Ibrutinib,a Bruton’s Tyrosine Kinase Inhibitor,exhibits Anti-Tumoral Activity And Induces Autophagy In Glioblastoma

[Background]:Glioblastoma(GBM)is the most common and aggressive primary brain tumor in adults.Ibrutinib,a Bruton’s tyrosine kinase(BTK)inhibitor,as a novel small inhibitor,has been found anti-cancer activity in several types of cancers.In this study,we aimed to determine the role of ibrutinib and ibrutinib-induced autophagy on GBM.[Methods]:(1)CCK8 assay evaluated cell viability for GBM cells with ibrutinib treatment in different concentrations and times.(2)Colony formation assay was used for the long time proliferation ability of GBM.(3)The ethynyl-2’-deoxyuridine(EdU)assays detected proliferation ability of GBM.(4)Cell cycle and cell apoptosis were analyzed by flow cytometry with different concentrations of ibrutinib treatment in GBM cells.(5)Western Blot assay was used for evaluating protein expression level from ibrutinib treatment cells or mice tumor tissues.(6)Cell migratory ability of GBM cells were evaluated by wound healing assays and Transwell migration assays.(7)Electron microscope was used for observing autophagic vacuole.(8)Immunocytochemistry also detected protein expression level from ibrutinib treatment cells.(9)Small interfering RNA(siRNA)was used for knocking down Atg7.(10)Constructed plasmid of constitutively active Akt(CA-Akt)and dominant-negative Akt(DN-Akt)plasmids was used for overexpressing p-Akt.(11)The U87 cells were injected subcutaneously into flanks of the nude mice to analyse status of tumor growth in every group.(12)Immunohistochemistry stained with H&E and the primary antibody and Western Blot from each group of mice sample to validate mechanism of ibrutinib in regulating GBM tumor development.[Results]:The results of CCK8 assay indicated ibrutinib can inhibit the GBM cell viability in a dose and time-dependent manner.Colony formation assay showed a reduced colonies number in LN229 and U87 cells.The results of EdU proliferation assay showed that ibrutinib obviously inhibit the proliferation ability of GBM cells.Moreover,the flow cytometric analysis confirmed ibrutinib induced G0/G1 phase arrest and apoptosis of GBM cells in a dose-dependent manner.We also confirmed ibrutinib depressed migratory ability of GBM cell from the wound healing assays and Transwell migration assays.The results of electron microscope,immunocytochemistry and Western Blot assay indicated that ibrutinib induced autophagy in GBM cells.Overexpression of the active Akt(CA-Akt)protein decreased ibrutinib-induced autophagy,while inhibiting Akt by LY294002 treatment enhanced ibrutinib-induced autophagy.These indicated that ibrutinib induced autophagy through targeting Akt/mTOR pathway.Specific inhibition of autophagy by 3-methyladenine(3MA)or Atg7 targeting with small interfering RNA(si-Atg7)enhanced the anti-GBM effect of ibrutinib in vitro and in vivo.[Conclusions]:Our results indicated that ibrutinib exerted a profound anti-tumor effect and induced autophagy through targeting Akt/mTOR signaling pathway in GBM cells.Autophagy inhibition promotes ibrutinib anti-tumor activity of GBM cell in vitro and in vivo.Our findings provide important insights into the development of new anticancer agent,mechanism of anti-chemotherapy and novel strategies to improve the response of cancer cells for malignant glioma.