Study Of Alantolactone Enhancing The Antitumor Effect Of Apatinib On Esophageal Squamous Cell Carcinoma Cells

Objective:This work aims to investigate the effect and possible mechanism of Alantolactone potentiating the activity of Apatinib against human esophageal squamous carcinoma cells lines TE-1 and ECA-109.Methods:CCK-8 test was used to detect the effects of different concentrations of Alantolactone and Apatinib on the proliferation of TE-1 and ECA-109 cells.The TE-1 and ECA-109 cells were divided in to 4 groups:Control group(Con,no drug),Alantolactone group(Ala),Apatinib group(Apa),Apatinib and Alantolactone group(Apa+Ala).The cell proliferation was observed by Colony formation experiment.Wound healing test t and Transwell test were used to observe the migration and invasion ability of cells.Hoechst33342 staining method was used to observe apoptotic cells under fluorescence microscope;The expression of proliferation related protein C-MYC,apoptosis related proteins BCL-2,BAX,PARP and related pathway egg VEGFR2/STAT3/BCL-2 were detected by Western blot;In addition,we established the xenograft tumor model in nude mice of ECA-109 cells to further explore the effect of Alantolactone potentiating the activity of Apatinib against esophageal squamous cell carcinoma in vivo.Results:1.CCK8 test showed that Apatinib inhibited the proliferation of human esophageal squamous carcinoma cells lines TE-1 and ECA-109 in a dose-dependent manner,respectively.When the concentration of Alantolactone was greater than3umol/L,Alantolactone also inhibited the TE-1 and ECA-109 cell proliferation in a dose-dependent manner.The TE-1 cell was treated with 3umol/L Alantolactone and30umol/L Apatinib,and the ECA-109 cell was treated with 3umol/L Alantolactone and 20umol/L Apatinib.The cell survival rate of group Apa lower than Con group.Moreover,the cell survival rate of group Apa+Ala lower than group Apa.As we expected,Colony formation experiment showed the same results.2.The results of Wound healing test showed that Apa group inhibited the migration ability of TE-1 and ECA-109 cells.Compared with Apa group,the combined group showed more obvious inhibition of migration ability.Transwell assay showed that the number of TE-1 and ECA-109 migrating cells in Apa group was lower than con group.Compared with Apa group,the combined group showed a lower number of migrating cells.3.Hoechst 33342 staining showed that the proportion of nuclear fragmentation and concentrated cells in combined group increased significantly.4.Western blot showed that the migration related protein MMP-9,anti apoptotic protein BCL-2 and total PARP in Apa group were significantly down-regulated,and the pro apoptotic protein BAX was significantly up-regulated.However,this change was most obvious in combined group.5.The tumor-bearing nude mice were employed to evaluate whether alantolactone can enhance the anti-tumor effect of apatinib in vivo.The results showed that Alantolactone could enhance the anticancer effect of of apatinib on xenograft tumor growth.Conclusions:Our findings suggested that Alantolactone enhance the antitumor effect of Apatinib on human esophageal squamous carcinoma cells lines TE-1 and ECA-109,and its mechanism might be related to the inhibition of VEGFR2/STAT3/BCL-2signaling pathway.