Effect Of Apatinib Standalone And Combine With Chemotherapeutics In Human Cervical Cancer

Objective:(1)To explore the efficacy of Apatinib on human cerical cancer.(2)To compare the difference of whole genomics expression before and after using Apatinib in human cervical cancer cell line SiHa.(3)To explore the efficacy of combination Apatinib with chemotherapy drugs on human cerical cancer.Method:(1)Cell proliferation ability was evaluated by CCK-8 assay and colony formation assay.(2)Cell cycle distribution and the apoptosis were determined by flow cytometry(FCM).(3)Westem-blot assay was used to investigate the expression of the protein related to cell cycle and apoptosis and the activities of VEGFR2 and its known downstream targets.(4)Detection the different expression of genes in SiHa before and after Apatinib deal with through expression profile chip screening,then further validate through RT-PCR.(5)CCK-8 assay was used to detect cytotoxicity of chemotherapy drugs(cisplatin and paclitaxel)combination with Apatinib on SiHa and Hcc94 cervical cancer cells.The synergistic index(CI)was calculated by CompuSyn.(6)A nude mouse model bearing Si Ha xenografts was established to test the anti-tumor effects of Apatinib standalone or combination with paclitaxel.Result:(1)As CCK-8 shown,cervical cancer cells were suppressed by dose dependent manner,IC50 values in SiHa?Hcc94?C33A and HeLa were 13.9±2.4 ?M,21.8±3.4 ?M,26.6±3.1 ?M,31.2±3.5 ?M,respectively.Colony-formation assay shown that Apatinib inhibit the ability of clone forming of SiHa and Hcc94(P<0.01 and P<0.01).(2)Further,the treatment of Apatinib induced significant G1-phase arrest in a dose-dependent manner in both SiHa and Hcc94 cells(P<0.01 and P=0.016).Apatinib could direct more SiHa and Hcc94 cells toward apoptosis(P<0.01 and P<0.01).(3)In both SiHa and Hcc94 cells,the 36 h treatment with Apatinib significantly inhibited the phosphorylation of VEGFR2,ERK and AKT,indicating its highly selective targeting and efficiency.(4)From gene expression profile,many genes involved in cell cycle progression(CDK1,CCNC and CCND3)and apoptosis(DAD1 and BAG1)were significantly downregulated Using KEGG analysis,we observed that p53,proteasome,TGF-beta,cell cycle,homologous recombination and mismatch repair signaling pathways were the most significantly regulated by Apatinib.In addition,GO analysis showed that the genes involved in mitotic cell cycle(both G1/S transition and G2/M transition),cell division,DNA repair,and cell proliferation were under the precise regulation of Apatinib.Using real time PCR,we confirmed the decreased expressions of CDK1,CCNC,CCND3.(5)Apatinib allowed for significant synergistic effects with Paclitaxel in both SiHa and Hcc94 cells.However,we found no synergy between Apatinib and cisplatin.(6)In ivo,according to the growth curve,the single use of Apatinib or Paclitaxel significantly suppressed the growth of SiHa xenografts,and the combination with the two drugs further augmented this effect(P=0.043,0.031 and <0.001).Tumor weights in the Apatinib group and Paclitaxel group were significant lower(P=0.045 and 0.024),and the tumor weights in the combination group were the lowest(P<0.01).The phosphorylation of VEGFR2 was positive in the control group and Paclitaxel group(P=0.033 and 0.019),but was notably inhibited in Apatinib group and combination group;in addition,Ki-67 was suppressed by the single treatment with Apatinib or Paclitaxel,and was further decreased in tumors treated with Apatinib plus Paclitaxel(P=0.041,0.029 and <0.01).Conclusion: Apatinib inhibit the proliferation of cervical cancer cell lines in vitro and vivo.Moreover,Apatinib synergized with Paclitaxel to achieve more significant suppression on tumor growth,proposing that Apatinib might be a potent drug for cervical cancer.