Department of Neurosurgery, Hunterian Neurosurgical Research Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
Department of Neurosurgery, School of Medicine - Catholic University of the Sacred Heart, Rome, Italy.
PLoS One. 2021 Nov 3;16(11):e0251957. doi: 10.1371/journal.pone.0251957. eCollection 2021.
Medulloblastoma (MB) is the most common brain malignancy in children, and is still responsible for significant mortality and morbidity. The aim of this study was to assess the safety and efficacy of Disulfiram (DSF), an FDA-approved inhibitor of Aldehyde-Dehydrogenase (ALDH), and Copper (Cu++) in human SSH-driven and Group 3 MB. The molecular mechanisms, effect on cancer-stem-cells (CSC) and DNA damage were investigated in xenograft models.
The cytotoxic and anti-CSC effects of DSF/Cu++ were evaluated with clonogenic assays, flow-cytometry, immunofluorescence, western-blotting. ONS76, UW228 (SHH-driven with Tp53m), D425med, D283 and D341 (Group 3) cell-lines were used. In vivo survival and nuclear protein localization protein-4 (NPL4), Ki67, Cleaved-Caspase-3, GFAP and NeuN expression were assessed in two Group 3 MB xenografts with immunohistochemistry and western-blotting.
Significant in vitro cytotoxicity was demonstrated at nanomolar concentrations. DSF/Cu++ induced cell-death through NPL4 accumulation in cell-nucleus and buildup of poly-ubiquitylated proteins. Flow-cytometry demonstrated a significant decrease in ALDH+, Nestin+ and CD133+ following treatment, anti-CSC effect was confirmed in vitro and in vivo. DSF/Cu++ prolonged survival, and increased nuclear NPL4 expression in vivo.
Our data suggest that this combination may serve as a novel treatment, as monotherapy or in combination with existing therapies, for aggressive subtypes of pediatric MB.
髓母细胞瘤（MB）是儿童中最常见的脑恶性肿瘤，仍然是导致高死亡率和发病率的主要原因。本研究旨在评估已获美国食品药品监督管理局（FDA）批准的乙醛脱氢酶（ALDH）抑制剂和铜（Cu++）二硫化物（DSF）在人源性 SHH 驱动型和 3 组 MB 中的安全性和疗效。在异种移植模型中研究了其分子机制、对癌症干细胞（CSC）和 DNA 损伤的影响。
采用集落形成实验、流式细胞术、免疫荧光、western blot 检测 DSF/Cu++的细胞毒性和抗 CSC 作用。使用 ONS76、UW228（SHH 驱动型，携带 Tp53m）、D425med、D283 和 D341（3 组）细胞系。采用免疫组化和 western blot 检测两种 3 组 MB 异种移植模型中的体内生存和核蛋白 4（NPL4）、Ki67、Cleaved-Caspase-3、GFAP 和 NeuN 表达。
在纳摩尔浓度下表现出显著的体外细胞毒性。DSF/Cu++通过 NPL4 在细胞核内积累和多泛素化蛋白的积累诱导细胞死亡。流式细胞术显示，经处理后 ALDH+、Nestin+和 CD133+显著减少，体外和体内均证实了抗 CSC 作用。DSF/Cu++延长了生存时间，并增加了体内核 NPL4 的表达。
我们的数据表明，该联合疗法可能作为一种新的治疗方法，单独或联合现有疗法，用于治疗小儿 MB 的侵袭性亚型。
