A HIF-independent, CD133-mediated mechanism of cisplatin resistance in glioblastoma cells.
A HIF-independent, CD133-mediated mechanism of cisplatin resistance in glioblastoma cells.
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DOI：
10.1007/s13402-018-0374-8
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发表时间：
2018-06
期刊：
影响因子：
--
通讯作者：
Grabowska A
中科院分区：
文献类型：
--
作者：
Ahmed EM;Bandopadhyay G;Coyle B;Grabowska A
胶质母细胞瘤（GBM）是成人中最常见的脑肿瘤。这些肿瘤中的细胞亚群，称为癌症干细胞（CSC），被认为介导了它们的化疗/放疗抗性。CD 133是用于鉴定和分离GBM CSC的细胞表面标志物。然而，其功能意义以及研究CD 133的相关微环境迄今仍不清楚。本研究探讨了低氧对CD 133表达及低氧相关因子HIF-1α和HIF-2α表达的影响，以及CD 133表达对GBM细胞获得化疗耐药性的潜在功能意义。在常氧和缺氧条件下，使用qRT-PCR对GBM细胞（U251、U87和SNB 19; 2D或3D培养物）进行CD 133、HIF-1α、HIF-2α、VEFG和（对照）HPRT mRNA表达分析。流式细胞术和qRT-PCR分别证实siRNA可下调GBM细胞中CD 133、HIF-1α和HIF-2α的表达。使用Alcohol Blue细胞活力测定结合Graphpad prism软件工具确定药物敏感性相关的IC 50值。我们发现在2D和3D GBM细胞培养模型中，在缺氧条件下CD 133的表达上调。此外，与常氧条件相比，在低氧条件下培养的GBM细胞中观察到对顺铂、替莫唑胺和依托泊苷的耐药性增加。siRNA介导的HIF-1α或HIF-2α的敲低导致CD 133表达减少，HIF-2α具有更长期的作用。我们还发现，HIF-2α下调使GBM细胞对顺铂的敏感程度大于HIF-1α，而CD 133敲低对顺铂敏感性的影响比任何一种HIF的敲低更显著，这表明存在由CD 133介导的HIF非依赖性顺铂耐药机制。同样的机制似乎不参与替莫唑胺耐药，因为我们发现HIF-1α下调，而不是HIF-2α或CD 133下调，使GBM细胞对替莫唑胺敏感。从我们的数据中，我们得出结论，缺氧诱导的CD 133介导的顺铂耐药的机制可能有助于设计新的GBM治疗策略。本文的在线版本（10.1007/s13402-018-0374-8）包含补充材料，可供授权用户使用。
Glioblastoma (GBM) is the commonest brain tumour in adults. A sub-population of cells within these tumours, known as cancer stem cells (CSCs), is thought to mediate their chemo-/radiotherapy resistance. CD133 is a cell surface marker that is used to identify and isolate GBM CSCs. However, its functional significance, as well as the relevant microenvironment in which to study CD133, have so far remained unknown. Here, we examined the effect of hypoxia on the expression of CD133 and on that of the hypoxia-related factors HIF-1α and HIF-2α, and the potential functional significance of CD133 expression on the acquisition of chemo-resistance by GBM cells. CD133, HIF-1α, HIF-2α, VEFG and (control) HPRT mRNA expression analyses were carried out on GBM cells (U251, U87 and SNB19; 2D or 3D cultures) under both normoxic and hypoxic conditions, using qRT-PCR. siRNA was used to downregulate CD133, HIF-1α and HIF-2α expression in the GBM cells, which was confirmed by flow cytometry and qRT-PCR, respectively. Drug sensitivity-related IC50 values were established using an Alamar Blue cell viability assay in conjunction with the Graphpad prism software tool. We found that the expression of CD133 was upregulated under hypoxic conditions in both the 2D and 3D GBM cell culture models. In addition, an increased resistance to cisplatin, temozolomide and etoposide was observed in the GBM cells cultured under hypoxic conditions compared to normoxic conditions. siRNA-mediated knockdown of either HIF-1α or HIF-2α resulted in a reduced CD133 expression, with HIF-2α having a more long-term effect. We also found that HIF-2α downregulation sensitized the GBM cells to cisplatin to a greater extent than HIF-1α, whereas CD133 knockdown had a more marked effect on cisplatin sensitisation than knockdown of either one of the HIFs, suggesting the existence of a HIF-independent cisplatin resistance mechanism mediated by CD133. This same mechanism does not seem to be involved in temozolomide resistance, since we found that HIF-1α downregulation, but not HIF-2α or CD133 downregulation, sensitized GBM cells to temozolomide. From our data we conclude that the mechanisms underlying hypoxia-induced CD133-mediated cisplatin resistance may be instrumental for the design of new GBM treatment strategies. The online version of this article (10.1007/s13402-018-0374-8) contains supplementary material, which is available to authorized users.
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