Ultrastructural Pathology, Beijing Neurosurgical Institute, Beijing, China.
Neuropathology, Beijing Neurosurgical Institute, Beijing, China.
Ultrastruct Pathol. 2023 Nov 2;47(6):529-539. doi: 10.1080/01913123.2023.2270676. Epub 2023 Nov 30.
Medulloblastoma (MB) is a frequently occurring malignant brain tumor in children, and many of these tumors are identified by the abnormal activation of the Sonic Hedgehog (SHH) pathway. Although the Shh inhibitor GDC0449 initially shows some effectiveness in certain tumors, they eventually recur due to drug resistance mechanisms, highlighting the need for new treatment options. In this study, we explore whether GDC0449 induces autophagy in the human MB cell lines. To investigate the ultrastructural pathology changes of GDC0449-treated Daoy and D283 cells, we employed Transmission Electron Microscopy (TEM) technology to identify the expression of autophagic vacuoles. Our results indicate that GDC0449 only increases autophagy in Daoy cells by increasing the LC3-II/LC3-I ratio and autophagosome formation.We also analyzed Beclin1, LC3, Bax, and Cleaved-caspase3 protein and mRNA expression levels of autophagic and apoptotic markers using fluorescence confocal microscopy, RT-PCR, and Western blot. We found that cell autophagy and apoptosis increased in a dose-dependent manner with GDC0449 treatment. Additionally, we observed increased mammalian target of rapamycin (mTOR) phosphorylation and decreased protein kinase B (AKT/PKB), Ribosomal Protein S6, eIF4E-binding protein (4EBP1) phosphorylation in GDC0449-treated Daoy cells. It was observed that inhibiting autophagy using Beclin1 siRNA significantly blocked the apoptosis-inducing effects of GDC0449, suggesting that GDC0449 mediates its apoptotic effects by inducing autophagy.Our data suggests that GDC0449 inhibits the growth of human MB Daoy cells by autophagy-mediated apoptosis. The mechanism of GDC0449-induced autophagy in Daoy cells may be related to the inhibition of the PI3K/AKT/mTOR signaling pathway.
髓母细胞瘤（MB）是儿童中常见的恶性脑肿瘤，其中许多肿瘤是由于 Sonic Hedgehog（SHH）途径的异常激活而识别的。尽管 Shh 抑制剂 GDC0449 最初在某些肿瘤中显示出一定的疗效，但由于耐药机制，它们最终会复发，这凸显了需要新的治疗选择。在这项研究中，我们探讨了 GDC0449 是否会诱导人 MB 细胞系发生自噬。为了研究 GDC0449 处理的 Daoy 和 D283 细胞的超微结构病理学变化，我们采用透射电子显微镜（TEM）技术来鉴定自噬空泡的表达。我们的结果表明，GDC0449 仅通过增加 LC3-II/LC3-I 比值和自噬体形成来增加 Daoy 细胞中的自噬。我们还使用荧光共聚焦显微镜、RT-PCR 和 Western blot 分析了自噬和凋亡标志物 Beclin1、LC3、Bax 和 Cleaved-caspase3 的蛋白和 mRNA 表达水平。我们发现，随着 GDC0449 处理，细胞自噬和凋亡呈剂量依赖性增加。此外，我们观察到 GDC0449 处理的 Daoy 细胞中哺乳动物靶标雷帕霉素（mTOR）磷酸化增加和蛋白激酶 B（AKT/PKB）、核糖体蛋白 S6、eIF4E 结合蛋白（4EBP1）磷酸化减少。用 Beclin1 siRNA 抑制自噬观察到显著阻断了 GDC0449 诱导的凋亡作用，表明 GDC0449 通过诱导自噬介导其凋亡作用。我们的数据表明，GDC0449 通过自噬介导的凋亡抑制人 MB Daoy 细胞的生长。GDC0449 诱导 Daoy 细胞自噬的机制可能与抑制 PI3K/AKT/mTOR 信号通路有关。
