Department of Pharmacy, Shanxi Medical University, Taiyuan, Shanxi, China.
Shanxi Traditional Chinese Medicine Hospital, Taiyuan, Shanxi, China.
PLoS One. 2023 May 19;18(5):e0285966. doi: 10.1371/journal.pone.0285966. eCollection 2023.
Ginsenoside 24-hydroxy-ginsengdiol (24-OH-PD), extracted from red ginseng, is a novel diol-type ginsenoside, strongly inhibits the growth of human T-cell acute lymphoblastic leukaemia (T-ALL) CCRF-CEM cells. Our research aimed at investigating the mechanism underlying this inhibition. Cell viability was determined using the cell counting kit-8 (CCK-8) assay, and NOD/SCID mice bearing CCRF-CEM cells were used to verify the therapeutic effect of 24-OH-PD on T-ALL in vivo. We equally analysed pathways related to 24-OH-PD in CCRF-CEM cells using RNA-Seq analysis. Cell apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and mitochondrial permeability transition pore (mPTP) levels were detected by flow cytometry. The activity of caspase3 and caspase9 was detected by enzyme activity detection kits. The expression levels of apoptosis-related proteins and mRNA were determined through western blotting and quantitative reverse-transcription PCR assays (qRT-PCR). CCK-8 assay and animal xenograft experiments confirmed that 24-OH-PD significantly inhibited T-ALL in a dose-dependent manner, both in vivo and in vitro. RNA-Seq results suggest that mitochondria-mediated apoptosis pathway plays an important role in this process. Furthermore, intracellular ROS levels increased, mPTP opened, and ΔΨm decreased following 24-OH-PD treatment. Pretreatment with the antioxidant, NAC, reversed the effects of 24-OH-PD on apoptosis and ROS generation. Moreover, 24-OH-PD treatment increased the expression of Bax and caspase family members, thereby releasing cytochrome c (Cytc) and inducing apoptosis. Our findings showed that, 24-OH-PD induces apoptosis in CCRF-CEM cells by activating the mitochondrial-dependent apoptosis pathway through ROS accumulation. This inhibitory effect implies that 24-OH-PD could be further developed as treatment of T-ALL.
人参二醇 24-羟基-原人参二醇（24-OH-PD），从红参中提取，是一种新型二醇型人参皂苷，强烈抑制人 T 细胞急性淋巴细胞白血病（T-ALL）CCRF-CEM 细胞的生长。我们的研究旨在探讨这种抑制作用的机制。使用细胞计数试剂盒-8（CCK-8）测定细胞活力，并用 NOD/SCID 小鼠携带 CCRF-CEM 细胞验证 24-OH-PD 在体内对 T-ALL 的治疗效果。我们同样使用 RNA-Seq 分析分析 CCRF-CEM 细胞中与 24-OH-PD 相关的途径。通过流式细胞术检测细胞凋亡、活性氧（ROS）、线粒体膜电位（ΔΨm）和线粒体通透性转换孔（mPTP）水平。通过酶活性检测试剂盒检测 caspase3 和 caspase9 的活性。通过 Western blot 和定量逆转录 PCR 分析（qRT-PCR）测定凋亡相关蛋白和 mRNA 的表达水平。CCK-8 测定和动物异种移植实验证实，24-OH-PD 以剂量依赖性方式显著抑制 T-ALL，无论是在体内还是体外。RNA-Seq 结果表明，线粒体介导的细胞凋亡途径在这个过程中起着重要作用。此外，24-OH-PD 处理后细胞内 ROS 水平升高，mPTP 开放，ΔΨm 降低。用抗氧化剂 NAC 预处理可逆转 24-OH-PD 对细胞凋亡和 ROS 生成的影响。此外，24-OH-PD 处理增加了 Bax 和 Caspase 家族成员的表达，从而释放细胞色素 c（Cytc）并诱导细胞凋亡。我们的研究结果表明，24-OH-PD 通过 ROS 积累激活线粒体依赖性凋亡途径诱导 CCRF-CEM 细胞凋亡。这种抑制作用表明，24-OH-PD 可进一步开发为治疗 T-ALL 的方法。
