N-acetylcysteine decreases malignant characteristics of glioblastoma cells by inhibiting Notch2 signaling
N-acetylcysteine decreases malignant characteristics of glioblastoma cells by inhibiting Notch2 signaling
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N-乙酰半胱氨酸通过抑制 Notch2 信号传导降低胶质母细胞瘤细胞的恶性特征
DOI：
10.1186/s13046-018-1016-8
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发表时间：
2019-01-03
影响因子：
11.3
通讯作者：
Cao, Xuan
中科院分区：
文献类型：
--
作者：
Deng, Jie;Liu, An-Dong;Cao, Xuan
研究背景多形性胶质母细胞瘤（GBM）是颅内最严重的原发性恶性肿瘤，目前临床上缺乏有效的治疗方法。Notch 2已被确定为预后标志物，并可能参与GBM恶性进展。N-乙酰半胱氨酸（NAC）是细胞内谷胱甘肽（GSH）的前体，广泛参与多种癌症的预防和治疗。然而，NAC在GBM中的作用尚不清楚，NAC是否具有抗氧化功能尚不清楚。方法采用RT-PCR和Western blot方法分别检测Notch家族及其他相关因子的mRNA和蛋白水平。流式细胞术检测细胞内活性氧（ROS）水平，CCK 8法检测细胞活力，PI染色法检测细胞周期。流式细胞仪Annexin V/PI检测细胞凋亡水平。通过伤口愈合和transwell侵袭实验评估细胞迁移和侵袭。最后，建立U87异种移植瘤模型，验证NAC是否能抑制肿瘤生长。同时，NAC对其下游靶基因Hes 1和Hey 1的mRNA和蛋白水平具有降低作用。NAC引起的这些效应与细胞内GSH和ROS水平无关。NAC介导的Notch 2减少的机制通过促进Notch 2通过痒依赖性溶酶体途径降解来阐明。此外，NAC可以阻止GBM细胞的增殖、迁移和侵袭，并可能通过靶向Notch 2诱导GBM细胞凋亡。结论NAC可通过非抗氧化剂依赖性的溶酶体途径促进Notch 2降解，从而减弱Notch 2在GBM细胞中的恶性信号传导。NAC抑制癌细胞增殖和肿瘤生长的显著能力可能暗示NAC在GBM治疗中的新应用。
BackgroundGlioblastomas multiforme (GBM) is the most devastating primary intracranial malignancy lacking effective clinical treatments. Notch2 has been established to be a prognostic marker and probably involved in GBM malignant progression. N-acetylcysteine (NAC), a precursor of intracellular glutathione (GSH), has been widely implicated in prevention and therapy of several cancers. However, the role of NAC in GBM remains unclear and the property of NAC independent of its antioxidation is largely unknown.MethodsThe mRNA and protein levels of Notch family and other related factors were detected by RT-PCR and western blot, respectively. In addition, intracellular reactive oxygen species (ROS) was measured by flow cytometry-based DCFH-DA. Moreover, cell viability was assessed by CCK8 and cell cycle was analyzed by flow cytometry-based PI staining. The level of apoptosis was checked by flow cytometry-based Annexin V/PI. Cell migration and invasion were evaluated by wound healing and transwell invasion assays. At last, U87 Xenograft model was established to confirm whether NAC could restrain the growth of tumor.ResultsOur data showed that NAC could decrease the protein level of Notch2. Meanwhile, NAC had a decreasing effect on the mRNA and protein levels of its downstream targets Hes1 and Hey1. These effects caused by NAC were independent of cellular GSH and ROS levels. The mechanism of NAC-mediated Notch2 reduction was elucidated by promoting Notch2 degradation through Itch-dependent lysosome pathway. Furthermore, NAC could prevent proliferation, migration, and invasion and might induce apoptosis in GBM cells via targeting Notch2. Significantly, NAC could suppress the growth of tumor in vivo.ConclusionsNAC could facilitate Notch2 degradation through lysosomal pathway in an antioxidant-independent manner, thus attenuating Notch2 malignant signaling in GBM cells. The remarkable ability of NAC to inhibit cancer cell proliferation and tumor growth may implicate a novel application of NAC on GBM therapy.