Abstract:

Objective To discuss the effect of multi-kinase inhibitor motesanib combined with enhancer of zeste homolog 2（EZH2）inhibitor GSK126　on the proliferation and apoptosis of the liver cancer Huh7 cells in vitro， and to clarify its related mechanism. Methods The Huh7 cells were treated with different concentrations （0，1，5，10，20，40，and 60 μmol·L^－1）of motesanib.The proliferation rates of Huh7 cells in various groups were detected by CCK-8 assay；Western blotting method was used to detect the expression levels of EZH2 and phosphorylated EZH2（p-EZH2） proteins in the Huh7 cells in different concentrations（0，2.5，5.0，10.0，20.0 μmol·L^－1） of motesanib groups.The Huh7 cells were treated with motesanib and GSK126 in various groups；the appropriate drug concentrations were selected by CCK-8 assay and colony formation assay.The Huh7 cells were divided into control group，motesanib（10 μmol·L^－1） group， GSK126（ 5 μmol·L^－1）group， and combination （motesanib+GSK126） group. The 5-ethynyl-2'-deoxyu ridine（EdU） fluorescence staining assay was used to detect the proliferation rates of Huh7 cells in various groups；the apoptotic rates of Huh7 cells in various groups were detected by flow cytometry； Western blotting method was used to detect the expression levels of extracellular regulated protein kinases（ERK）， phosphorylated ERK （p-ERK），protein kinase B（AKT），and phosphorylated AKT（p-AKT） proteins in the Huh7 cells in various groups. Results The CCK-8 assay results showed that proliferation rate of Huh7 cells in different concentrations of motesenib groups were decreased gradually with the increasing of drug concentrations； compared with motesenib （0 μmol·L^－1） group， the proliferation rates of the Huh7 cells in 20， 40， and 60 μmol·L^－1 motesenib groups were decreased significantly （P<0.05 or P<0.01）.The Western blotting results showed that compared with 0 μmol·L^－1 motesanib group， the expression levels of EZH2 and p-EZH proteins in the Huh7 cells in 5，10， and 20 μmol·L^－1 motesanib groups were increased （P<0.05 or P<0.01）.The CCK-8 results and colony formation experiment results showed that 10 μmol·L^－1 motesanib and 5 μmol·L^－1 GSK126 could be used for the following experiment.Compared with control group，the proliferation rates of the Huh7 cells in motesanib group，GSK126 group，and combination group were significantly decreased（P<0.05 or P<0.01）； compared with motesanib group and GSK126 group， the proliferation rate of the cells in combination group was decreased （P<0.01），and the apoptotic rate was increased （P<0.01）.Compared with control group，motesanib group and GSK126 group， the expression levels of p-AKT and p-ERK proteins in the Huh7 cells in combination group were significantly decreased （P<0.05 or P<0.01）. Conclusion Motesanib alone has no significantly inhibitory effect on the proliferation and promoting effect on the apoptosis of the liver cancer Huh7 cells， which may be related to the increasing of EZH2 leading to the cell drug resistance. The combination of motesanib and GSK126 enhances the proliferation inhibition and pro-apoptotic effects of motesanib on the Huh7 cells by inhibiting AKT and ERK signaling pathways.

Key words: Motesanib, Hepatocellular carcinoma, Cell proliferation, Apoptosis, Enhancer of zeste homolog 2, GSK126