a Life Science and Biology Pharmacy College, Shenyang Pharmaceutical University , Shenyang , PR China.
Free Radic Res. 2019 Jul;53(7):714-726. doi: 10.1080/10715762.2019.1603376. Epub 2019 Jul 3.
The antitumor effects of silibinin are of increasing interest, though its mechanism is not yet clear. The goal of this study was to clarify the mechanism of silibinin-induced cell death in the A431 human epidermoid carcinoma cell line. We used a cell viability assay, flow cytometry, nitric oxide (NO) assay, and western blotting to examine relationships between silibinin, NO generation and apoptosis in A431 cells. Silibinin inhibited A431 cell growth in a dose-dependent manner, inducing mitochondrial damage, and apoptosis at a high dose. At the same time, high dose silibinin increased NO levels in A431 cells and the endothelial nitric oxide synthase (eNOS) inhibitor NG-nitro-L-arginine methylester (L-NAME) attenuated silibinin-induced cell growth inhibition. By western blotting, silibinin caused increased eNOS phosphorylation in the mitochondria. The AMP-activated protein kinase inhibitor compound C significantly decreased p-eNOS expression, while blocking eNOS did not affect p-AMPK levels, suggested that AMPK acted upstream of eNOS. This study showed that silibinin increased NO levels in A431 cells by activating the AMPK-eNOS pathway, leading to mitochondrial dysfunction and apoptosis. In this mechanism of action, mitochondrial eNOS played an important role. The results provided new understanding of the functions of intracellular NO.
水飞蓟宾的抗肿瘤作用越来越受到关注，但作用机制尚不清楚。本研究旨在阐明水飞蓟宾诱导 A431 人表皮癌细胞系细胞死亡的机制。我们使用细胞活力测定、流式细胞术、一氧化氮（NO）测定和 Western blot 来研究水飞蓟宾、NO 生成和 A431 细胞凋亡之间的关系。水飞蓟宾以剂量依赖性方式抑制 A431 细胞生长，在高剂量下诱导线粒体损伤和凋亡。同时，高剂量水飞蓟宾增加了 A431 细胞中的 NO 水平，内皮型一氧化氮合酶（eNOS）抑制剂 NG-硝基-L-精氨酸甲酯（L-NAME）减弱了水飞蓟宾诱导的细胞生长抑制。通过 Western blot，水飞蓟宾导致线粒体中 eNOS 的磷酸化增加。AMP 激活的蛋白激酶抑制剂化合物 C 显著降低了 p-eNOS 的表达，而阻断 eNOS 不影响 p-AMPK 水平，表明 AMPK 位于 eNOS 的上游。本研究表明，水飞蓟宾通过激活 AMPK-eNOS 通路增加 A431 细胞中的 NO 水平，导致线粒体功能障碍和细胞凋亡。在这种作用机制中，线粒体 eNOS 发挥了重要作用。该结果为理解细胞内 NO 的功能提供了新的认识。
