Integrated proteomics and metabolomics reveal phytosesquiterpene lactones DET and DETD-35 inhibit triple-negative breast cancer cell activity by depleting ATP synthesis and reprogramming primary metabolism

Phytosesquiterpene lactones DET and DETD-35 are reported to induce oxidative stress towards inhibiting the activities of triple-negative breast cancer (TNBC) cells. However, how DET and DETD-35 affect mitochondrial proteome and associated oxidative phosphorylation in TNBC cells is unclear. In this study, the mechanisms through which DET and DETD-35 inhibit MDA-MB-231 TNBC cells were dissected. DET and DETD-35 promoted mitochondrial superoxide production by upregulating expression of SOD1 and SOD2, and induced permeability transition pore opening, and attenuated intracellular ATP levels. Neither compound interfered with mitochondrial respiration/bioenergetics in normal mammary MCF-10A cells. Comparative mitochondria proteome and bioinformatic analyses showed significant deregulation of proteins related to the oxidative phosphorylation, depolarization of mitochondria, and apoptosis signaling in DET- or DETD-35-treated TNBC cells, and primary metabolomics revealed that both compounds deregulated metabolites dynamics and the corresponding metabolic pathways in TNBC cells. Knockdown of the PRKCA gene/protein involved in inducing mitochondrial toxicity in TNBC cells reversed cytotoxicity and apoptosis and decreased the levels of several metabolites induced by DET or DETD-35 in the cancer cells. Integrated Pearson’s correlation and IPA network analyses of differentially expressed proteins and metabolites revealed the networks of ATP synthesis, energy homeostasis, and mitochondrial activities (respiration, depolarization, and transmembrane potential) highly correlated to the compound effects. Notable, DET/DETD-35 inhibited mitochondrial ATPase activity, and molecular modeling further predicted the binding sites of either compound with ATP synthase at the subunits α/βand c/a interfaces. In summary, the mechanisms through which these phytosesquiterpene lactones disrupt mitochondrial biogenetics to inhibit TNBC cell activities were delineated.

植物倍半萜内酯（Phytosesquiterpene lactones）DET与DETD-35已被报道可通过诱导氧化应激，抑制三阴性乳腺癌（triple-negative breast cancer, TNBC）细胞的增殖活性。然而，DET与DETD-35如何调控TNBC细胞的线粒体蛋白质组及相关氧化磷酸化过程，目前仍不明晰。
本研究解析了DET与DETD-35抑制MDA-MB-231型TNBC细胞的具体作用机制。实验结果表明，DET与DETD-35可通过上调超氧化物歧化酶1（SOD1）与超氧化物歧化酶2（SOD2）的转录与表达，促进线粒体超氧化物的生成，诱导线粒体通透性转换孔开放，并显著降低细胞内ATP水平。两种化合物均不会对正常乳腺MCF-10A细胞的线粒体呼吸/生物能学过程产生任何干扰。
对比线粒体蛋白质组学与生物信息学分析结果显示，经DET或DETD-35处理的TNBC细胞中，与氧化磷酸化、线粒体去极化及凋亡信号通路相关的蛋白质均出现显著表达失调；代谢组学分析进一步表明，两种化合物均可扰乱TNBC细胞内的代谢物动态平衡及对应代谢通路。
敲低TNBC细胞中参与诱导线粒体毒性的PRKCA基因与蛋白，可有效逆转DET或DETD-35诱导的细胞毒性与凋亡反应，并降低癌细胞中该两种化合物诱导产生的多种代谢物水平。
对差异表达蛋白质与代谢物开展Pearson相关性分析及Ingenuity通路分析（IPA）的整合网络研究，揭示了与化合物作用高度相关的调控网络，涵盖ATP合成、能量稳态及线粒体活动（呼吸、去极化及跨膜电位）相关的通路。值得注意的是，DET与DETD-35均可抑制线粒体ATP合酶的活性，分子建模实验进一步预测了两种化合物分别与ATP合酶α/β亚基及c/a亚基界面的结合位点。
综上，本研究阐明了这类植物倍半萜内酯通过扰乱线粒体生物发生，进而抑制TNBC细胞活性的完整作用机制。