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

Glioma,short for neurogioma,is one of the most common primary intracranial neoplasms.At present,comprehensive therapy mainly consists of tumour excision by surgery,auxiliary assisted chemotherapy,immunotherapy and/or biological treatment,which usually delay tumor recurrence and prolong patient survival,but without getting satisfying curative effect.It is still imperative to develop successful therapeutic agents.Macrocyclic lactones with high and extended-spectrum efficiency,some studies have showed that they could serve as potential effective multidrug reversal(MDR)agents,and we also found that they were partially effective in killing non drug resistant tumor cells.More importantly,macrocyclic lactones have less side-effect and well curative effects.Now there is no much study about macrocyclic lactones could serve as anti-tumor drugs,due to lacking of relevant research foundation.Hence,this study aimed to explore the effects of macrocyclic lactones like ivermectin and moxidectin on viability of glioma cells,and to understand the inhibitory of two drugs on glioma cells.On the basis,the contents of cell apoptosis rate and cell cycle arrest were detected by using flow cytometry.Morphologic characteristics was observed by using transmission electron microscope.The expression levels of cycle-related and mitochondrial apoptosis-associated proteins were analyzed by using western blotting and RT-PCR.Moreover,in our animal study,cell proliferation,apoptosis analyses and its mechanisms were carried out by HE,TUNEL,Ki67,Cleaved caspase-3 and Cleaved caspase-9 immunostaining assay.The aim of study is to develop effective antitumor drugs for the treatment of glioma.Results are as follows:(1)The anti-proliferative activity of ivermectin and moxidectin in glioma cellsCell viability was performed in vitro by MTT assay according to the manufacturer’s instructions.As shown in results,IVM/MOX treatment dramatically decreased the cell viability of glioma cells in a dose-dependent manner compared with normal glioma cells.Next,C6 and U251 cell clonogenic capacity were employed,we found that IVM/MOX significantly inhibited colony formation and induced significant decreases in the colony formation ratio.(2)The effects and mechanisms of ivermectin and moxidectin induced G0/G1 arrest in glioma cellsThe cell cycle distribution was detected by PI staining and flow cytometry,these results indicated that the G0/G1 phase in 48 h reflects an upward trend,and the S phase shows a decreasing trend,compared with control group,which was subject to the dose(P < 0.001).The expression levels of cell cycle-associated proteins were detected by using western blotting analysis.These results suggested that IVM/MOX down-regulated the expression of cell cycle proteins like Cyclin D1,Cyclin E,CDK2,CDK4 and CDK6 in a does dependent manner.(3)The effects and mechanisms of ivermectin and moxidectin induced apoptosis of glioma cellsThe effect on the induction of cell apoptosis on glioma cells was examined by TUNEL in vivo and the flow cytometry in vitro.TUNEL assay demonstrated the evident increase in positive cells proportion in IVM/MOX-treated tumor tissues.Annexin-V-FITC/PI double staining assay showed that treatment with different concentrations of IVM/MOX for 48 h increased the percentage of apoptotic cells in a dose dependent,compared with control group.What’s more,the activation of caspase-3 and caspase-9 augmented in a dose dependent manner after IVM/MOX treating.Subsequently,the expression levels of apoptosis-associated proteins were detected by using western blotting analysis.Our results showed that the expression of pro-apoptotic Bax was obviously increased,and apoptosis inhibitory protein Bcl-2 was significantly decreased,caused the release of Cytochrome-c in the cytosol,which resulted the activation of caspase-3 and caspase-9 in a does dependent manner.(4)Ivermectin and moxidectin induced ultrastructural morphologic changes in glioma cellsThe transmission electron microscope results demonstrated that the treated cells with IVM/MOX presented typical apoptotic features: cell shrinkage,apoptotic chromatin condensation,pervacuolization of cytoplasm most of mitochondria and formation of apoptotic bodies and autophagic vacuoles.(5)The effects and mechanisms of ivermectin and moxidectin repressed xenografts growth in vivoMacroscopically,xenografts treated with IVM/MOX grew at a slower rate than those treated with saline,with a significant difference(P < 0.01).Strikingly,tumor growth curves in mice treated with IVM/MOX had a relatively slow trend.No significant difference in the weights of the mice was observed between the test group after all measured days(P > 0.05).Simultaneously,Ki67 staining and demonstrated more dead cells in apoptosis proportion in IVM/MOX-treated tumor tissues.Immunohistochemistry also demonstrated the increasing in IVM/MOX-treated tumor tissues that stained positively for Cleaved caspase-3 and Cleaved caspase-9(P < 0.01).In conclusion,the present study demonstrated that IVM/MOX have an inhibitory effect on viability of glioma cells in vitro and in vivo by inducing mitochondria-mediated apoptosis and G0/G1 cell cycle arrest.We propose that IVM and MOX might be the potent and promising agents to combat glioma.