Anthocyanins from Lycium ruthenicum Murray inhibit HCC tumor angiogenesis and promote autophagic apoptosis through AMPK/PI3K/AKT/mTOR pathway
Authors: Wei Tingting , Cheng Jiajie , Fan Hongli , Cao Xue , Ding Shuqin , Kang Fei , Li Fangye , Wang Zhisheng 
Published: September 2023

Abstract
Purpose
Angiogenesis is a crucial factor in the initiation, progression and metastasis of tumor. The exploration for novel anti-angiogenic agents from natural sources has garnered significant attention. Our previous study confirmed the inhibitory effects of anthocyanins obtained from Lycium ruthenicum Murray (ALR) on the growth and metastasis of HepG2 cells, yet its potential to suppress neovascularization in hepatocellular carcinoma (HCC) remains unknown. This research aimed to assess the anti-angiogenic effect of ALR in HCC and elucidate its underlying mechanism.
Methods
Cell viability, colony formation, migration, invasion, and tubule formation were performed to evaluate the effect of ALR on EA.hy926 cells. Transcriptomics sequencing was performed to analyze the functional enrichment and pathway prediction of differentially expressed genes (DEGs). The formation of autophagosomes and autophagy-related proteins level were observed by AO staining, MDC staining, GFP-LC3-Adenovirus infection and western blot analysis. The relationship between autophagy and cell proliferation, angiogenesis, as well as AMPK/PI3K/AKT/mTOR pathway were analyzed in EA.hy926 cells treated with ALR in the presence or absence of 3-Methyladenine (3-MA, autophagy inhibitor). The transplanted hepatocellular carcinoma (HCC) model was established using HepG2 cells, and the therapeutic effect and underlying mechanism of ALR on HCC were assessed by H&E staining, TUNEL assay, immunofluorescence and immunohistochemistry analysis.
Results
Our findings demonstrated that ALR significantly suppressed the viability, proliferation, migration, invasion and angiogenesis of EA.hy926 cells in a dose-dependent pattern. The transcriptomic sequencing results revealed a close association between ALR inhibition of angiogenesis and autophagy, primarily enriched in the PI3K/AKT, AMPK and mTOR signaling pathways. In vitro outcomes further validated that ALR significantly augmented the presence of autophagosomes, enhanced the levels of Beclin-1, LC3-II/LC3-I, and p-AMPK while concurrently suppressing the levels of p62, p-PI3K, p-AKT, and p-mTOR. Notably, these effects were effectively counteracted using 3-Methyladenine. In vivo xenograft results demonstrated that ALR significantly inhibited the growth of HCC tumors. Mechanistically, ALR activated autophagy to suppress the expression of CD31, VEGF, and VEGFR2 in tumor tissues. Additionally, ALR induced autophagy-dependent apoptosis by up-regulating the levels of Bax, Cleaved caspase-3, and Cleaved caspase-9 while down-regulating the Bcl-2 level. Furthermore, it was discovered that ALR regulates autophagy by activating AMPK and suppressing the activity of PI3K/AKT/mTOR axis.
Conclusion
In combination, our data suggest that ALR executes its anti-HCC activity by inhibiting tumor angiogenesis and promoting autophagic apoptosis through regulation of the AMPK/PI3K/AKT/mTOR signaling cascade. The utilization of ALR as a natural adjuvant against tumors may hold immense potential in the foreseeable future.