High-Mobility Group Box 1 Promotes Colon Cancer Cell Migration By Down-Regulating E-Cadherin/p120-Catenin

I. ObjectiveThe purpose of this study is to detect whether HMGB1 can modulate the migration of colon cancer cells by decreasing E-cadherin and p120-catenin and to provide a potential therapeutic target for colon cancer. II. MethodsIn this study, Lo Vo cells, an immortalized human colon cancer cell line, were exposed to recombinant human HMGB1. Wound healing assay, transwell system and aggregation assay were used to investigate cell migration and adhesion. The effect of HMGB1 on adherens junction proteins was determined by Western blot and RT-PCR. Immunofluorescence assay was used to visualize the localization of p120-catenin and E-cadherin. Src family kinase specific inhibitor PP2 was employed to investigate the role of Src in HMGB1-induced cell migration and disassembly of junction proteins. III. Results 1. The influence of exogenous HMGB1 on Lo Vo cell migrationWound healing assay and transwell migration assay showed that migration was remarkably enhanced in HMGB1 group compared with the control group. 2. The effect of exogenous HMGB1 on cell-cell adhesionFollowing incubation with r HMGB1, Lo Vo cells displayed a marked down-regulation in cell–cell adhesion. 3. Expressions of E-cad and p120 were changed in HMGB1 treated cells(1) We treated the Lo Vo cells with HMGB1 at 40, 200, and 1000 ng/ml in culture medium for 24 h to detect the expression of p120 and E-cad. There was an HMGB1 dose-dependent decrease of p120 and E-cad protein expression.(2) Lo Vo cells were stimulated with HMGB1(1000 ng/ml) at 0, 6, 12, and 24 h, and the protein levels of E-cad and p120 were determined using Western blot analysis. It was observed that p120 and E-cad decreased in a time-dependent manner. To clarify whether HMGB1 regulated p120-catenin expression at the transcriptional level, we examined p120-catenin m RNA expression in Lo Vo cells and found that the content of p120-catenin m RNA was not changed significantly.(3) Immunofluorescence assay was used to visualize the localization of p120-catenin and E-cadherin. In control cells, p120-catenin and E-cadherin were localized at cell-cell contact sites in an intact and continuous linear pattern. In contrast, HMGB1(1000 ng/ml) exposure induced a weak fluorescence in cytoplasm. 4. The effect of PP2 on HMGB1-induced migration and down-regulation of E-cadherin/catenin complex.PP2 pretreatment of Lo Vo cells rescued E-cadherin and p120-catenin expression and inhibited the cell migration. IV. ConclusionsHMGB1 may down-regulate E-cadherin and p120-catenin via Src family kinase, which decreases cell-cell adhesion and promotes migration of colon cancer cells.