胶质母细胞瘤患者常常对替莫唑胺化疗产生耐药性。低氧环境有助于化疗耐药性，使胶质母细胞瘤干细胞（GSC）扩张，从而促进肿瘤复发。胶质母细胞瘤组织由于鞘脂代谢异常，导致促生存的鞘脂酸-1-磷酸丰富，而促凋亡的神经酰胺含量降低。而鞘脂酰胺激酶（SK）1是将后者代谢生成鞘脂酸-1-磷酸的酶，其在胶质母细胞瘤中过度表达。小分子化合物SKI-II抑制SK和二氢鞘脂酰胺脱饱和酶1，后者将二氢鞘脂酰胺代谢生成鞘脂酸。我们先前的研究表明，在正氧气条件下，SKI-II与替莫唑胺联合使用能诱导卡他蛋白依赖性细胞死亡，该细胞死亡过程在二氢神经酰胺脂类积累和自噬之前发生。本研究探讨了正氧气条件和低氧气条件下替莫唑胺和SKI-II低剂量联合对胶质母细胞瘤细胞和患者源性GSCs的影响。我们使用Chou-Talalay方法分析药物协同作用。采用Sulforhodamine B比色法测定了每种药物的剂量-效应曲线。通过免疫荧光、流式细胞术和蛋白质免疫印迹对细胞死亡机制和自噬进行了分析；通过质谱和基因表达分析了鞘脂代谢变化。我们利用极限稀释法确定了GSCs的自我更新能力，利用三维球体模型检测了胶质母细胞瘤细胞的侵袭能力。在低氧环境下，胶质母细胞瘤细胞对替莫唑胺产生的耐药性增加。然而，在低氧环境下，替莫唑胺（48µM）与SKI-II（2.66µM）的联合使用对胶质母细胞瘤细胞的生长有协同抑制作用，并且相对于单药治疗，能够增强胶质母细胞瘤细胞的死亡。这种低剂量联合方案并未导致二氢神经酰胺脂类的积累，但却导致了鞘脂酸及其代谢产物的降低。该联合方案引起了氧化和内质网应激，并且触发了卡他蛋白非依赖性细胞死亡。该联合方案削弱了对替莫唑胺耐药的GSCs的自我更新能力，尤其在低氧环境下。此外，该联合方案降低了胶质母细胞瘤球体的侵袭能力。这项体外研究为鞘脂代谢与癌症侵袭及癌症干细胞的关系提供了新的见解。它展示了将调节鞘脂代谢与一线药物替莫唑胺联合使用以克服肿瘤生长和复发，通过减少低氧环境诱导的化疗耐药性并同时靶向分化和干性胶质母细胞瘤细胞的治疗潜力。© 2023. BioMed Central Ltd., part of Springer Nature.

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.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.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.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.© 2023. BioMed Central Ltd., part of Springer Nature.