Next, we investigated whether ectopic Lcn2 expression by SH-J1 cells significantly inhibited wound closure (Fig. 4A, right panels). Cells harboring stable transfectants were significantly less likely to migrate to the wounded area compared with parent or vector control cells. In a modified Boyden chamber assay, stable transfectants penetrated the matrix and colonized the bottom surface of the Matrigel-coated membrane to DNA Synthesis inhibitor a lesser extent than vector control cells; this was true for both SH-J1 (Fig. 4B, left panels) and SH-J1-luc stable transfectants (Fig. 4B, right panels).

Lcn2 expression therefore appears to inhibit the migration and invasiveness of cells in vitro. We also evaluated the effects of EGF and TGF-β1 treatment on the ability of Lcn2-positive or -negative cell lines to close wounds (Supporting Fig. S11). EGF and TGF-β1 treatment MG-132 datasheet significantly enhanced the wound closure ability of HCC cells endogenously

expressing Lcn2, but not that of HCC cells ectopically expressing Lcn2. These results suggested that Lcn2 functions downstream of EGF and TGF-β1 signaling. Next, to determine the functional role of Lcn2 in HCC cell metastasis, we used SH-J1-luc cells expressing luciferase and established Lcn2-expressing SH-J1-luc cells (Lcn2-luc) (Fig. 4C, left panels). The metastatic phenotype of the Lcn2-luc cells was examined by injection of the cells into the tail veins (200 μL of 5 × 105 cells) of nude mice, followed by detection of multiple metastatic nodules in the lungs (Fig. 4C, right panel). Vector control cells first colonized and then continued growing in the lungs with many metastatic nodules, whereas Lcn2-expressing cells formed far fewer metastatic nodules in the lungs. Sufficient bioluminescence data were obtained 40 days postinjection (Fig. 4D). These results suggest that Lcn2

plays a critical role in inhibiting metastasis and invasion in HCC. Twist expression has been shown to promote migration and invasion in HCC.[31] MCE We found that Twist1 protein expression was significantly down-regulated in stable transfectants expressing Lcn2 (Fig. 5A) and in cells transduced with Lcn2-expressing adenovirus (Fig. 5B; Supporting Fig. S12). In contrast, Twist2, Slug, and Snail expression did not change (Supporting Fig. S13). Next, to investigate whether Twist1 down-regulation is dependent on transcriptional regulation, we examined Lcn2-mediated Twist1 mRNA expression by real-time PCR analysis in HEK293T (Supporting Fig. S14, left panel) and SH-J1 cells (Fig. 5C, left panel). We found that Twist1 down-regulation was associated with a decrease in transcript levels of this gene. Furthermore, a promoter assay revealed that Lcn2 effectively decreased Twist1 promoter activity in HEK293T (Supporting Fig. S14, right panel) and SH-J1 cells transfected with a Twist1-luc construct containing the human Twist1 promoter linked to a luciferase reporter gene (Fig. 5C, right panel).