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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.
摘要：
肝细胞癌（HCC）是一种高度血管化的癌，靶向其新血管形成代表了一种有效的治疗方法。我们先前的研究表明，杆状病毒介导的内皮抑素和血管抑素融合蛋白（BDS-hEA）有效抑制血管内皮细胞的血管生成和HCC肿瘤的生长。然而,其抗血管生成作用的潜在机制尚不清楚.越来越多的证据表明，自噬对血管内皮细胞的功能和对癌症治疗的反应具有重要影响。因此，本研究的目的是探讨BDS-hEA诱导的血管生成抑制与自噬,以及潜在的监管机制。我们的结果表明BDS-hEA在EA中诱导自噬。hy926细胞，自噬体和活性氧数量的增加证明了这一点，伴随着Beclin-1，LC3-II/LC3-I的上调，和p62蛋白表达。使用3-甲基腺嘌呤抑制自噬减弱了BDS-hEA诱导的EA的功能。hy926细胞，包括生存能力，扩散,入侵,迁移,和血管生成。此外，BDS-hEA通过下调CD31、VEGF、和VEGFR2，以及磷酸化蛋白激酶B（p-AKT）和磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR），同时上调磷酸化AMP激活蛋白激酶（p-AMPK）。体内结果进一步表明，氯喹对自噬的抑制作用显着阻碍了BDS-hEA抑制小鼠HCC肿瘤生长的能力。机械上,BDS-hEA显著促进肿瘤组织的自噬凋亡,降低肿瘤组织中ki67、CD31、VEGF、MMP-9,p-AKT,和p-mTOR，同时增强p-AMPK表达。总之,我们的研究结果表明，BDS-hEA通过调节AMPK/AKT/mTOR信号通路诱导自噬作为细胞毒性反应，从而对HCC发挥抗血管生成作用。
