Anti-tumor Effect And Mechanism Of Apatinib On Biliary Tract Cancer Cells

Background and objectiveBiliary tract cancer(BTC)is a group of rare heterogeneous tumors in the biliary system.It can be divided into Gallbladder Neoplasms,Intrahepatic cholangiocarcinoma(iCCA),Hilar cholangiocarcinoma(pCCA),distal cholangiocarcinoma(dCCA).In actual clinical work,this type of disease is diagnosed at the advanced stage,and the 5-year survival rate is less than 5%.Surgery is the only treatment that may cure the biliary tract cancer,but no more than one-third of patients can accept surgical excision.Systemic chemotherapy is the main treatment for patients with advanced cholangiocarcinoma.Anti-tumor effect of cisplatin combined with gemcitabine is poor which is recommended by NCCN as the first-line chemotherapy.Currently,There is no effective targets drugs for advanced BTC.Apatinib,is a small molecule TKI that inhibits VEGFR-2,also moderately inhibits tyrosine agonisms such as PDGFR-?,c-Kit,Ret,and c-src.It have achieved good anti-tumor responses and have been approved for the treatment of advanced gastric adenocarcinoma or adenocarcinoma of the gastro-esophageal junction as third-line and beyond therapy.Good anti-tumor efficacy has been found in breast cancer,and non-small cell cells,Lung cancer,liver cancer,etc.Therefore,it is of far-reaching significance to explore the pathogenesis of cholangiocarcinoma and to seek targeted therapy.Preclinical studies found that two patients shows PFS more than 6 months with treatment of apatinib.In this experiment,the effects of apatinib on proliferation,apoptosis,migration,invasion,and colony formation of biliary tumor cell lines were investigated by MTT assay,Annexin V-Flow cytometry,scratch test,transwell assay,and colony formation.Through Western blot,the changes of signal pathway protein level were preliminarily detected,and the possible signaling pathways were analyzed.We hope to provide a theoretical basis for the experimental and clinical trials in vivo,and provide a new therapeutic optitial for patients with biliary tract cancer.Methods1.The human biliary tumor cell lines GBC-SD,Hccc-9810,and RBE cells were treated with different concentrations of apatinib and respectively examined proliferation,apoptosis,migration,invasion,and colony formation of cholangiocarcinoma cells by MTT assay,Annexin V-Flow cytometry,scratch test,transwell assay,and colony formation.2.The expression of related proteins such as VEGFR2-RAS-RAF-MEK-ERK pathway,P-SRC,E-cadherin,matrix metalloproteinases and apoptotic regulating proteins were detected by Western blot test after different concentrations of Apatinib treatment.Results1.The proliferative rate of human biliary tumor cell lines GBC-SD,Hccc-9810,and RBE cells was increased over time within 1-5 days.Proliferation was inhibited by apatinib in a concentration-dependent manner.2.Apatinib can promote the apoptosis of human biliary tumor cell lines GBC-SD,Hccc-9810,and RBE cells in a concentration-dependent manner.The effect of apoptosis was increased with the increase of the concentration of apatinib,but it was not obvious effect to the apoptosis of Hccc-9810.3.Apatinib can inhibit the invasion and migration of human biliary tumor cell lines GBC-SD,Hccc-9810,and RBE cells in a concentration-depen-dent manner.With the increase of apatinib concentration,the degree of inhibition increased.4.Apatinib reduced colony formation of human biliary tumor cells in a concentration-dependent manner.5.Apatinib can down-regulate the expression of p-VEGFR2 in the human biliary tumor cell lines GBC-SD,Hccc-9810,and RBE cells,and then down-regulate the expression of downstream signals p-ERK1/2 and k-ras in a concentration-dependent manner.With increasing concentration of apatinib,the degree of inhibition was enhanced,and apatinib inhibited proliferation through the VEGFR-2/ERK/K-ras pathway.6.The SRC expression was observed in the human biliary tumor cell lines GBC-SD,Hccc-9810,and RBE cells.Apatinib can up-regulate E-cadherin,down-regulate p-src and MMP9 in a concentration-dependent manner.With the increase of apatinib concentration,the inhibitory effect was enhanced,and finally it can inhibit invasion and migration.It was showed that apatinib did not regulate E-cadherin and matrix metalloproteinase changes by targeting SRC.7.Apatinib can upregulate human biliary tumor cell lines GBC-SD,Hccc-9810,and RBE cells pro-apoptotic protein p21,Bax,caspase3,down-regulate anti-apoptotic protein Bcl-2,concentration-dependent,There was no significant change in the level of p53 protein in the concentration dependence.ConclusionsApatinib can inhibit the proliferation,invasion,migration,and colony formation of human biliary tumor cell lines GBC-SD,Hccc-9810,and RBE cells,and at the same time promote tumor cell apoptosis.It inhibits proliferation through the VEGFR-2/K-RAS/RAF/MEK/ERK/pathway,by up-regulation of apoptotic proteins p21,Bax,caspase3,down-regulation of anti-apoptotic protein Bcl-2 to apoptosis.It was showed that apatinib could up-regulate E-cadherin and down-regulate matrix metalloproteinase to inhibit invasion and migration.