The Effect And Mechanism Of Anlotinib On Glioblastoma

Background and objectiveGliomas are the most common primary intracranial tumor,accounting for eighty percent of primary central nervous system malignant tumors.More than half of all gliomas in adults are malignant.Additionally,more than 80% of gliomas are Glioblastoma(GBM).Current standard treatment for GBM consists of surgery and combined radiation therapy,concurrent chemotherapy temozolomide(TMZ),and adjuvant chemotherapy.However,the therapeutic effects are still deemed unsatisfactory owing to the resistance to chemotherapies.In 2020 national comprehensive cancer network(NCCN)guidelines,regorafenib,a multi-targeting tyrosine protein kinases inhibitor(TKI),was performed in recurrent GBM.Anlotinib,a novel multi-targeted tyrosine kinase inhibitor targets vascular endothelial growth factor receptor A,fibroblast growth factor receptor 1,platelet-derived growth factor receptor β,and the stem cell factor receptor which is developed in China.Furthermore,anlotinib has been approved for the third-line treatment for various tumors including non-small cell lung cancer and colon cancer in China.Preclinical research has been reported that anlotinib can suppress the proliferation of U87.But its basic research remains to be uncovered.The purpose of this study is to explore the effect of anlotinib on glioblastoma cell lines in proliferation,apoptosis,autophagy,migration,invasion and angiogenesis.Then the changes of VEGFA and its upstream JAK2/STAT3 signaling pathway were evaluated.Furthermore,we demonstrated that anlotinib suppresses glioblastoma via mediation of JAK2/STAT3 signaling pathwayMethods(1)Cells(A172,U87 and U251)were treated with anlotinib(0,1.25,2.5,5,10 and20 μM)for 24,48,or 72 hours.Next,the cell viability was evaluated by CCK-8assay.According to drawing “S” curve and calculating IC50,the suitable concentrations of anlotinib were performed in subsequent vitro experiments.Colony-forming assay was used to evaluate cell viability.Cell migration and invasion were assessed by wound-healing,Transwell migration,and Matrigel invasion assays.Cellular apoptosis and cell cycle analysis were determined by flow cytometry.(2)A172,U87 and U251 cells were treated with anlotinib for 24 h.Then protein was extracted.Western blot assays were performed to measure the levels of apoptosisrelated proteins(Bcl-2,BAX and caspase-3),the autophagy-related proteins(Beclin-1 and LC3B),and the JAK2/STAT3 pathway components.The changes of LC3 was evaluated by immunofluorescence staining when 3-MA,an inhibitor of autophagy,was combined with anlotinib,or each drug used alone.(3)VEGFA concentrations in the supernatants from U87 cells were determined using human VEGFA ELISA kits.Then human umbilical vein endothelial cells(HUVECs)were cultured with supernatant collected from U87 lines treated with or without anlotinib.(4)After inhibited STAT3 by S3I-201,the above experiments were repeated and the effects of anlotinib on glioblastoma cell lines in growth,motion and angiogenesis were detected.(5)Transfected U87MG-Luc cells were implanted in nude mice brain.After 7 days of implantation,the mice were treated with different concentrations of anlotinib.Moreover,the Caliper IVIS Spectrum was used for detecting tumor volumes at 7,14 and 28 days.After 14 days of transplantation,immunohistochemical(Ki-67)was detected and western blot assays was used to evaluate the levels of JAK2 and STAT3Results(1)Anlotinib restricted the proliferation,migration,and invasion of glioblastoma cells in a dose-dependent manner.Furthermore,anlotinib induced G2/M phase arrest and promoted apoptosis in glioblastoma cells.(2)Western blot confirmed that anlotinib decreased the expression of Bcl-2 and upregulated BAX,Caspase-3,LC3-Ⅱ/Ⅰ,and Beclin-1 expression.Immunofluorescence staining suggested that 3-MA decreased the elevation of LC3 induced by anlotinib.The results suggested that anlotinib induced autophagy and apoptosis.(3)Anluotinib restrained excreted VEGFA from U87 and tubular formation of HUVECs.(4)Compared to vehicle alone,the suppressing effects of anlotinib on growth,motion and angiogenesis were enhanced by combining S3I-201.(5)Anlotinib also suppressed the growth of glioblastoma in orthotopic GBM modelConclusionAnlotinib presents excellent therapeutic effects in glioblastoma.In detail,it can greatly suppress proliferation,invasion,migration and angiogenesis,and promote autophagy and apoptosis.Furthermore,the JAK2/STAT3 signaling pathway possibly plays a vital role in the process.Collectively,it is indicated the potential application of anlotinib as a treatment option for glioblastoma.