# Baculovirus-mediated endostatin and angiostatin activation of autophagy through the AMPK/AKT/mTOR pathway inhibits angiogenesis in hepatocellular carcinoma

## TL;DR

This study shows that a baculovirus-based protein inhibits liver cancer growth by triggering autophagy through a key signaling pathway.

## Contribution

The novel contribution is identifying autophagy as a mechanism for anti-angiogenic effects of BDS-hEA in HCC via AMPK/AKT/mTOR pathway modulation.

## Key findings

- BDS-hEA induces autophagy in endothelial cells, marked by increased autophagosomes and Beclin-1/LC3-II expression.
- Autophagy inhibition reduces BDS-hEA's anti-angiogenic and anti-tumor effects in vitro and in vivo.
- BDS-hEA modulates AMPK/AKT/mTOR signaling to suppress angiogenesis and tumor growth in HCC.

## Abstract

Hepatocellular carcinoma (HCC) is a highly vascularized carcinoma, and targeting its neovascularization represents an effective therapeutic approach. Our previous study demonstrated that the baculovirus-mediated endostatin and angiostatin fusion protein (BDS-hEA) effectively inhibits the angiogenesis of vascular endothelial cells and the growth of HCC tumors. However, the mechanism underlying its anti-angiogenic effect remains unclear. Increasing evidence suggests that autophagy has a significant impact on the function of vascular endothelial cells and response to cancer therapy. Hence, the objective of this research was to investigate the correlation between BDS-hEA-induced angiogenesis inhibition and autophagy, along with potential regulatory mechanisms. Our results demonstrated that BDS-hEA induced autophagy in EA.hy926 cells, as evidenced by the increasing number of autophagosomes and reactive oxygen species, accompanied by an upregulation of Beclin-1, LC3-II/LC3-I, and p62 protein expression. Suppression of autophagy using 3-methyladenine attenuated the functions of BDS-hEA-induced EA.hy926 cells, including the viability, proliferation, invasion, migration, and angiogenesis. Moreover, BDS-hEA induced autophagy by downregulating the expression of CD31, VEGF, and VEGFR2, as well as phosphorylated protein kinase B (p-AKT) and phosphorylated mammalian target of rapamycin (p-mTOR), while concurrently upregulating phosphorylated AMP-activated protein kinase (p-AMPK). The in vivo results further indicated that inhibition of autophagy by chloroquine significantly impeded the ability of BDS-hEA to suppress HCC tumor growth in mice. Mechanistically, BDS-hEA prominently facilitated autophagic apoptosis in tumor tissues and decreased the levels of ki67, CD31, VEGF, MMP-9, p-AKT, and p-mTOR while simultaneously enhancing the p-AMPK expression. In conclusion, our findings suggest that BDS-hEA induces autophagy as a cytotoxic response by modulating the AMPK/AKT/mTOR signaling pathway, thereby exerting anti-angiogenic effects against HCC.