition of glioma cell growth and glioma cell apoptosis in vitro and in vivo.Therefore,doramectin may be the next potentially effective chemotherapy drug for the treatment of glioma.