The lipid homeostasis regulation study of arenobufagin in zebrafish HepG2 xenograft model and HepG2 cells using integrated lipidomics-proteomics approach

Abstract

Ethnopharmacological relevance: Arenobufagin (ArBu) is an important anti-tumor ingredient of Chan'su which has long been used as traditional Chinese medicine in clinic for tumor therapy in China.

Aim of the study: The purpose of our study is to investigate the lipid homeostasis regulation effects of ArBu on zebrafish model of liver cancer and hepatoma cells, and to provide a reference for further clarifying its active mechanisms.

Materials and methods: The zebrafish xenograft model was established by injecting HepG2 cells stained with CM-Dil red fluorescent dye. Both the xenograft model and HepG2 cells were used to evaluate the anti-hepatoma activity of ArBu. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was the main method to study lipidomics, proteomics and the semiquantification of endogenous metabolites. Bioinformatics was used as an assistant tool to further explore the antitumor mechanism of ArBu.

Results: The lipidomics analysis revealed that ArBu caused differential lipids changes in a dose-dependent manner, including PCs, PEs, TGs, SMs, DGs, Cer and PA. PCs, PEs, SMs and TGs were markedly altered in both two models. The influence of glycerophospholipid metabolism was the major and commonly affected pathway. Notably, DGs and Cer were significantly changed only in HepG2 cells. Furthermore, the proteomics research in HepG2 cells fished the target proteins related to lipid homeostasis abnormalities and tumor suppression. ArBu reduced the expression of 65 differential proteins associated with the lipid metabolism, apoptosis and autophagy, such as LCLAT1, STAT3, TSPO and RPS27. Meanwhile, 7 amino acids of 29 determined metabolites were significantly changed, including tyrosine, glutamate, glutamine, leucine, threonine, arginine and isoleucine.

Conclusion: ArBu has a significant anti-hepatoma effect in vitro and a therapeutic effect on zebrafish xenograft model. It regulated the lipid homeostasis. Activated SM synthase and arginine deiminase, inhibited sphingomyelinase, amino acid supply and JAK-STAT3 signaling pathway, and the affected glycerophospholipid metabolism might explain these results.

Keywords: Amino acid; Anti-hepatoma; Arenobufagin; Lipid homeostasis; Lipidomics; Proteomics.