Metabolic Profiling Reveals Sphingosine-1-Phosphate Kinase 2 and Lyase as Key Targets of (Phyto-) Estrogen Action in the Breast Cancer Cell Line MCF-7 and Not in MCF-12A

To search for new targets of anticancer therapies using phytoestrogens we performed comparative metabolic profiling of the breast cancer cell line MCF-7 and the non-tumorigenic breast cell line MCF-12A. Application of gas chromatography-mass spectrometry (GC-MS) revealed significant differences in the metabolic levels after exposure with 17ß-estradiol, genistein or a composition of phytoestrogens within a native root flax extract. We observed the metabolites 3-(4-hydroxyphenyl)-lactic acid, cis-aconitic acid, 11-beta-hydroxy-progesterone, chenodeoxycholic acid and triacontanoic acid with elevated levels due to estrogen action. Particularly highlighted were metabolites of the sphingolipid metabolism. Sphingosine and its dihydro derivate as well as ethanolaminephosphate were significantly altered after exposure with 1 nM 17ß-estradiol in the cell line MCF-7, while MCF-12A was not affected. Treatment with genistein and the flax extract normalized the sphingosine concentrations to the basic levels found in MCF-12A cells. We could further demonstrate that the expression levels of the sphingosine metabolizing enzymes: sphingosine-1-phosphate kinase (Sphk) and lyase (S1P lyase) were significantly influenced by estrogens as well as phytoestrogens. The isoform Sphk2 was overexpressed in the tumorigenic cell line MCF-7, while S1P lyase was predominantly expressed in the non-tumorigenic cell line MCF-12A. Importantly, in MCF-7 the weak S1P lyase expression could be significantly increased after exposure with 10 µM genistein and 1 µg/ml root flax extract. Here, we present, for the first time, an analysis of metabolic response of phytoestrogens to breast cancer cell lines. The contrasting regulation of sphingolipid enzymes in MCF-7 and MCF-12A render them as preferred targets for future anticancer strategies.

本研究为探寻植物雌激素(phytoestrogens)的抗癌治疗新靶点，对乳腺癌细胞系MCF-7与非致瘤性乳腺细胞系MCF-12A开展了比较代谢谱分析。采用气相色谱-质谱联用法(GC-MS)检测发现，经17β-雌二醇、染料木黄酮或天然亚麻根提取物中的植物雌激素复合成分处理后，两组细胞的代谢物水平存在显著差异。我们观察到，3-(4-羟苯基)-乳酸、顺式乌头酸、11-β-羟孕酮、鹅脱氧胆酸与三十烷酸因雌激素作用而水平升高。其中尤为值得关注的是鞘脂代谢相关代谢物。在MCF-7细胞系中，经1 nM 17β-雌二醇处理后，鞘氨醇及其二氢衍生物与乙醇胺磷酸的水平发生显著改变，而MCF-12A细胞未受影响。染料木黄酮与亚麻根提取物处理可将鞘氨醇浓度恢复至MCF-12A细胞中的基础水平。我们进一步证实，鞘氨醇代谢相关酶——鞘氨醇-1-磷酸激酶(Sphk)与S1P裂解酶(S1P lyase)的表达水平，会显著受雌激素与植物雌激素的调控。鞘氨醇激酶2(Sphk2)同工型在致瘤性细胞系MCF-7中过表达，而S1P裂解酶则主要在非致瘤性细胞系MCF-12A中表达。值得注意的是，在MCF-7细胞中，经10 μM染料木黄酮与1 μg/ml天然亚麻根提取物处理后，原本较弱的S1P裂解酶表达水平可显著升高。本研究首次对植物雌激素作用于乳腺癌细胞系的代谢应答进行了分析。鞘脂代谢酶在MCF-7与MCF-12A细胞中的差异化调控，使其成为未来抗癌治疗策略的优选靶点。