Antitumor Effects Of Bevacizumab Combined With Apatinib In The Treatment Of Lung Carcinoma

Objective:The aim of this study was to investigate the therapeutic effect of combining the anti-VEGF-A antibody bevacizumab?Bev?with apatinib?Apa?,a VEGF receptor-2?VEGFR-2?-targeting tyrosine kinase inhibitor,in lung cancer.Methods:Proliferation and apoptosis of Lewis lung carcinoma?LLC?cells were measured using the MTT assay and Annexin V staining respectively.Tumor-bearing models were established by subcutaneous injecting administration of C57BL/6 mice intra-muscularlysubcutaneous with LLC cells,and 84 tumor-bearing mice were randomly divided into 7 groups?n=12 per group?that were treated as follows for 14 days:1)Control group?NS 0.1ml?,2)PTX group?10 mg/kg/day?,3)Apa group?100 mg/kg/day?,4)Bev group?5 mg/kg/day?,5)Apa+PTX group,6)Bev+PTX group and 7)Apa+Bev group.PTX and Bev were administered intraperitoneally,and Apa was administered intra-gastrically.The dose and schedule of the combination group were the same as for the single drug group.Tumor dimensions were measured with Vernier calipers every two days during treatment,then draw the tumor growth curve and calculate the tumor inhibition rate.On the second day after the end of treatment,fluorine-18-fluorode oxyglucose?18F-FDG?micro positron emission tomography?PET?/CT images were taken using the Inveon micro PET/CT animal scanner,and the maximum value of the normalized uptake value?SUVmax?in the ROI were calculated.The animals were sacrificed after 14 days,and intra-tumor angiogenesis and apoptosis were respectively analyzed by CD31,VEGF and VEGFR-2 immunostaining and TUNEL assay.In addition,heart,liver,lung and kidney tissues were removed after treatment and examined by HE staining for observeing the toxicity of the organs in each group.Five mice per group were monitored till natural death,and survival curves were created.Results:In vivo,treatment with Apa inhibited the proliferation of LLC cells in a dose-dependent manner.The inhibitory effects of the combination of Apa and Bev was significantly enhanced compared to Bev monotherapy?P<0.05?.In addition,the combination treatment resulted in significantly higher percentage of apoptotic cells compared to the corresponding concentration of Bev monotherapy.In the LLC xenograft mouse model,the Apa+Bev group had stronger antitumor effect than the other groups.All monotherapies and drug combinations significantly decreased the tumor volume compared to the untreated control,and the combined drug group has a more significant inhibitory effect than the single drug group.Among them,the Apa+Bev group had the most significant antitumor effect.In addition,the survival duration of the Apa+Bev group was significantly longer compared to that of either monotherapy groups?P<0.05 vs Apa?Bev group?.Taken together,the combination of Apa and Bev was highly effective in controlling tumor growth,acted in a synergistic manner.Thus,Apa and Bev prolonged the survival of tumor-bearing mice.The microvessel density?MVD?,as determined by the in situ CD31 expression levels,was significantly decreased by Apa and Bev monotherapies compared to that of both the untreated control and the PTX group.However,the different combinations of Apa,Bev,and PTX achieved lower MVD values,with the lowest MVD observed in the Apa+Bev group?P<0.01 vs other groups?.To further elucidate the mechanisms underlying Apa and Bev-mediated angiogenesis inhibition,the expression of VEGF and VEGFR-2in mouse xenografts was analyzed.The control group had a higher positive expression rate of VEGF and VEGFR-2.The lowest percentage of VEGF-positive cells were was detected in the Apa+Bev group?6.92±0.33%,P<0.01 vs other groups?.Similarly,the percentage of VEGFR-2 positive cells was significantly reduced after Apa treatment.The Apa+Bev group had the lowest VEGFR-2 positive rate?P<0.05 vs other groups?.Micro PET/CT scan results shown,compared to the control group,the SUV_maxax values of the all treatment groups were significantly lower,while the lowest SUV_max value was observed in the Apa+Bev group?P<0.01 vs Apa?Bev group?.Compared to the untreated control,all therapies resulted in a significant increase in the percentage of apoptotic cells?P<0.05?,and the apoptosis rate was significantly higher in the Apa+Bev group compared with that of all other groups?P<0.01?.Conclusion:In conclusion,the findings of the present study revealed that the combination of Apa and Bev exhibited a strong inhibitory effect on the growth,metastasis potential,and angiogenesis in lung cancer cells and in a xenograft model through attenuation of the VEGF and VEGFR-2 signaling pathways.Our data provide prospective evidence for the clinical application of Apa+Bev in the therapeutic management of NSCLC.