Department of Neurosurgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA.
Department of Neurosurgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA.
Cancer Lett. 2017 Aug 1;400:161-174. doi: 10.1016/j.canlet.2017.04.015. Epub 2017 Apr 24.
The anticancer agent 3-bromopyruvate (3-BP) is viewed as a glycolytic inhibitor that preferentially kills glycolytic cancer cells through energy depletion. However, its cytotoxic activity is dependent on cellular drug import through transmembrane monocarboxylate transporter 1 (MCT-1), which restricts its anticancer potential to MCT-1-positive tumor cells. We created and characterized an MCT-1-independent analog of 3-BP, called NEO218. NEO218 was synthesized by covalently conjugating 3-BP to perillyl alcohol (POH), a natural monoterpene. The responses of various tumor cell lines to treatment with either compound were characterized in the presence or absence of supplemental pyruvate or antioxidants N-acetyl-cysteine (NAC) and glutathione (GSH). Drug effects on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzyme activity were investigated by mass spectrometric analysis. The development of 3-BP resistance was investigated in MCT-1-positive HCT116 colon carcinoma cells in vitro. Our results show that NEO218: (i) pyruvylated GAPDH on all 4 of its cysteine residues and shut down enzymatic activity; (ii) severely lowered cellular ATP content below life-sustaining levels, and (iii) triggered rapid necrosis. Intriguingly, supplemental antioxidants effectively prevented cytotoxic activity of NEO218 as well as 3-BP, but supplemental pyruvate powerfully protected cells only from 3-BP, not from NEO218. Unlike 3-BP, NEO218 exerted its potent cytotoxic activity irrespective of cellular MCT-1 status. Treatment of HCT116 cells with 3-BP resulted in prompt development of resistance, based on the emergence of MCT-1-negative cells. This was not the case with NEO218, and highly 3-BP-resistant cells remained exquisitely sensitive to NEO218. Thus, our study identifies a mechanism by which tumor cells develop rapid resistance to 3-BP, and presents NEO218 as a superior agent not subject to this cellular defense. Furthermore, our results offer alternative interpretations of previously published models on the role of supplemental antioxidants: Rather than quenching reactive oxygen species (ROS), supplemental NAC or GSH directly interact with 3-BP, thereby neutralizing the drug's cytotoxic potential before it can trigger ROS production. Altogether, our study introduces new aspects of the cytotoxic mechanism of 3-BP, and characterizes NEO218 as an analog able to overcome a key cellular defense mechanism towards this drug.
抗癌剂 3-溴丙酮酸（3-BP）被视为一种糖酵解抑制剂，通过能量耗竭优先杀死糖酵解癌细胞。然而，其细胞毒性活性取决于通过跨膜单羧酸转运蛋白 1（MCT-1）的细胞内药物导入，这将其抗癌潜力限制在 MCT-1 阳性肿瘤细胞中。我们创建并表征了 3-BP 的一种不依赖 MCT-1 的类似物，称为 NEO218。NEO218 通过将 3-BP 与薄荷醇（POH）共价连接合成，POH 是一种天然的单萜。在存在或不存在补充丙酮酸或抗氧化剂 N-乙酰半胱氨酸（NAC）和谷胱甘肽（GSH）的情况下，表征了各种肿瘤细胞系对任一化合物处理的反应。通过质谱分析研究了药物对甘油醛 3-磷酸脱氢酶（GAPDH）酶活性的影响。在体外研究了 MCT-1 阳性 HCT116 结肠癌细胞中 3-BP 耐药性的发展。我们的结果表明，NEO218：（i）在其 4 个半胱氨酸残基上丙酮酸化 GAPDH 并关闭酶活性；（ii）严重降低细胞内 ATP 含量低于维持生命的水平，以及（iii）引发快速坏死。有趣的是，补充抗氧化剂可有效阻止 NEO218 和 3-BP 的细胞毒性活性，但补充丙酮酸仅能有力地保护细胞免受 3-BP 的侵害，而不受 NEO218 的侵害。与 3-BP 不同，NEO218 发挥其强大的细胞毒性活性而与细胞 MCT-1 状态无关。用 3-BP 处理 HCT116 细胞会导致 MCT-1 阴性细胞的出现，从而迅速产生耐药性。NEO218 则不是这种情况，高度耐 3-BP 的细胞仍然对 NEO218 高度敏感。因此，我们的研究确定了肿瘤细胞对 3-BP 快速产生耐药性的机制，并将 NEO218 作为一种不受此细胞防御影响的优越药物。此外，我们的结果提供了对先前发表的关于补充抗氧化剂作用模型的替代解释：补充 NAC 或 GSH 不是通过清除活性氧（ROS），而是直接与 3-BP 相互作用，从而在 3-BP 引发 ROS 产生之前中和其细胞毒性潜力。总之，我们的研究介绍了 3-BP 细胞毒性机制的新方面，并将 NEO218 作为一种能够克服该药物关键细胞防御机制的类似物进行了表征。
