Effects Of 8-bromo-7-methoxychrysin On STAT3/Twist Signal Axis Of Hepatic Stellate Cell And Inducing Sphere Formation Of SMMC-7721 Cell Line

Objective:To determine the effect of 8-bromo-7-methoxychrysin(BrMC)on the activation of hepatic stellate cells and the properties of SMMC-7721 stem-like cell induced by conditional medium of liver cancer-associsted stellate cell,and to explore whether its mechanism is involved in blocking the STAT3/Twist signal axis.Methods:Human hepatocellular carcinoma SMMC-7721 cell line was cultured in vitro.Culture and expansion of liver cancer stem-like cells(LCSLCs)was used by ultra-low-adhesion plate suspension culture with stem cell condition medium.Treatments of LCSLCs with BrMC(0?5?10?20?mol/L)were used to prepare the conditional medium of liver cancer stem-like cells(LCSLC-CM)and LCSLC-CM derived from BrMC treatment(BrMC/LCSLC-CM).Human hepatic stellate cell LX-2 cell line was cultured in vitro.Treatment of LX-2 cells with LCSLC-CM act as liver cancer-associated stellate cell(LCAHSCs).And conditional medium of LCAHSC(LCAHSC-CM)and LCAHSC-CM derived from BrMC/LCSLC-CM treatment(BrMC/LCAHSC-CM)were prepared.Cellular immunofluorescence staining and Western-blot analysis were used to detect the expression of a-SMA and FAP in LX-2 cells and LCAHSC.LX-2 and LCAHSC cells were treated with cucurbitacin(JSI-124),a STAT3-specific inhibitor,and the expression of p-STAT3 and Twist protein were detected by western blot.The self-renewal ability,epithelial mesenchymal transition(EMT)and cancer stem cell marker protein expressions,migration and invasion of SMMC-7721 cells were measured by tumor sphere formation assay,Western blot analysis,scratching and Transwell invasion assay.Results:LCSLC-CM could activate the LX-2 cells to become LCAHSCs that expressed higher level of FAP than LX-2 cells.LCAHSC-CM effectively induced ability of self-renewal,cancer stem cell markers(CD 133,CD44 and ALDH1)expressions,epithelial mesenchymal transition,migration and invasion in SMMC-7721 cell.BrMC/LCSLC-CM prepared by treating LCSLCs with BrMC could inhibit the expression of FAP protein of LCAHSCs in a concentration-dependent manner but had no significant effect on that of a-SMA.In contrast to LCAHSC-CM,BrMC/LCAHSC-CM inhibited cancer stem cell markers(CD133,CD44 and ALDH1)expressions,sphere formation,epithelial mesenchymal transition,cell migration and invasion of SMMC-7721 cells induced by LCAHSC-CM,in a concentration-dependent manner.JSI-124 reduced STAT3 phosphorylation and Twist expression in LCAHSCs in a concentration-dependent manner.Furthermore,JSI-124 and BrMC synergistically antagonized cancer stem cell markers(CD133,CD44 and ALDH1)expressions,sphere formation,epithelial mesenchymal transition,cell migration and invasion of SMMC-7721 cells induced by LCAHSC-CM.Conclusion:1.LCSLC-CM pathologically activate the hepatic stellate cell LX-2 cell line and make it exhibits tumor-associated fibroblast phenotype,LCAHSC-CM induces characteristics of stem-like cells in SMMC-7721 cell line.2.BrMC/LCSLC-CM prepared by treatment LCSLCs with BrMC effectively inhibit the pathological activation of hepatic stellate cells induced by LCSLC-CM,and reduces the characteristics of liver cancer stem-like cells induced by LCAHSC-CM.3.The mechanism underlying BrMC inhibited hepatic stellate cell pathologic activation by LCSLC-CM and reduced the characteristics of liver cancer stem-like cells induced by LCAHSCs may be related to blocking of STAT3/Twist1 signaling pathway in LCAHSCs.