Effect Of Apatinib,a Small Molecular Tyrosine Kinase Inhibitor,on Radiosensitivity Of Glioblastoma

Part Ⅰ.Effect of apatinib radiosensitivity of glioblastoma cell lines.Objectives1.To investigate the effect of small molecular tyrosine kinase inhibitor apatinib mesylate on radiosensitivity of glioblastoma cell lines.2.To investigate the mechanism of the effect of small molecular tyrosine kinase inhibitor apatinib mesylate on radiosensitivity of glioblastoma cell lines.Methods1.Cell grouping and irradiation:glioblastoma cell lines were divided into blank control group,apatinib group,irradiation group and combination group.Using Varian linear accelerator（Source skin distance:100cm;Dose rate:200c Gy/min）2.The effects of different concentrations of apatinib（5,10,20,40,80μmol/L）on cell proliferation by CCK8 method.3.The effect of apatinib on migration and invasion of glioblastoma cells was detected by scratch wound assay and invasion assay.4.The apoptosis rates of glioblastoma cell lines were detected by flow cytometry.5.The protein expressions of Bax and Bcl-2 were detected by western blot.6.Statistical analysis:The data were analyzed by SPSS 24.0 software.Graph Pad Prism 7.0 was used to fit the cell survival curve and radiobiological parameters.The survival rate,invasion ability,apoptosis rates and the relative expression of Bax and Bcl-2 in each group were compared by one-way ANOVA.Results1.Apatinib significantly inhibited the proliferation of glioma cell lines.2.Compared with the irradiation group,combination group could significantly inhibit cell proliferation,migration and invasion（P<0.05）.The apoptosis rate of U87MG and U251 cells in combination group was significantly higher than that in other groups（P<0.05）.3.Compared with irradiation group,the D₀,D_q and SF₂ of combination group decreased,and the radiosensitive enhancement ratio(SER_D0)was 1.30,1.64,respectively.4.When U87MG and U251 cells were treated with apatinib and irradiation,the expression of Bax protein increased and the expression of Bcl-2 protein decreased,and the change in the combination group was more significant.Conclusion1.Apatinib can inhibit the proliferation of glioblastoma cell lines and enhance the inhibition of radiation on cell proliferation.2.Apatinib can increase the radiosensitivity of glioblastoma cell lines（U87MG and U251）,and its mechanism may be related to inhibiting cell invasion and migration,promoting apoptosis and regulating the expression of related proteins.Part Ⅱ.Clinical observation of apatinib in the treatment of recurrent glioblastoma.Objectives1.To observe the efficacy and side effects of small molecular tyrosine kinase inhibitor apatinib in the treatment of temozolomide-resistant recurrent glioblastoma（GBM）.2.The changes of immune factors in the process of treatment were analyzed,and their effects on the therapeutic effect were analyzed.Methods1.Case collection:From June1,2018 to November1,2020,51 patients with recurrent GBM were treated in our hospital.2.Treatment method:Patients received apatinib 500 mg/day,for 2 weeks（days1–14）,then rested for 1 week,and started oral TMZ（150 mg/m²/day）for 5consecutive days（days 22–26）,with a cycle of 28 days.3.Immune factors:Before starting treatment,5–10 m L peripheral blood was collected on an empty stomach.After starting treatment,peripheral blood was collected every cycle on an empty stomach.Expression of interleukin（IL）-6 and IL-10 in serum and expression of soluble PD-1（s PD-1）and soluble PD-L1（s PD-L1）in plasma were measured using ELISA.4.Tumor volume measurement:（1）Image acquisition:all patients underwent a plain MRI scan and an enhanced scan before and after treatment.MRI sequences downloaded in DICOM format.（2）Volume measurement:we used medical image analysis software and visualization developed using 3D Slicer 4.10.2.Two radiation therapists processed the images and measured tumor volume.5.Evaluation criteria:MRI examinations were repeated every cycle during treatment for 4 cycles.Patients were followed up every 3months.6.Statistical analysis:The data were analyzed by SPSS 24.0 software.Kaplan-Meier method calculated median PFS,median OS and 95%CI,and drew survival curve.Log-rank test was used to compare the data between groups,and Spearman correlation analysis was performed.Results1.Short-term curative:51 patients included two with CR,twenty with SD,and fourteen with PD.The ORR was 33.3%,and the DCR was 72.5%.2.Long-term survival:The m PFS was 5.9months（95%CI:5.345-6.455）,the m OS was 10.5 months（95%CI:9.958-11.042）,the 6-month PFS rate was 44.4%,and the2-year OS rate was 24.9%.3.Analysis of related factors:TERT promoter and MGMT methylation status were independent prognostic factors for overall survival.4.Adverse reactions:The side effects were tolerable.There were 14 patients with hypertension（25.4%）,10 patients with fatigue（17.8%）,9 patients with nausea and vomiting（16.1%）,6 patients with myelosuppression（10.7%）,6 patients with diarrhea（10.7%）,5 patients with abnormal transaminase（8.9%）,4 patients with hand and foot syndrome（7.2%）,and 2 patients with proteinuria（3.6%）.5.Changes of immune factors:There was significant correlation between the change of expressions（s PD-1,s PD-L1,IL-6）and the change of tumor volume（r=-0.846,p=0.001;r=0.874,p<0.001;r=0.769,p=0.003）.A weak,not significant correlation（r=0.538,p=0.071）was found between the change of IL-6 expression and the change of tumor volume.Conclusion1.Apatinib combined with temozolomide was safe and effective in the treatment of recurrent GBM,and the side effects can be tolerated.The changes of immune factors were related to the curative effect.2.MGMT and TERT status were independent prognostic factors for overall survival.