Burgess Eleanor R, Praditi Citra, Phillips Elisabeth, Vissers Margreet C M, Robinson Bridget A, Dachs Gabi U, Wiggins George A R
Mackenzie Cancer Research Group, Department of Pathology and Biomedical Science, University of Otago Christchurch, Christchurch, New Zealand.
Department of Immunobiochemistry, Mannheim Institute for Innate Immunology (MI3), Heidelberg University, Medical Faculty Mannheim, Mannheim, Germany.
J Cell Biochem. 2025 Jan;126(1):e30658. doi: 10.1002/jcb.30658. Epub 2024 Oct 9.
The most common and aggressive brain cancer, glioblastoma, is characterized by hypoxia and poor survival. The pro-tumour transcription factor, hypoxia-inducible factor (HIF), is regulated via HIF-hydroxylases that require ascorbate as cofactor. Decreased HIF-hydroxylase activity triggers the hypoxic pathway driving cancer progression. Tissue ascorbate accumulates via the sodium-dependent vitamin C transporter-2 (SVCT2). We hypothesize that glioblastoma cells rely on SVCT2 for ascorbate accumulation, and that knockout of this transporter would disrupt the regulation of the hypoxic pathway by ascorbate. Ascorbate uptake was measured in glioblastoma cell lines (U87MG, U251MG, T98G) by high-performance liquid chromatography. CRISPR/Cas9 was used to knockout SVCT2. Cells were treated with cobalt chloride, desferrioxamine or 5% oxygen, with/without ascorbate, and key hypoxic pathway proteins were measured using Western blot analysis. Ascorbate uptake was cell line dependent, ranging from 1.7 to 11.0 nmol/10 cells. SVCT2-knockout cells accumulated 90%-95% less intracellular ascorbate than parental cells. The hypoxic pathway was induced by all three stimuli, and ascorbate reduced this induction. In the SVCT2-knockout cells, ascorbate had limited effect on the hypoxic pathway. This study verifies that intracellular ascorbate is required to suppress the hypoxic pathway. As patient survival is related to an activated hypoxic pathway, increasing intra-tumoral ascorbate may be of clinical interest.
最常见且侵袭性最强的脑癌——胶质母细胞瘤，其特征为缺氧和低生存率。促肿瘤转录因子缺氧诱导因子（HIF）通过需要抗坏血酸作为辅因子的HIF羟化酶进行调节。HIF羟化酶活性降低会触发驱动癌症进展的缺氧途径。组织中的抗坏血酸通过钠依赖性维生素C转运蛋白2（SVCT2）积累。我们假设胶质母细胞瘤细胞依赖SVCT2来积累抗坏血酸，并且敲除该转运蛋白会破坏抗坏血酸对缺氧途径的调节。通过高效液相色谱法测量胶质母细胞瘤细胞系（U87MG、U251MG、T98G）中的抗坏血酸摄取。使用CRISPR/Cas9敲除SVCT2。细胞用氯化钴、去铁胺或5%氧气处理，添加或不添加抗坏血酸，然后使用蛋白质印迹分析测量关键缺氧途径蛋白。抗坏血酸摄取具有细胞系依赖性，范围为1.7至11.0 nmol/10个细胞。SVCT2敲除细胞内的抗坏血酸积累比亲代细胞少90% - 95%。所有三种刺激均诱导了缺氧途径，而抗坏血酸减少了这种诱导。在SVCT2敲除细胞中，抗坏血酸对缺氧途径的影响有限。这项研究证实细胞内抗坏血酸是抑制缺氧途径所必需的。由于患者生存率与激活的缺氧途径相关，增加肿瘤内抗坏血酸可能具有临床意义。
