Kanamori Yuta, Via Lisa Dalla, Macone Alberto, Canettieri Gianluca, Greco Antonio, Toninello Antonio, Agostinelli Enzo
Department of Biochemical Sciences 'A. Rossi Fanelli', Sapienza University of Rome, I-00185 Rome, Italy.
Department of Pharmaceutical and Pharmacological Sciences, University of Padua, I-35131 Padua, Italy.
Exp Ther Med. 2020 Feb;19(2):1511-1521. doi: 10.3892/etm.2019.8383. Epub 2019 Dec 27.
Aged garlic extract (AGE) has been demonstrated to have therapeutic properties in tumors; however its mechanisms of action have not yet been fully elucidated. A previous study revealed that AGE exerts an anti-proliferative effect on a panel of both sensitive [wild-type (WT)] and multidrug-resistant (MDR) human cancer cells. Following treatment of the cells with AGE, cytofluorimetric analysis revealed the occurrence of dose-dependent mitochondrial membrane depolarization (MMD). In this study, in order to further clarify the mechanisms of action of AGE, the effects of AGE on mitochondria isolated from rat liver mitochondria (RLM) were also examined. AGE induced an effect on the components of the electrochemical gradient (Δµ ), mitochondrial membrane potential (ΔΨ) and mitochondrial electrochemical gradient (ΔpH). The mitochondrial membrane dysfunctions of RLM induced by AGE, namely the decrease in both membrane potential and chemical gradient were associated with a higher oxidation of both the endogenous glutathione and pyridine nucleotide content. To confirm the anti-proliferative effects of AGE, experiments were performed on the human neuroblastoma (NB) cancer cells, SJ-N-KP and the MYCN-amplified IMR5 cells, using its derivative S-allyl-L-cysteine (SAC), with the aim of providing evidence of the anticancer activity of this compound and its possible molecular mechanism as regards the induction of cytotoxicity. Following treatment of the cells with SAC at 20 mM, cell viability was determined by MTT assay and apoptosis was detected by flow cytometry, using Annexin V-FITC labeling. The percentages of cells undergoing apoptosis was found to be 48.0% in the SJ-N-KP and 50.1% in the IMR5 cells. By cytofluorimetric analysis, it was suggested that the target of SAC are the mitochondria. Mitochondrial activity was examined by labeling the cells with the probe, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylimidacarbocyanine iodide (JC-1). Following treatment with SAC at 50 mM, both NB cell lines exhibited a marked increase in MMD. On the whole, the findings of this study indicate that both natural products, AGE and SAC, cause cytotoxicity to tumor cells via the induction of mitochondrial permeability transition (MPT).
老化大蒜提取物（AGE）已被证明在肿瘤治疗中具有一定特性；然而其作用机制尚未完全阐明。先前的一项研究表明，AGE对一组敏感的[野生型（WT）]和多药耐药（MDR）人类癌细胞均具有抗增殖作用。用AGE处理细胞后，细胞荧光分析显示出现剂量依赖性线粒体膜去极化（MMD）。在本研究中，为了进一步阐明AGE的作用机制，还检测了AGE对从大鼠肝线粒体（RLM）分离出的线粒体的影响。AGE对电化学梯度（Δµ）、线粒体膜电位（ΔΨ）和线粒体电化学梯度（ΔpH）的组成部分产生了影响。AGE诱导的RLM线粒体膜功能障碍，即膜电位和化学梯度的降低，与内源性谷胱甘肽和吡啶核苷酸含量的更高氧化有关。为了证实AGE的抗增殖作用，使用其衍生物S-烯丙基-L-半胱氨酸（SAC）对人神经母细胞瘤（NB）癌细胞SJ-N-KP和MYCN扩增的IMR5细胞进行了实验，目的是提供该化合物抗癌活性及其诱导细胞毒性可能的分子机制的证据。用20 mM的SAC处理细胞后，通过MTT法测定细胞活力，并使用膜联蛋白V-FITC标记通过流式细胞术检测细胞凋亡。发现SJ-N-KP细胞中发生凋亡的细胞百分比为48.0%，IMR5细胞中为50.1%。通过细胞荧光分析表明，SAC的作用靶点是线粒体。用探针5,5',6,6'-四氯-1,1',3,3'-四乙基咪唑羰花青碘化物（JC-1）标记细胞来检测线粒体活性。用50 mM的SAC处理后，两种NB细胞系均表现出MMD显著增加。总体而言，本研究结果表明，天然产物AGE和SAC均通过诱导线粒体通透性转变（MPT）对肿瘤细胞产生细胞毒性。
