| Objective: To investigate the effects of 8-bromo-7-methoxychrysin(Br MC) on polarized phenotype of liver tumor-associated macrophage(TAM) and the liver cancer stem-like cell characteristics such as self-renewal, expressions of stem cell markered protein, migration and invasion in SMMC-7721 cell line.Furthmore, to explore the relevance of regulating TAM NF-κB expression between reversing TAM polarized phenotype and inhibiting TAM induced liver cancer stem-like cell characteristics of SMMC-7721 cells by Br MC.Methods: The third generation of sphere forming cells(SFCs) derived from SMMC-7721 cell were obtained to serve as liver cancer stem-like cell(LCSLCs) model and collected LCSLCs-conditioned medium(LCSLC-CM). THP-1 monocyte cell line were activated by Phorbol Myristate Acetate(PMA) as THP-1 derived macrophage, TAM were obtained from THP-1 drived macrophage incubated by LCSLC-CM or co-cultured with LCSLCs in Transwell insert system. M1 phenotype macrophages were treated by LPS and INF-γas control. LCSLCs or LCSLCs/THP-1 derived macrophage co-cuture system was treated with various concentrations of Br MC(0、5、10、20 μmol/L) to observe the effect of drug intervention. SN50, a specific inhibitor of the NF-κB pathway, was used to explore the mechanism underlying Br MC pharmacological effects. Self-renewal capacity was analyzed by sphere formation assay, expressions of stem cell markers CD133 and CD44 were analyzed by western blot analysis,cell migration and invasiveness were measured by wound-healing and invasion assay.Expressions of macrophage surface marker CD68 and M2 phonetype macrophage, i.e.TAM, surface marker CD163 were analyzed by immunofluorescence staining and western blotting.Concentration of IL-10, IL-12, VEGF and MMP-9 in TAM-CM was analyzed by ELISA assay. NO was detected by Giess assay.Results: 1. Compared with parental cells, the third generation of SFCs derived from human hepatocellular carcinoma SMMC-7721 cell line over-expressed stem cell marker CD133 and CD44 and had higher sphere forming rate, which exhibited liver cancer stem like properties. 2. LCSLC-CM or co-cultured with liver cancer stem like cell effectively induced THP-1 macrophage transdifferentiation to TAM in vitro. Compared with M1 phonetype macrophage, TAM over-expressed CD163 protein; cell cytokine profiles was IL-10high、IL-12low、VEGF high、MMP-9 high, and NO decreased release, which exhibited M2 phonetype polarization. 3. Conditional medium derived from TAM or LCSLCs/THP-1 derived macrophage co-culture significantly induced SMMC-7721 cells self-renewal capacity, stem cell marker expression, cell migration and invasion in vitro. 4. Br MCreduced TAM CD163 expression and reversed TAMs M2-like phenotype cytokine profiles, in a dose-dependent manner. 5. Br MC reduced TAM-induced SMMC-7721 cell stem cell marker CD133 and CD44 protein expression, sphere-forming and cell migration and invasion in vitro, in a dose-dependent manner(P<0.05). 6. SN50, a specific inhibitor of the NF-κB, reduced expression level of NF-κB(p65) protein in TAM(P<0.05). Meanwhile, SN50 and Br MC synergistically inhibited TAM-CM or LCSLCs/THP-1 derived macrophage co-culture-CM induced SMMC-7721 cells self-renewal capacity, stem cell marker CD133 and CD44 expression, cell migration and invasion in vitro.Conclusions:1. LCSLC-CM or LCSLCs/THP-1 derived macrophage co-culture contribute to M2 phonetype polarization of THP-1 derived-macrophages. 2. LCSLC-CM or LCSLCs/THP-1 derived macrophage co-culture induce liver stem like cell characteristics such as self-renewal, stem cell marker protein expression, migration and invasion in SMMC-7721 cell line. 3. Br MC reverses M2 phonetype polarization of TAM. 4. Br MC inhibits TAM-induced SMMC-7721 cell line liver cancer stem like characteristics. 5. Br MC reverses M2 phonetype polarization and inhibits TAM-induced SMMC-7721 cell line liver cancer stem like characteristics, which is involved in downregulation of NF- κB(p65) protein expression in TAM. |
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