Synergistic Effect Of Apatinib Combined With Trastuzumab On Gastric Cancer Cell Lines

Gastric cancer(GC)is one of the most common cancer and the third leading cause of cancer-related death worldwide.Chemotherapy is the basic option for the treatment of most patients with metastatic or recurrent GC,although treated with optimal chemotherapy regimens,their median overall survival(OS)still limited within 1 year According to the result of ToGA study-a phase III,open-label,randomized controlled study-demonstrated that the patients with HER2-positive oesophagogastric junction(EGJ)or advanced gastric cancer(AGC)receiving Trastuzumab plus capecitabine/5FU and cisplatin achieved a better outcomes compared to them who treated with chemotherapy alone.Therefore,the European Union and FDA approved the combination of Trastuzumab with cisplatin and fluoropyrimidine as a standard treatment in patients with advanced gastric or EGJ HER2-positive adenocarcinomas.However,there are still some limitations in the regimen of Trastuzumab with chemotherapy due to lacking of sensitivity of Trastuzumab to most patients with GC.A few studies reported the efficacy and tolerance in patients with advanced GC treated with Trastuzumab and apatinib recently.However,it remains unclear that the certain mechanism of above combined pattern works in patients with GCObjective:To observe and understand the effect and mechanism of apatinib combining with Trastuzumab on NCI-N87 cells,which may provide a preclinic theory for clinic practiceMethods:NCI-N87 cells were treated with apatinib,Trastuzumab monotherapy and combination,respectively.The cellular preliferation in each group was detected by MTT assay,CCK-8 assay,plate colony respectively.Their interaction effect was assessed by q value.Cell cycle and apoptosis of each group of NCI-N87 cells were detected by Flow cytometry.Western blotting was utilized to detect the expression of HER2,VEGFR2,BAX,Bcl-2 and p53 in each group Results:1.MTT result showed that monotherapy of Trastuzumab or apatinib inhibited NCI-N87 cells proliferation with a both concentration-and time-dependent manner(P<0.05).2.According to the result of CCK-8 assay,when compared with the monotherapy,it revealed that apatinib with Trastuzumab significantly inhibited NCI-N87 cells proliferation(P<0.05).Based on the result of q-value,when combined Trastuzumab(0.1 μg/ml)with apatinib(1 μmol/L),it indicated that a synergic effect on proliferation inhabitation of NCI-N87 cells at the time of 24h and 48h,and an additivity effect at the time of 72h.3.plate colony result showed that compared with the monotherapy,combined Trastuzumab(0.1μg/ml)with apatinib(1 μmol/L)significantly inhibited colony formation abality of NCI-N87 cells(P<0.01)4.Annexin-V/PI double staining assay result showed that both monotherapy and combination of Trastuzumab with apatinib induced apoptosis of NCI-N87 cells significantly.Interestingly,the count of apoptosis NCI-N87 cells were increased obviously when treat with apatinib and Trastuzumab compared with monotherapy(P<0.05).When detected the cell cycle of each group,it uncovered that monotherapy of apatinib and Trastuzumab blocked NCI-N87 cells in the phase G1(P<0.05),and combination of apatinib and Trastuzumab blocked more cells than monotherapy in the phase G1(P<0.01).5.When combined Apatinib with Herceptin,the level of HER2 was lower than that of control and monotherapy groups,respectively(P<0.05).Similarly,this combination decreased the expression Bcl-2(P<0.05),and showed a lower Bcl-2 to BAX ratio(P<0.01).Trastuzumab combined with apatinib significantly increased the expression of p53 in NCI-N87 cells(P<0.01).Nevertheless,there was no significant difference in VEGFR2 expression among each groupConclusion:Our findings suggest that there is an obviously synergistic effect in inhibiting gastric cancer cells proliferation when treated with apatinib and Trastuzumab The potential mechanism of this effect may result from regulating HER2 expression and promoting cellular apoptosis via regulating Bcl-2 and BAX,as well as p53.