Nikodijević Danijela D, Jovankić Jovana V, Cvetković Danijela M, Anđelković Marko Z, Nikezić Aleksandra G, Milutinović Milena G
University of Kragujevac, Faculty of Science, Department of Biology and Ecology, Radoja Domanovića 12, 34000, Kragujevac, Serbia.
University of Kragujevac, Faculty of Science, Department of Biology and Ecology, Radoja Domanovića 12, 34000, Kragujevac, Serbia.
Eur J Pharmacol. 2021 Nov 5;910:174466. doi: 10.1016/j.ejphar.2021.174466. Epub 2021 Sep 3.
This study evaluated the potential of antitumor activity of snake venom from Vipera ammodytes and L-amino acid oxidase from Crotalus adamanteus on different colorectal cancer cell lines through determination of cytotoxic activity by MTT assay, pro-apoptotic activity by acridine orange/ethidium bromide staining, and concentrations of redox status parameters (superoxide, reduced glutathione, lipid peroxidation) by colorimetric methods. The expression of genes involved in the biotransformation process and metabolite efflux was determined by qPCR method, while protein expression of glutathione synthetase and P-glycoprotein were analysed by immunocytochemistry. The analysis of cell death shows that snake venom dominantly leads cells to necrosis. Induction of apoptosis by L-amino acid oxidase was in correlation with oxidative disbalance in cancer cells. Gene expression profile of membrane transporters and CYP genes were different in each cell line and in correlation with their sensitivity of treatment. Our results show that L-amino acid oxidase from snake venom is a potent cytotoxic substance with pronounced pro-apoptotic activity. The inhibition of P-glycoprotein suggests that L-amino acid oxidase is a good substance for furter research of antitumor effect, with unexpressed potential for occurrence of drug resistance in vitro.
本研究通过MTT法测定细胞毒性活性、吖啶橙/溴化乙锭染色法测定促凋亡活性以及比色法测定氧化还原状态参数（超氧化物、还原型谷胱甘肽、脂质过氧化）的浓度，评估了蝰蛇（Vipera ammodytes）蛇毒和金刚背响尾蛇（Crotalus adamanteus）的L-氨基酸氧化酶对不同结肠癌细胞系的抗肿瘤活性潜力。通过qPCR法测定参与生物转化过程和代谢物外排的基因表达，同时通过免疫细胞化学分析谷胱甘肽合成酶和P-糖蛋白的蛋白表达。细胞死亡分析表明，蛇毒主要导致细胞坏死。L-氨基酸氧化酶诱导的凋亡与癌细胞中的氧化失衡相关。每种细胞系中膜转运蛋白和CYP基因的基因表达谱不同，且与其治疗敏感性相关。我们的结果表明，蛇毒中的L-氨基酸氧化酶是一种具有显著促凋亡活性的强效细胞毒性物质。对P-糖蛋白的抑制表明，L-氨基酸氧化酶是进一步研究抗肿瘤作用的良好物质，在体外具有未表达的耐药发生潜力。
