The Combination Of Apatinib And Chemotherapy Enhances Anti-Tumor And Anti-Angiogenesis Effects In A549 Cell Line And Zebrafish Xenograft Model Of Nonsmall Cell Lung Cancer

Background:Apatinib is thought to inhibit angiogenesis through inhibiting VEGF-mediated endothelial cells migration and proliferation thus blocking new blood vessels formation in tumor tissues.Several studies have found that Apatinib may be useful in increasing the intracellular concentrations of the antineoplastic agents in several chemotherapy-resistant cancer cell lines.And these studies suggested that Apatinib will be potentially effective in combination therapies with conventional antineoplastic agents especially in cases where resistance to chemotherapy exists.Purpose:Whether Apatinib combined with conventional antineoplastic drugs could enhance anti-tumor and anti-angiogenic effects in advanced NSCLC is unclear.The present study aimed to answer this question and identify an optimal treatment regimen for the combination of Apatinib and conventional antineoplastic drugs in a zebrafish tumor model of NSCLC.In addition,this study will explore the potential molecular mechanisms of the synergistic effect of this therapeutic strategy so as to guide future clinical therapies more efficiently than existing protocols.Methods:In vitro,Apatinib was named drug A and cytotoxic agents were named drug B（Gemcitabine,Paclitaxel,Pemetrexed,were named B1,B2,B3,respectively）for the convenience of discussion.There are four combination proportions of Apatinib and cytotoxic agents in our experiment（（1）4/5A+1/5B,（2）3/5A+2/5B,（3）2/5A+3/5B,（4）1/5A+4/5B）.The initial concentration of each agent was chosen depending on the value of its IC50 for these four combination proportions.Then the A549 cell viability were measured at every gradient descending concentration of each combination by MTT assay.Finally,combination index（CI）were used to estimate the combination effects.In the second part of this study,to determine the MNLC and LC₅₀0 of lead compounds,zebrafish were treated from2-dpf to 5-dpf and mortality was recorded every 24 h.Dead zebrafish was defined as the absence of heartbeat under a dissecting stereomicroscope.Mortality curves were generated using GraphPad Prism 5.0 and the MNLC was determined with logistic regression.In the third part of this study,to evaluate blood vessels formation in zebrafish embryos,22hpf embryos were distributed into 12-well plates（thirty embryos per well）for a treatment period of 26h.The positive control for this assay was 5μM PTK787,a VEGFR antagonist,and the negative control was 0.1%dimethyl sulfoxide（DMSO）.Different concentration of Apatinib（1,2.5,5 and 10μM）and PTK787（5μM）were diluted in 0.1%DMSO.Then DMSO containing drug of interest was soaking in fish water and zebrafish can take the drug orally.Finally,the number of complete intersegmental vessels（ISVs）were counted.In the fourth part of this study,A549 cells were stained with red fluorescent cell tracer CM-Dil fluorescence.By means of microinjection,A549 cells were grafted into the yolk sac of zebrafish embryo without immunosuppressant treatment.We evaluated the tumor xenografts by fluorescence microscopy immediately after injection.Those embryos with red fluorescence were moved to 6-well plate and cultured in fish water containing drugs of interest（5μmol Apatinib with or without 70.5μmol Pemetrexed,70.5μmol Pemetrexed）.Then we evaluated tumor growth at the 4th day post injection（dpi）by fluorescence microscopy and measured the relative fluorescence intensity（RFI）to estimate tumor xenografts volume in control and drug treated animals.In the fifth part of this study,in order to identify molecular mechanism of the combination effect of Apatinib and Pemetrexed in zebrafish,we examined mRNA expression levels of VEGFaa-VEGFR,DLL4-Notch-hey2,Efnb2a,Slit-Robo,FGFR,PTP-RB,COX-2,VE-cadherin and PIK3R2 genes in different treatment groups which is same as the above zebrafish A549 xenograft experiments using qRT-PCR experiment.Results:1.The MTT results showed that Apatinib single-agent had a slight inhibitory effect on the viability of A549 lung cancer cells with the IC50 values ranging from 2.693 to5.384μmol.However,the IC50 value of Apatinib single-agent is higher than those of the three cytotoxic agents（B1 0.75μmol,B2 0.48nmol,B3 0.88μmol）,suggesting that Apatinib had a low cytotoxicity compared with chemotherapy agents,and apatinib may not mainly depend on its cytotoxicity to exerted antitumor effect.Apatinib combined with Pemetrexed at the proportion of 3/5A+2/5B3（CI=0.42）can give an optimal antitumor effect in vitro.2.Pemetrexed is toxic to zebrafish embryos with LC50 value of 218.5μmol and MNLC value of 70.5μmol.Compared with Pemetrexed,Apatinib is less toxic to zebrafish embryos with LC50 value of 404.9μmol and Maximum non-lethal concentration（MNLC）value of70.5μmol.3.Apatinib was administrated at 1,2.5,5 and 10μmol and the inhibition rates of ISV were 0,62.68%,94.57%and 100%,respectively.When micro-injected with 10μmol of Apatinib,the number of ISV was decreased to 0,which was similar to the PTK787 group.4.The inhibition rates of tumor mass in Apatinib group,Pemetrexed group and combination group were 23.42%,28.87%and 45.71%respectively.Compared with control group,there were significant statistical differences in Apatinib group,Pemetrexed group and combination group(P_Apa<0.05,P_pem<0.01,P_Apa+pem<0.0001).Compared with Aptinib group,the combination of Apatinib and Pemetrexed can enhance antitumor effect（P<0.05）,but compared with Pemetrexed group,the combination of Apatinib and Pemetrexed didn’t significantly enhance antitumor effect（P>0.05）.These results clearly revealed that Apatinib had definite antitumor activity and suggested that the combination of apatinib and Pemetrexed may be a potential alternative therapy for lung cancer.5.Apatinib combined with Pemetrexed can synergistically inhibited the expression of VEGFR2,Robo4,FGFR4gene.Compared with control group,there were significant statistical differences(P_VEGFR2<0.0001,P_Robo4<0.05,P_FGFR4<0.0001).Apatinib single agent can induce compensatory upregulation of Efnb2a gene,but Apatinib combined with Pemetrexed can significantly inhibited the expression of Efnb2a gene（P<0.0001）.Conclusion:Apatinib is a kind of low toxicity,safe and efficient angiogenesis inhibitor.Apatinib combined with Pemetrexed,enhanced anti-tumor effects compared with either one in zebrafish model of NSCLC,which may be involved the synergistic inhibition of VEGFR2,Efnb2a,Robo4 and FGFR4 pathways.