4-Nerolidylcatechol induces autophagy in human glioblastoma cells

ABSTRACT Gliomas account for the majority of primary malignant brain tumors and present invasive behavior into adjacent healthy tissue. While 4-NC had previously shown to induce apoptotic cell death in a melanoma model, for the glioma model described in this paper 4-NC is cytotoxic for the cells with the induction of the autophagic pathway. Trypan blue exclusion assay showed that 4-NC was cytotoxic in a dose-dependent manner for A172 and T98G cell lines. IC10 and IC50 values were at 32 µM and 41 µM for A172 and T98G respectively. Inhibition of cell proliferation was observed by total cell counts and by cell cycle analysis by flow cytometry, with cell cycle arrest of A172 and T98G cell lines respectively in the G1/G0 and S phases of the cell cycle. 4-NC induced up-regulation of autophagic pathways, as shown by immunoblotting for LC3-I/II, Real-Time PCR for ATG-7 and Beclin-1 genes, and by fluorescence microscopy observation of autophagic vacuoles in cells transfected with GFP-LC3 and electron microscopy. Glioma cells concomitantly treated with 4-NC and 3-MA, an inhibitor of the autophagic process, are more sensible to cell death, suggesting that autophagy protects the cells from the action of 4-NC.

摘要：胶质瘤（Gliomas）占原发性恶性脑肿瘤的绝大多数，且具有向邻近健康组织侵袭的特性。此前已有研究表明4-NC可在黑色素瘤模型中诱导凋亡性细胞死亡，但在本文所研究的胶质瘤模型中，4-NC对细胞的细胞毒性作用伴随自噬通路的激活。台盼蓝排斥试验（Trypan blue exclusion assay）结果显示，4-NC对A172与T98G细胞系呈现剂量依赖性的细胞毒性。A172细胞与T98G细胞的IC10及IC50值分别为32 μM与41 μM。通过总细胞计数法与流式细胞术（flow cytometry）开展的细胞周期分析显示，细胞增殖受到抑制：A172与T98G细胞系分别在细胞周期的G1/G0期与S期出现周期阻滞。4-NC可诱导自噬通路上调，具体证据包括：通过免疫印迹法检测LC3-I/II的蛋白表达、实时荧光定量PCR（Real-Time PCR）检测ATG-7与Beclin-1基因的转录水平，以及对转染了GFP-LC3的细胞进行荧光显微镜观察与电子显微镜观察以识别自噬空泡。当胶质瘤细胞同时接受4-NC与自噬过程抑制剂3-MA处理时，其对细胞死亡的敏感性更高，这提示自噬可保护细胞免受4-NC的杀伤作用。