Antiproliferative activity of marine stingray Dasyatis sephenvenom on human cervical carcinoma cell line

Abstract Background Venoms comprise mixtures of numerous bioactive compounds that have a wide range of pharmacologic actions. Toxins from venomous animals have attracted the attention of researchers because of their affinity for primary sites responsible for lethality and their efficacy at extremely low concentrations. The venoms of marine stingrays have not been extensively studied and limited data is available on them. The present study aims to evaluate the antiproliferative and biochemical properties of the venom obtained from a species of marine stingray (Dasyatis sephen) on human cervical cancer cell line HeLa. Methods The antiproliferative effect of D. sephen venom was determined by MTT assay, and the oxidative stress was determined by lipid peroxidation method along with assessment of changes in the enzymatic and non-enzymatic antioxidant status. We observed intracellular reactive oxygen species (ROS) levels by DCFH-DA method, mitochondrial membrane potential alterations by rhodamine 123 staining and apoptotic morphological changes by acridine orange/ethidium bromide dual staining method. Results D. sephen venom enhances lipid peroxidative markers such as thiobarbituric acid reactive substance, conjugated diene, and lipid hydroperoxide in HeLa cell lines. Stingray venom enhances the ROS levels, which is evidenced by the increased 2–7-diacetyl dichlorofluorescein fluorescence. Further, D. sephen venom treatment altered the mitochondrial membrane potential in HeLa cells. Additionally, we observed increased apoptotic morphological changes in D. sephen venom-treated groups. Conclusions Dasyatis sephen venom exhibits potent antiproliferative effect on HeLa cell line and upon further purification it could be a promising antiproliferative agent.

摘要
背景：毒液是由多种生物活性化合物组成的混合物，具备广泛的药理学活性。有毒动物来源的毒素因其可与致死性关键靶点结合、且在极低浓度下即可发挥功效，而备受研究者关注。目前海洋魟鱼毒液的相关研究尚不够深入，公开数据较为有限。本研究旨在评估一种海洋魟（Dasyatis sephen）毒液对人宫颈癌细胞系HeLa的抗增殖活性与生化特性。
方法：采用MTT比色法（MTT assay）检测该魟鱼毒液的抗增殖活性；通过脂质过氧化法（lipid peroxidation method）检测氧化应激水平，并同时评估酶促与非酶促抗氧化系统的状态变化。采用DCFH-DA法检测细胞内活性氧（reactive oxygen species, ROS）水平，通过罗丹明123（rhodamine 123）染色检测线粒体膜电位改变，采用吖啶橙（acridine orange）/溴化乙锭（ethidium bromide）双染色法观察细胞凋亡形态学变化。
结果：该魟鱼毒液可提升HeLa细胞系中的脂质过氧化标志物水平，包括硫代巴比妥酸反应物质、共轭二烯与脂质过氧化物。魟鱼毒液可升高ROS水平，这一现象可通过2',7'-二乙酰二氯荧光素荧光强度的增强得以证实。此外，该魟鱼毒液处理可改变HeLa细胞的线粒体膜电位。同时，该魟鱼毒液处理组的细胞凋亡形态学变化显著增加。
结论：鸢𫚉毒液对HeLa细胞系具有显著的抗增殖活性，经进一步纯化后有望成为极具潜力的抗增殖制剂。