Pharmaceutical College, Guangxi Medical University, Nanning, China; Department of Pharmacy, Liuzhou Traditional Chinese Medical Hospital, Liuzhou, China.
Guangxi Institute For Food and Drug Control, Nanning, China.
Int J Biol Macromol. 2019 Mar 1;124:1264-1273. doi: 10.1016/j.ijbiomac.2018.11.060. Epub 2018 Nov 30.
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.
从中华眼镜蛇蛇毒中分离得到的一种新胱氨酸丰富分泌蛋白（CRISP）家族成员 Natrin 已被证明具有抗癌活性。然而，其潜在的分子机制仍需进一步阐明。本研究采用 MTT 法评价细胞活力，透射电镜观察细胞凋亡形态，首次采用整合超高效液相色谱-四极杆飞行时间质谱（UPLC-Q/TOF MS）联用技术对 Natrin 诱导分化的 SMMC-7721 肝癌细胞凋亡引起的代谢紊乱进行代谢组学研究，并用实时荧光定量 PCR（FQ-PCR）检测 Natrin 诱导肝癌细胞凋亡过程中与凋亡相关的 mRNA 变化，探讨其可能的作用机制。Natrin 呈剂量依赖性显著抑制细胞增殖。主成分分析（PCA）和偏最小二乘判别分析（PLS-DA）结果表明，Natrin 能明显影响细胞的代谢轮廓。多元统计分析结果显示，共有 13 种代谢物被鉴定为与 Natrin 诱导的肝癌细胞凋亡密切相关的潜在生物标志物，涉及到包括鞘脂代谢、脂肪酸生物合成、脂肪酸代谢、甘油磷脂代谢和糖脂生物合成等 5 条代谢通路的变化。此外，与对照组相比，Natrin 诱导的肝癌细胞凋亡导致线粒体途径中 Bax/Bcl-2 比值增加。该研究表明，快速、整体的细胞代谢组学与分子生物学方法相结合可能是评估 Natrin 诱导细胞凋亡的潜在机制的有力工具，有助于深入了解 Natrin 的抗肝癌机制，并为 Natrin 在未来的临床应用提供参考。
