Hepatology. could be a potential target for HCC chemotherapy. in Methods for details) of Hus, HepG2, Hep3B, Huh7, and PLC/PRF/5 cells. ***p 0.001 versus Hus cells. C. Western blotting analysis of L-FABP and VEGF-A expression in Hus/L-FABP and Hus/Vector (vector-only control) cells. *p 0.05 versus Hus/Vector control. D. angiogenic potential (score: see panel B) of Hus/L-FABP and Hus/Vector cells. Angiogenic vascular tube was imaged at 8 h. ***p 0.001 versus Hus/Vector control. E. angiogenic activity of Hus/L-FABP and Hus/Vector cells assessed using a Matrigel plug assay. a: Matrigel plugs recovered from mice injected with Hus/Vector and Hus/L-FABP cells. Arrows indicate infiltration of blood vessels. b: Larotaxel Immunohistochemical (IHC) staining of CD31 (angiogenesis marker) in Matrigel plugs showed that Hus/L-FABP promoted angiogenesis, and the positively stained vessels are indicated by arrows. *p 0.05 versus Hus/Vector control (n = 3). To examine the effects of L-FABP on VEGF-A expression, we generated L-FABP-overexpressing stable clones with Hus cells and we used Huh7 cells to produce L-FABP shRNA knockdown clones. As shown in Figure ?Figure2C,2C, VEGF-A expression (at both the mRNA and protein levels) was higher in Hus/L-FABP cells than Larotaxel in control cells, whereas the expression of VEGF-A decreased markedly in Huh7/L-FABP shRNA cells relative to the control (Supplementary Figure 2A). Angiogenesis was also significantly higher in Hus/L-FABP cells than in control cells (Figure ?(Figure2D),2D), i.e., it decreased in Huh7/L-FABP shRNA cells (Supplementary Figure 2B). To further examine whether L-FABP promotes angiogenesis angiogenic activity (score: see in Methods for details) of Hus/L-FABP and Hus/Vector cells treated with rapamycin or cyclohexamide (doses identical to those in D) for 12 h. ***p 0.001 versus Hus/Vector control. To confirm our findings, full-length and successive 5 deletion (D1CD3) constructs of the VEGF-A gene promoter were cloned into pGL4.22 luciferase reporter vectors, and a RGS20 luciferase reporter assay was conducted (Figure 5C, a). Results showed that VEGF-A transcriptional activity was elevated ~16.5-fold in L-FABP-overexpressing Hus cells compared with that in control cells, whereas deletion of the HIF-1 binding site (D1-D3) reduced this activity to ~2.5 fold that of the control (Figure 5C, b). Additionally, the chromatin immunoprecipitation assay demonstrated that the association between HIF-1 and the VEGF-A promoter was enhanced in Hus/L-FABP cells (Figure ?(Figure5D).5D). To further address the regulation of VEGF-A expression, Hus/L-FABP cells were treated with rapamycin (mTOR inhibitor) or cyclohexamide (translation inhibitor); consequently, decreased VEGF-A expression (Figure 5E, a) and concentration-dependent inhibition of angiogenic potential (Figure ?(Figure5F)5F) were observed. Treatment of Hus/Vector cells with the proteasome inhibitor MG132 also indicated that L-FABP-induced VEGF-A expression did not occur via inhibition of protein degradation (Figure 5E, b). Taken together, these data suggest that L-FABP-induced VEGF-A expression was regulated via the Akt/mTOR/P70S6K/4EBP1 pathway in a HIF-1-dependent manner. L-FABP promotes tumor growth and metastasis (Figure ?(Figure6C).6C). In an tumor metastasis assay, the number of metastatic nodules formed in the lungs of NOD/SCID mice after 60 days was 3.9-fold higher in the Hus/L-FABP-injected group relative to the control group (Figure ?(Figure6D),6D), and angiogenic vessel formation was increased in these nodules (Figure ?(Figure6E).6E). The results of these experiments support the correlation between L-FABP and VEGF-A expression. Open in a separate window Figure 6 L-FABP promotes tumor growth and metastasis angiogenic activity (score: see in Methods for details) of mutants. Images represent amino acid substitutions: (a) L-FABP (wild type), (b) L-FABP (F3 to W), (c) L-FABP (K20 to E), (d) Larotaxel L-FABP (K31 to E), and (e) L-FABP (T94 to A). ***p 0.001 versus wild-type. C. Migration activity of the mutants. Images (aCe) represent the amino acid substitutions described in (B). **p 0.01, ***p 0.001 versus wild-type. D. Migration activity of Hus/L-FABP cells treated with MCD (cholesterol depletion agent: 5, 10, or 20 mM) for 12 h. **p 0.01 versus water-treated control group. E. Western blot analysis of Hus/L-FABP cells treated with MCD (5, 10, or 20 mM) for 6 h. *p 0.05, **p 0.01, ***p 0.001 versus water-treated control group. DISCUSSION HCC is characterized by its aggressiveness and angiogenic capability; thus, the angiogenic factor VEGF is considered to be a target for HCC therapy [1, 3]. Here, we reported for the first time that overexpression of L-FABP plays an important role in VEGF-A expression and cell migration in HCC. Furthermore, we demonstrated that L-FABP associates with VEGFR2 in the cell membrane, which leads to activation of VEGFR2-related signaling (i.e., Src/FAK/cdc42 and Akt/mTOR/HIF-1 signaling). Additionally, we showed that the T94A mutation of L-FABP, which is related to cholesterol association activity, significantly decreases.