Research On The Mechanism Of AURKA Inhibitor Alisertib In Combination With Lenvatinib In Hepatocellular Carcinoma

Purpose:First,using human HCC(hepatocellular carcinoma)cell lines,we investigated that the effects of Aurora kinase A inhibitor Alisertib and Lenvatinib combination on the morphological changes,proliferation,clone formation,migration,cell cycle,apoptosis,DNA damage and the molecular mechanisms in vitro.Then,the anti-proliferation and pro-apoptotic effects of the combined drug were verified in vivo.Method:After 48 hours of treatment with Alisertib alone,Lenvatinib alone,and their combination,the morphological changes of HCC cells were observed by inverted fluorescence microscope;the proliferation ability of HCC cells was detected using cell counting kit-8 and clone formation experiment;scratch healing experiments were used to observe the changes in the migration and invasion ability of HCC cells;flow cytometry and laser confocal imaging were able to detect cell cycle and apoptosis;westernblot could test the expression level of related DNA damageproteins;the number of foci focus counting by laser confocal imaging represented the situation of DNA damage response.The results in vitro were verified through establishing a subcutaneous tumor xenograft in nude mice,measuring tumor size,analyzing changes in body weight of nude mice,and performing related experiments in vivo such as immunohistochemistry and TUNEL fluorescent probe detection.Results:1.Compared with the control group and the drug alone group,we observed that the combined application of Alisertib and Lenvatinib significantly changed the cell morphology and enhanced the inhibitory effect on cell proliferation and migration and healing in various human HCC cell lines.Cell cycle analyses after 48 hr of treatment with Alisertib alone or in combination with Lenvatinib demonstrated a significant increase in the percentage of G2 cells and G2/M phase was blocked.Discontinuous spindles and missegregated chromosomes in HCC cells treated with Alisertib in combination with Lenvatinib were observed.Western blot analysis demonstrated Alisertib inhibited the expression of DNA damage pathway protein with the combined treatment,as compared to single agent treatments.2.In addition,Hep G2 cell xenograft models demonstrated an enhanced anti-tumor activity for Alisertib in combination with Lenvatinib,as compared to single agent treatments.Conclusion:1.The combination of Alisertib and Lenvatinib can obviously change the morphology of HCC cell lines,significantly inhibit the proliferation,migration and healing ability,induce arresting of cell cycle,promote cell apoptosis,and activate related DNA damage repair signal pathway.2.The combined application of Alisertib and Lenvatinib can significantly inhibit the growth of transplanted tumors carried by nude mice and induce apoptosis,suggesting that the combination treatment scheme provides a new potential treatment direction for patients with HCC.