Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, One John Marshall Drive, Huntington, WV 25755, United States.
Biomed Pharmacother. 2017 Feb;86:502-513. doi: 10.1016/j.biopha.2016.12.056. Epub 2016 Dec 23.
INTRODUCTION: Accumulation of hypoxia inducible factor-1 alpha (HIF-1α) in malignant tissue is known to contribute to oncogenic progression and is inversely associated with patient survival. Ascorbic acid (AA) depletion in malignant tissue may contribute to aberrant normoxic activity of HIF-1α. While AA supplementation has been shown to attenuate HIF-1α function in malignant melanoma, the use of dehydroascorbic acid (DHA) as a therapeutic means to increase intracellular AA and modulate HIF-1α function is yet to be evaluated. Here we compared the ability of AA and DHA to increase intracellular vitamin C content and decrease the malignant potential of human melanoma by reducing the activity of HIF-1α. METHODS: HIF-1α protein accumulation was evaluated by western blot and transcriptional activity was evaluated by reporter gene assay using a HIF-1 HRE-luciferase plasmid. Protein expressions and subcellular localizations of vitamin C transporters were evaluated by western blot and confocal imaging. Intracellular vitamin C content following AA, ascorbate 2-phosphate (A2P), or DHA supplementation was determined using a vitamin C assay. Malignant potential was accessed using a 3D spheroid Matrigel invasion assay. Data was analyzed by One or Two-way ANOVA with Tukey's multiple comparisons test as appropriate with p<0.05 considered significant. RESULTS: Melanoma cells expressed both sodium dependent vitamin C (SVCT) and glucose (GLUT) transporters for AA and DHA transport respectively, however advanced melanomas responded favorably to AA, but not DHA. Physiological glucose conditions significantly impaired intracellular vitamin C accumulation following DHA treatment. Consequently, A2P and AA, but not DHA treated cells demonstrated lower HIF-1α protein expression and activity, and reduced malignant potential. The ability of AA to regulate HIF-1α was dependent on SVCT2 function and SVCT2 was not significantly inhibited at pH representative of the tumor microenvironment. CONCLUSIONS: The use of ascorbic acid as an adjuvant cancer therapy remains under investigated. While AA and A2P were capable of modulating HIF-1α protein accumulation/activity, DHA supplementation resulted in minimal intracellular vitamin C activity with decreased ability to inhibit HIF-1α activity and malignant potential in advanced melanoma. Restoring AA dependent regulation of HIF-1α in malignant cells may prove beneficial in reducing chemotherapy resistance and improving treatment outcomes.
简介：已知缺氧诱导因子-1 ɑ（HIF-1ɑ）在恶性组织中的积累有助于致癌进展，并与患者的生存呈负相关。恶性组织中抗坏血酸（AA）的耗竭可能导致 HIF-1ɑ 的异常正常氧活性。虽然已经证明 AA 补充可以减弱恶性黑色素瘤中 HIF-1ɑ 的功能，但尚未评估使用脱氢抗坏血酸（DHA）作为增加细胞内 AA 并调节 HIF-1ɑ 功能的治疗手段。在这里，我们比较了 AA 和 DHA 通过降低 HIF-1ɑ 的活性来增加细胞内维生素 C 含量和降低人黑色素瘤恶性潜能的能力。 方法：通过 Western blot 评估 HIF-1ɑ 蛋白积累，通过使用 HIF-1 HRE-荧光素酶质粒的报告基因测定评估转录活性。通过 Western blot 和共聚焦成像评估维生素 C 转运蛋白的蛋白表达和亚细胞定位。通过维生素 C 测定法确定 AA、抗坏血酸 2-磷酸（A2P）或 DHA 补充后的细胞内维生素 C 含量。使用 3D 球体 Matrigel 侵袭测定法评估恶性潜能。通过 One 或 Two-way ANOVA 分析数据，并根据需要使用 Tukey 的多重比较检验，p<0.05 被认为具有统计学意义。 结果：黑色素瘤细胞分别表达用于 AA 和 DHA 转运的钠依赖性维生素 C（SVCT）和葡萄糖（GLUT）转运体，然而晚期黑色素瘤对 AA 反应良好，但对 DHA 无反应。生理葡萄糖条件显著削弱了 DHA 处理后的细胞内维生素 C 积累。因此，与 DHA 处理的细胞相比，A2P 和 AA 处理的细胞表现出更低的 HIF-1ɑ 蛋白表达和活性，并降低了恶性潜能。AA 调节 HIF-1ɑ 的能力取决于 SVCT2 功能，并且在代表肿瘤微环境的 pH 值下，SVCT2 没有被显著抑制。 结论：作为辅助癌症治疗的抗坏血酸的使用仍在研究中。虽然 AA 和 A2P 能够调节 HIF-1ɑ 蛋白积累/活性，但 DHA 补充导致细胞内维生素 C 活性降低，抑制 HIF-1ɑ 活性和晚期黑色素瘤恶性潜能的能力降低。在恶性细胞中恢复 AA 依赖性的 HIF-1ɑ 调节可能有助于降低化疗耐药性并改善治疗结果。
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