Natrin, a new member of the cysteine-rich secretory protein (CRISP) family purified from the snake venom of Naja naja atra, has been demonstrated to have anticancer activity. However, the underlying molecular mechanisms need further elucidation. In this study, MTT was used to evaluate cell viability. Apoptotic cells were analyzed by employing a transmission electron microscope (TEM). Metabolomic study of the metabolic perturbations caused by natrin-induced apoptosis in differentiated SMMC-7721 cells was performed for the first time by using integrative ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS). To investigate the possible mechanism in the mitochondrial pathway of natrin-induced apoptosis, we measured apoptosis-related mRNA changes using real-time fluorescent quantitative PCR (FQ-PCR). Cell proliferation was significantly inhibited after treatment with natrin in a dose-dependent manner. Principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) clearly demonstrated that metabolic profiles were affected by natrin. The results of multivariate statistical analysis showed that a total of 13 metabolites were characterized as potential biomarkers highly implicated in natrin-induced apoptosis, which corresponded to fluctuations of five pathways, including sphingolipid metabolism, fatty acid biosynthesis, fatty acid metabolism, glycerophospholipid metabolism and glycosphingolipid biosynthesis. Furthermore, natrin-induced apoptosis showed an increase in the Bax/Bcl-2 ratio in the mitochondrial pathway compared with controls. This study illustrated that rapid and holistic cell metabolomics combining molecular biological approaches might be a powerful tool for evaluating the underlying mechanisms of natrin-induced apoptosis, which would help to deepen specific insights into the anti-hepatoma mechanisms of natrin and facilitate the clinical application of natrin in the future.
Keywords: Apoptosis; Hepatocellular carcinoma (HCC); Metabolomics; Natrin; UPLC-Q/TOF MS.
Copyright © 2018. Published by Elsevier B.V.