School of Pharmacy, Shaanxi University of Chinese Medicine, Xianyang, China.
Department of Chinese Materia Medica and Natural Medicines, Key Laboratory of Gastrointestinal Pharmacology of Chinese Materia Medica of the State Administration of Traditional Chinese Medicine, School of Pharmacy, Air Force Medical University, Xi'an, China.
J Biochem Mol Toxicol. 2022 Mar;36(3):e22973. doi: 10.1002/jbt.22973. Epub 2021 Dec 29.
Many glioma patients develop resistance to temozolomide (TMZ) treatment, resulting in reduced efficacy and survival rates. TMZ-resistant cell lines SHG44R and U87R, which highly express O -methylguanine DNA methyltransferase (MGMT) and P-gp, were established. CN-3, a new asterosaponin, showed cytotoxic effects on TMZ-resistant cells in a dose- and time-dependent manner via reactive oxygen species (ROS)-mediated apoptosis and autophagy. Transmission electron microscopy and monodansylcadaverine (MDC) staining showed turgidity of the mitochondria and autophagosomes in CN-3-treated SHG44R and U87R cells. The autophagy inhibitor 3-methyladenine was used to confirm the important role of autophagy in CN-3 cytotoxicity in TMZ-resistant cells. The ROS scavenger N-acetyl- l-cysteine (NAC) attenuated the levels of ROS induced by CN-3 and, therefore, rescued the CN-3 cytotoxic effect on the viability of SHG44R and U87R cells by Cell Counting Kit-8 assays and JuLI-Stage videos. MDC staining also confirmed that NAC rescued an autophagosome increase in CN-3-treated SHG44R and U87R cells. Western blotting revealed that CN-3 increased Bax, cleaved-caspase 3, cytochrome C, PARP-1, LC3-Ⅱ, and Beclin1, and decreased P-AKT, Bcl-2, and p62. Further rescue experiments revealed that CN-3 induced apoptosis and autophagy through ROS-mediated cytochrome C, cleaved-caspase 3, Bcl-2, P-AKT, PARP-1, and LC3-Ⅱ. In addition, CN-3 promoted SHG44R and U87R cells sensitive to TMZ by reducing the expression of P-gp, MGMT, and nuclear factor kappa B p65, and it had a synergistic cytotoxic effect with TMZ. Moreover, CN-3 disrupted the natural cycle arrest and inhibited the migration of SHG44R and U87R cells by promoting cyclin E1 and D1, and by decreasing P21, P27, N-cadherin, β-catenin, transforming growth factor beta 1, and Smad2.
许多神经胶质瘤患者对替莫唑胺（TMZ）治疗产生耐药性，导致疗效和生存率降低。建立了高度表达 O-甲基鸟嘌呤 DNA 甲基转移酶（MGMT）和 P-糖蛋白的 TMZ 耐药细胞系 SHG44R 和 U87R。新型甾体皂苷 CN-3 通过活性氧（ROS）介导的细胞凋亡和自噬，以剂量和时间依赖的方式对 TMZ 耐药细胞表现出细胞毒性作用。透射电子显微镜和单丹磺酰尸胺（MDC）染色显示 CN-3 处理的 SHG44R 和 U87R 细胞线粒体和自噬体肿胀。自噬抑制剂 3-甲基腺嘌呤用于确认自噬在 CN-3 对 TMZ 耐药细胞的细胞毒性中的重要作用。ROS 清除剂 N-乙酰-L-半胱氨酸（NAC）减弱了 CN-3 诱导的 ROS 水平，并通过细胞计数试剂盒-8 测定和 JuLI-Stage 视频挽救了 NAC 对 SHG44R 和 U87R 细胞活力的 CN-3 细胞毒性作用。MDC 染色也证实了 NAC 挽救了 CN-3 处理的 SHG44R 和 U87R 细胞中自噬体的增加。Western blot 显示 CN-3 增加了 Bax、cleaved-caspase 3、细胞色素 C、PARP-1、LC3-Ⅱ和 Beclin1，降低了 P-AKT、Bcl-2 和 p62。进一步的挽救实验表明，CN-3 通过 ROS 介导的细胞色素 C、cleaved-caspase 3、Bcl-2、P-AKT、PARP-1 和 LC3-Ⅱ诱导细胞凋亡和自噬。此外，CN-3 通过降低 P-gp、MGMT 和核因子 kappa B p65 的表达，使 SHG44R 和 U87R 细胞对 TMZ 敏感，并与 TMZ 具有协同细胞毒性作用。此外，CN-3 通过促进细胞周期蛋白 E1 和 D1 并降低 P21、P27、N-钙粘蛋白、β-连环蛋白、转化生长因子β 1 和 Smad2，破坏 SHG44R 和 U87R 细胞的自然周期阻滞并抑制其迁移。
