背景:胶质母细胞瘤患者通常对替莫唑胺化疗产生耐药性。缺氧,支持化疗抵抗,有利于胶质母细胞瘤干细胞(GSC)的扩增,导致肿瘤复发。因为鞘脂代谢失调,胶质母细胞瘤组织含有高水平的促存活鞘氨醇-1-磷酸和低水平的促凋亡神经酰胺。后者可以通过在胶质母细胞瘤中过表达的鞘氨醇激酶(SK)1代谢为鞘氨醇-1-磷酸。小分子SKI-II抑制SK和二氢神经酰胺去饱和酶1,其将二氢神经酰胺转化为神经酰胺。我们先前报道了SKI-II联合替莫唑胺诱导半胱天冬酶依赖性细胞死亡,在常氧中,二氢鞘脂积累和自噬。在本研究中,我们研究了替莫唑胺和SKI-II低剂量联合在常氧和缺氧条件下对胶质母细胞瘤细胞和患者来源的GCSs的影响.
方法:用Chou-Talalay组合指数法分析药物协同作用。用磺酰罗丹明B比色测定法测定每种药物的剂量-效应曲线。通过免疫荧光分析细胞死亡机制和自噬,流式细胞术和蛋白质印迹;鞘脂代谢改变通过质谱和基因表达分析。使用极端有限稀释测定和使用3D球体模型对胶质母细胞瘤细胞的侵袭来确定GSC自我更新能力。
结果:缺氧条件下胶质母细胞瘤细胞对替莫唑胺的耐药性增加。然而,替莫唑胺(48µM)与SKI-II(2.66µM)的组合协同抑制胶质母细胞瘤细胞生长,并相对于缺氧下的单一治疗增强胶质母细胞瘤细胞死亡。这种低剂量组合不会诱导双氢鞘脂的积累,而是神经酰胺及其代谢物的减少。它诱导氧化和内质网应激,并引发不依赖caspase的细胞死亡。它损害了替莫唑胺抗性GSC的自我更新能力,尤其是在缺氧的情况下.此外,它减少了胶质母细胞瘤细胞球体的侵袭。
结论:这项体外研究为鞘脂代谢和侵袭之间的联系提供了新的见解,癌症的标志,和癌症干细胞,癌症的关键驱动因素。它证明了将鞘脂代谢的调节与一线药物替莫唑胺结合在一起的方法的治疗潜力,可以通过减少缺氧诱导的对化疗的抗性以及靶向分化和干胶质母细胞瘤细胞来克服肿瘤生长和复发。
BACKGROUND: Glioblastoma patients commonly develop resistance to temozolomide chemotherapy. Hypoxia, which supports chemotherapy resistance, favors the expansion of glioblastoma stem cells (GSC), contributing to tumor relapse. Because of a deregulated sphingolipid metabolism, glioblastoma tissues contain high levels of the pro-survival sphingosine-1-phosphate and low levels of the pro-apoptotic ceramide. The latter can be metabolized to sphingosine-1-phosphate by sphingosine kinase (SK) 1 that is overexpressed in glioblastoma. The small molecule SKI-II inhibits SK and dihydroceramide desaturase 1, which converts dihydroceramide to ceramide. We previously reported that SKI-II combined with temozolomide induces caspase-dependent cell death, preceded by dihydrosphingolipids accumulation and autophagy in normoxia. In the present study, we investigated the effects of a low-dose combination of temozolomide and SKI-II under normoxia and hypoxia in glioblastoma cells and patient-derived GCSs.
METHODS: Drug synergism was analyzed with the Chou-Talalay Combination Index method. Dose-effect curves of each drug were determined with the Sulforhodamine B colorimetric assay. Cell death mechanisms and autophagy were analyzed by immunofluorescence, flow cytometry and western blot; sphingolipid metabolism alterations by mass spectrometry and gene expression analysis. GSCs self-renewal capacity was determined using extreme limiting dilution assays and invasion of glioblastoma cells using a 3D spheroid model.
RESULTS: Temozolomide resistance of glioblastoma cells was increased under hypoxia. However, combination of temozolomide (48 µM) with SKI-II (2.66 µM) synergistically inhibited glioblastoma cell growth and potentiated glioblastoma cell death relative to single treatments under hypoxia. This low-dose combination did not induce dihydrosphingolipids accumulation, but a decrease in ceramide and its metabolites. It induced oxidative and endoplasmic reticulum stress and triggered caspase-independent cell death. It impaired the self-renewal capacity of temozolomide-resistant GSCs, especially under hypoxia. Furthermore, it decreased invasion of glioblastoma cell spheroids.
CONCLUSIONS: This in vitro study provides novel insights on the links between sphingolipid metabolism and invasion, a hallmark of cancer, and cancer stem cells, key drivers of cancer. It demonstrates the therapeutic potential of approaches that combine modulation of sphingolipid metabolism with first-line agent temozolomide in overcoming tumor growth and relapse by reducing hypoxia-induced resistance to chemotherapy and by targeting both differentiated and stem glioblastoma cells.