Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong 510282, China; Department of Molecular Biology, State Administration of Traditional Chinese Medicine of the People's Republic of China, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, Guangdong 510515, China.
Department of Neurosurgery/Neuro-Oncology, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-Sen University Cancer Center, Guangzhou, Guangdong 510060, China.
Phytomedicine. 2023 Jun;114:154764. doi: 10.1016/j.phymed.2023.154764. Epub 2023 Mar 16.
Our previous study found that XHP could induce GBM cells to undergo apoptosis. A lot of evidence suggests that glioma stem-like cells (GSCs) are key factors that contribute to disease progression and poor prognosis of glioblastoma multiforme (GBM). Traditional Chinese medicine has been applied in clinical practice as a complementary and alternative therapy for glioma.
To evaluate the effect and the potential molecular mechanism of Xihuang pill (XHP) on GSCs.
UPLC-QTOF-MS analysis was used for constituent analysis of XHP. Using network pharmacology and bioinformatics methods, a molecular network targeting GSCs by the active ingredients in XHP was constructed. Cell viability, self-renewal ability, apoptosis, and GSC markers were detected by CCK-8 assay, tumor sphere formation assay and flow cytometry, respectively. The interrelationship between GSC markers (CD133 and SOX2) and key proteins of the EGFR/Akt/mTOR signaling pathway was evaluated using GEPIA and verified by western blot. A GBM cell line stably overexpressing Akt was constructed using lentivirus to evaluate the role of Akt signaling in the regulation of glioma stemness. The effect of XHP on glioma growth was analyzed by a subcutaneously transplanted glioma cell model in nude mice, hematoxylin-eosin staining was used to examine pathological changes, TUNEL staining was used to detect apoptosis in tumor tissues, and the expression of GSC markers in tumor tissues was identified by western blot and immunofluorescence.
Bioinformatics analysis showed that 55 matched targets were related to XHP targets and glioma stem cell targets. In addition to causing apoptosis, XHP could diminish the number of GBM 3D spheroids, the proportion of CD133-positive cells and the expression level of GSC markers (CD133 and SOX2) in vitro. Furthermore, XHP could attenuate the expression of CD133, EGFR, p-Akt, p-mTOR and SOX2 in GBM spheres. Overexpression of Akt significantly increased the expression level of SOX2, which was prohibited in the presence of XHP. XHP reduced GSC markers including CD133 and SOX2, and impeded the development of glioma growth in xenograft mouse models in vivo.
We demonstrate for the first time that XHP down-regulates stemness, restrains self-renewal and induces apoptosis in GSCs and impedes glioma growth by down-regulating SOX2 through destabilizing the CD133/EGFR/Akt/mTOR cascade.
我们之前的研究发现，熊去氧胆酸（XHP）可诱导脑胶质瘤细胞发生凋亡。大量证据表明，神经胶质瘤干细胞（GSCs）是导致多形性胶质母细胞瘤（GBM）疾病进展和预后不良的关键因素。中药已作为一种补充和替代疗法应用于临床治疗胶质瘤。
评估西黄丸（XHP）对 GSCs 的作用及潜在分子机制。
采用 UPLC-QTOF-MS 分析方法对 XHP 进行成分分析。利用网络药理学和生物信息学方法，构建了 XHP 中活性成分靶向 GSCs 的分子网络。通过 CCK-8 检测、肿瘤球形成实验和流式细胞术分别检测细胞活力、自我更新能力、细胞凋亡和 GSC 标志物。利用 GEPIA 评估 GSC 标志物（CD133 和 SOX2）与 EGFR/Akt/mTOR 信号通路关键蛋白之间的相互关系，并通过 Western blot 进行验证。利用慢病毒构建 Akt 稳定过表达的 GBM 细胞系，评估 Akt 信号在调控胶质瘤干细胞特性中的作用。通过裸鼠皮下移植胶质瘤细胞模型分析 XHP 对胶质瘤生长的影响，苏木精-伊红染色观察病理变化，TUNEL 染色检测肿瘤组织中的细胞凋亡，Western blot 和免疫荧光鉴定肿瘤组织中 GSC 标志物的表达。
生物信息学分析表明，55 个匹配靶点与 XHP 靶点和神经胶质瘤干细胞靶点相关。XHP 不仅能诱导细胞凋亡，还能减少 GBM 3D 球体数量、CD133 阳性细胞比例以及体外 GSC 标志物（CD133 和 SOX2）的表达水平。此外，XHP 还能减弱 GBM 球体中 CD133、EGFR、p-Akt、p-mTOR 和 SOX2 的表达。Akt 的过表达显著增加了 SOX2 的表达水平，而 XHP 的存在则抑制了这一过程。XHP 降低了 CD133 和 SOX2 等 GSC 标志物的表达，并抑制了体内异种移植小鼠模型中胶质瘤的生长。
我们首次证明，XHP 通过破坏 CD133/EGFR/Akt/mTOR 级联反应，下调 SOX2 的表达，下调干细胞特性，抑制自我更新并诱导 GSCs 凋亡，从而抑制胶质瘤的生长。
