Impact of shikonin on the malignant biological activity of liver cancer cells by regulating Notch signaling pathway / 天津医药

ABSTRACT

Objective To investigate the impact of shikonin(SHI)on the malignant biological activity of liver cancer cells by regulating Notch signaling pathway.Methods Western blot assay was used to detect the expression of Notch,Hairy mitosis-related enhancer-1(Hes1),hairy-related transcription factor-1(HEY1)protein in liver cancer tissue,paracancerous tissue,hepatoma cells(HepG2 cells,Hep3B cells,HCCLM3 cells,Huh-7 cells and SMMC-7721 cells)and normal liver cells(HL-7702 cells).Huh-7 cells were divided into the control group,the L-SHI group(1μmol/L SHI),the M-SHI group(2μmol/L SHI),the H-SHI group(4μmol/L SHI),the DAPT group(50μmol/L Notch signal inhibitor DAPT)and the H-SHI+VPA group[4μmol/L SHI and 8 mmol/L Notch pathway activator Valproic acid(VPA)].The proliferation of Huh-7 cells was detected by CCK-8 method and plate cloning test.The apoptosis and cell cycle of Huh-7 cells were detected by flow cytometry.Cell scratch test and Transwell invasion test were used to detect migration and invasion of Huh-7 cells.Western blot assay was used to detect the expression of epithelial-mesenchymal transformation(EMT)and apoptosis related proteins.Results The expression levels of Notch,HES1 and HEY1 were obviously increased in liver cancer tissue and cells,and Huh-7 cells showed the most obvious difference,therefore,Huh-7 cells were taken as the research object.Compared with the control group,the protein levels of Notch,HES1,HEY1 and Bcl-2 decreased,and the proportions of S phase and G2 phase cells,OD450 value,number of clones,migration rate,number of invasive cells and levels of N-cadherin and Vimentin decreased significantly in the L-SHI group,the M-SHI group,the H-SHI group and the DAPT group(P＜0.05).The proportion of G1/G0 phase cells,apoptosis rate and levels of Bax,cleaved Casase-3,and E-cadherin increased obviously(P＜0.05).The effect of SHI was dose-dependent.Compared with the H-SHI group,the above indexes showed the opposite trend in the H-SHI+VPA group.VPA attenuated the effect of SHI on reducing the malignant biological activity of liver cancer cells.Conclusion SHI may inhibit the proliferation,migration and invasion of Huh-7 cells and promote apoptosis of Huh-7 cells by inhibiting Notch signal pathway.