Effects Of Combination With Sorafenib And 8-bromo-7-methoxychrysin On Properties Of Liver Cancer Stem-like Cells In SMMC-7721 Cell Line

Objective: To investigate Effects of combination with the8-bromo-7-methoxychrysin( Br MC) and Sorafenib(SO) on properties of liver cancer stem-like cell( LCSLCs) in SMMC-7721 cell line,which whether involved in regulating the expression of HIF-1alpha,Twist and NF-κB protein.Methods: Sphere forming cells(SFCs) were cultured and amplified from SMMC-7721 cell line with stem cell conditioned medium using ultra-low adhesion plate. In parental cells, SFCs or the SFCs treated by Br MC(5mol/L) or SO(5, 15, 45mol/L) or both,tumorsphere formation assay was used to determine the ability to self renew, Western blot was used to analyze the protein expression of stem cell markers(CD133, CD44, ALDH1), scratch assays cell was used to examine migration of cells in vitro, the Transwell chamber invasion assay was used to test the the of ability to invasive of cell in vitro, Western blot was used to analyze expression of E-cadherin and N-cadherin protein, the cell apoptosis detection kit was used to determine apoptosis rate, so as to identification of LCSLCs properties and evaluating the effects of combination with Br MC and SO; Western blot was used to analyze expression of HIF-1alpha, Twist and NF- κB(p65) for exploring the mechanism underlying these actions.Results: Culture in suspension with stem cell culture medium using ultra-low adhesion plate for 5 days, obtained SFCs from human hepatocellular carcinoma SMMC-7721 cell line. SFCs possess characteristics of LCSLCs, as evidenced by higher tumorsphere formation rate(P≤0.05) and the expression of stem cell markers(CD133, CD44, ALDH1)(P≤0.05), stronger ability of cell migration and invasion in vitro(P≤0.05), downregulation of the E-cadherin protein expression(P≤ 0.05), at the same time, upregulation of the expression of N-cadherin protein(P≤ 0.05), the low rate of spontaneous apoptosis compared with the parental cells.Treatment of LCSLCs with Br MC(5 mol/L) or SO(5, 15 and 45 mol/L) or in combination caused a synergistic inhibition of the self-renewal capacity(P≤0.05), decreased expression of stem cell markers CD133, CD44 and ALDH1(P≤0.05), inhibited the migration and invasion of cell(P≤0.05), downregulated the expression of N-cadherin protein(P≤0.05) and upregulated the E-cadherin protein(P≤0.05) and promoting cell apoptosis(P≤0.05). Western blot analysis showed that the Br MC and SO cooperatively downregulated expression of HIF-1alpha and Twist1 protein of LCSLCs(P≤0.05) and Br MC antagonismed the effects of the upregulation of NF- κ B(p65) protein expression induced by SO in LCSLCs(P≤0.05).Conclusion:1. SFCs of human hepatocellular carcinoma SMMC-7721 cell line possess properties of LCSLCs.2. BrMC and SO synergistically inhibit the characteristics of LCSLCs.3. BrMC enhances the inhibitory effect of SO on the characteristics of LCSLCs, which is involved in downregulation of HIF-alpha and Twist1 protein expression by that Br MC cooperate with SO and antagonism of upregulation of NF- κB(p65) protein expression induced by SO.