Angiopoietin-like protein 4 suppresses liver tumor growth and metastases by inhibition of angiogenesis and cell invasion

Abstract

Background and aim: Although surgical procedures are the first front treatments for liver cancer, tumor recurrence or metastasis remains to be the major problem, which significantly affects the long-term disease-free survival. Therefore, development of novel adjuvant therapies targeting for liver cancer growth, recurrence/metastasis will be essential. Angiopoietin-like protein 4 (ANGPLT-4) has been recently demonstrated to be able to inhibit tumor invasiveness by modification of cytoskeleton organization. In the present study, we aim to investigate the effect of ANGPLT-4 on liver cancer growth and metastasis in an orthotopic nude mice liver cancer model with metastatic potentials. Materials and methods: The orthotopic liver tumor nude mice model with distant metastatic potential was applied. 5×106 MHCC97L cells labeled with luciferase gene were injected subcutaneously into the right flank of the mice. Once the tumor reached 1 cm in diameter, it was removed and cut into about 1-2 mm cubes which were implanted into the left liver lobe of another group of nude mice. Ad-ANGPLT-4 (1×108) (treatment group) or Ad-luciferase (control group) was injected via portal vein after the tumor implantation. Tumor growth and metastasis were monitored by Xenogen in vivo imaging system. Hepatic stellate cell (HSC) activation and tumor-associated macrophage (TAM) in the tumor tissue were detected by α-SMA and ED1/ED2 staining. Tumor micro-vessel density (MVD) was also compared. Protein expression of ROCK (invasion) and VEGF (angiogenesis) of tumor tissues was detected by Western blot. Tumor-non-tumor margin was examined under electron microscopy. In vitro functional studies including tube formation, cell signaling linking to invasion and angiogenesis, lamellipodia formation in cell surface and stress fiber formation inside tumor cell were also compared between the MHCC97L cells with or without administration of ANGPLT-4. Results: The tumor growth was significantly inhibited by ANGPLT-4 treatment at different time points accompanied by lower incidence of lung/prostate metastasis compared to that of the control group. The observation of Xenogen IVIS was confirmed by histopathological examination. The HSC activation by α-SMA staining in the liver tumors was suppressed by ANGPLT-4 treatment accompanied with less TAM infiltration. Protein expression of ROCK and VEGF was down-regulated in the treatment group. Tumor vascular endothelial cell damage was found in the treatment group under electron microscopy. Consistent with in vivo study, in vitro functional study also demonstrated that ANGPLT-4 significantly inhibited tube formation and down-regulated cell invasion signaling pathways. Conclusion: ANGPLT-4 treatment significantly inhibited liver tumor growth and metastasis by inhibition of angiogenesis and down-regulation of cell invasion pathways.