Zhao Boxian, Shen Chen, Zheng Zhixing, Wang Xiaoxiong, Zhao Wenyang, Chen Xin, Peng Fei, Xue Linmeng, Shu Mengting, Hou Xu, Wang Kaikai, Zhong Chen, Sun Jingxian, Wan Jinzhao, Zhao Shiguang
Department of Neurosurgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China.
Institute of Brain Science, Harbin Medical University, Harbin, China.
Cell Physiol Biochem. 2018;51(4):1566-1583. doi: 10.1159/000495646. Epub 2018 Nov 29.
BACKGROUND/AIMS: Glioblastoma multiforme (GBM) is the most devastating and widespread primary central nervous system tumour in adults, with poor survival rate and high mortality rates. Existing treatments do not provide substantial benefits to patients; therefore, novel treatment strategies are required. Peiminine, a natural bioactive compound extracted from the traditional Chinese medicine Fritillaria thunbergii, has many pharmacological effects, especially anticancer activities. However, its anticancer effects on GBM and the underlying mechanism have not been demonstrated. This study was conducted to investigate the potential antitumour effects of peiminine in human GBM cells and to explore the related molecular signalling mechanisms in vitro and in vivo Methods: Cell viability and proliferation were detected with MTT and colony formation assays. Morphological changes associated with autophagy were assessed by transmission electron microscopy (TEM). The cell cycle rate was measured by flow cytometry. To detect changes in related genes and signalling pathways in vitro and in vivo, RNA-seq, Western blotting and immunohistochemical analyses were employed.
Peiminine significantly inhibited the proliferation and colony formation of GBM cells and resulted in changes in many tumour-related genes and transcriptional products. The potential anti-GBM role of peiminine might involve cell cycle arrest and autophagic flux blocking via changes in expression of the cyclin D1/CDK network, p62 and LC3. Changes in Changes in flow cytometry results and TEM findings were also observed. Molecular alterations included downregulation of the expression of not only phospho-Akt and phospho-GSK3β but also phospho-AMPK and phospho-ULK1. Furthermore, overexpression of AKT and inhibition of AKT reversed and augmented peiminine-induced cell cycle arrest in GBM cells, respectively. The cellular activation of AMPK reversed the changes in the levels of protein markers of autophagic flux. These results demonstrated that peiminine mediates cell cycle arrest by suppressing AktGSk3β signalling and blocks autophagic flux by depressing AMPK-ULK1 signalling in GBM cells. Finally, peiminine inhibited the growth of U251 gliomas in vivo.
Peiminine inhibits glioblastoma in vitro and in vivo via arresting the cell cycle and blocking autophagic flux, suggesting new avenues for GBM therapy.
背景/目的：多形性胶质母细胞瘤（GBM）是成人中最具破坏性且分布广泛的原发性中枢神经系统肿瘤，生存率低且死亡率高。现有治疗方法未给患者带来实质性益处，因此需要新的治疗策略。浙贝母碱是从传统中药浙贝母中提取的一种天然生物活性化合物，具有多种药理作用，尤其是抗癌活性。然而，其对GBM的抗癌作用及潜在机制尚未得到证实。本研究旨在探讨浙贝母碱在人GBM细胞中的潜在抗肿瘤作用，并在体外和体内探索相关分子信号机制。方法：采用MTT法和集落形成试验检测细胞活力和增殖。通过透射电子显微镜（TEM）评估与自噬相关的形态学变化。用流式细胞术测量细胞周期率。为检测体外和体内相关基因及信号通路的变化，采用了RNA测序、蛋白质免疫印迹和免疫组化分析。
浙贝母碱显著抑制GBM细胞的增殖和集落形成，并导致许多肿瘤相关基因和转录产物发生变化。浙贝母碱潜在的抗GBM作用可能涉及通过细胞周期蛋白D1/细胞周期蛋白依赖性激酶（CDK）网络、p62和微管相关蛋白1轻链3（LC3）表达的改变使细胞周期停滞和自噬流受阻。还观察到流式细胞术结果和TEM结果的变化。分子改变包括不仅磷酸化蛋白激酶B（Akt）和磷酸化糖原合成酶激酶3β（GSK3β）表达下调，而且磷酸化腺苷酸活化蛋白激酶（AMPK）和磷酸化unc-51样自噬激活激酶1（ULK1）表达下调。此外，Akt的过表达和抑制分别逆转和增强了浙贝母碱诱导的GBM细胞周期停滞。AMPK的细胞活化逆转了自噬流蛋白标志物水平的变化。这些结果表明，浙贝母碱通过抑制Akt-GSk3β信号介导细胞周期停滞，并通过抑制GBM细胞中的AMPK-ULK1信号阻断自噬流。最后，浙贝母碱在体内抑制了U251胶质瘤的生长。
浙贝母碱通过使细胞周期停滞和阻断自噬流在体外和体内抑制胶质母细胞瘤，为GBM治疗提供了新途径。
