Study On The Mechanism Of Amantadine Inhibiting Glioma Growth By Inducing Apoptosis And Autophagy

Objective: Glioma is a common primary malignant tumor of the nervous system,and its therapeutic effect is poor,so a new therapeutic strategy is urgently needed.Amantadine,as an antiviral agent and a therapeutic agent for Parkinson’s disease,has been found to inhibit tumor growth recently.The main purpose of this study is to study the effect of amantadine on the growth and proliferation of glioma cells,to verify that amantadine can inhibit glioma both in vivo and in vitro,to explore how amantadine affects apoptosis and autophagy,and to research its molecular mechanism.Methods: 1.Glioma cells U87 and U251 were treated according to the set amantadine concentration gradient,and we verified the effects of amantadine on the viability and proliferation of glioma cells by CCK-8,colony formation assay,Ed U proliferation assay as well as lactate dehydrogenase(LDH)release assay.2.By immunoblot analysis,flow cytometry,ROS generation assay and JC-1staining kit,the changes of ROS levels as well as mitochondrial membrane potential and the effect of amantadine on apoptosis of glioma cells were verified.The effects of amantadine on autophagy and autophagy flow of glioma cells were determined by immunoblot analysis and immunofluorescence assay.3.A subcutaneous graft tumor model was constructed in nude mice.After amantadine was administered in vivo,tumor specimens were observed and tumor weight as well as volume were measured.HE staining was used to observe tumor cell morphology and number,Ki67 expression level was detected by immunohistochemistry,and the inhibitory effect of amantadine in vivo was verified.The body weight of nude mice with tumor was measured and curves were drawn.Important viscera such as heart,liver,spleen,lung and kidney were taken for HE staining to detect the toxic and side effects of amantadine in vivo.4.Immunoblot analysis and immunohistochemical assay were performed on tumor tissues to detect the expression levels of cleaved-caspase3 and LC3 II,so as to clarify the apoptosis and autophagy of glioma in vivo.Results: 1.Amantadine inhibited the activity and proliferation of glioma cells U87 and U251 in a concentration-dependent manner in vitro.2.Amantadine induced the accumulation of ROS and the damage of mitochondria in glioma cells,which promoted apoptosis through the activation of caspase protein family cascade reaction.Amantadine increased the expression of ATG5,ATG7 and P62 in glioma cells and promoted the transformation of LC3 I to LC3 II,which induced initiation of autophagy and blocked autophagy flow.3.Amantadine showed strong inhibitory ability in vivo without significant side effects.4.Amantadine overexpressed cleaved-caspase3 as well as LC3 II in vivo,and then induced apoptosis and autophagy.Conclusion: Amantadine inhibits the growth and proliferation of glioma by inducing apoptosis and autophagy in vivo and in vitro.