Copper Binding Agents Acting as Copper Ionophores Lead to Caspase Inhibition and Paraptotic Cell Death in Human Cancer Cells

We report a quantitative structure−activity relationship study of a new class of pyrazole-pyridine copper complexes that establishes a clear correlation between the ability to promote copper accumulation and cytotoxicity. Intracellular metal accumulation is maximized when ligand lipophilicity allows the complex to rapidly cross the membrane. Copper and ligand follow different uptake kinetics and reach different intracellular equilibrium concentrations. These results support a model in which the ligand acts as an ionophore for the metal ion, cycling between intra- and extracellular compartments as dissociated or complexed entities. When treating cancer cells with structurally unrelated disulfiram and pyrazole-pyridine copper complexes, as well as with inorganic copper, the same morphological and molecular changes were reproduced, indicating that copper overload is responsible for the cytotoxic effects. Copper-based treatments drive sensitive cancer cells toward paraptotic cell death, a process hallmarked by endoplasmic reticulum stress and massive vacuolization in the absence of apoptotic features. A lack of caspase activation, as observed in copper-treated dying cells, is a consequence of metal-mediated inhibition of caspase-3. Thus, copper acts simultaneously as an endoplasmic reticulum (ER) stress inducer and a caspase-3 inhibitor, forcing the cell into caspase-independent paraptotic death. The establishment of a mechanism of action common to different copper binding agents provides a rationale for the exploitation of copper toxicity as an anticancer tool.

本报告阐述了一种新型吡唑-吡啶铜配合物定量结构-活性关系研究，该研究确立了促进铜积累能力与细胞毒性之间的明确关联。当配体亲脂性允许配合物快速穿越细胞膜时，细胞内金属积累达到最大值。铜和配体遵循不同的摄取动力学，达到不同的细胞内平衡浓度。这些结果支持了一种模型，其中配体作为金属离子的离子载体，在细胞内和细胞外空间以解离或复合状态循环。在用结构上不相关的双硫仑和吡唑-吡啶铜配合物，以及无机铜治疗癌细胞时，产生了相同的形态和分子变化，表明铜过量是细胞毒效应的原因。基于铜的治疗促使敏感癌细胞向凋亡性细胞死亡转变，这一过程以内质网应激和大量空泡化为特征，而不具备凋亡特征。在铜处理死亡的细胞中观察到caspase激活缺失，这是金属介导的caspase-3抑制的结果。因此，铜同时作为内质网（ER）应激诱导剂和caspase-3抑制剂，迫使细胞进入caspase非依赖性凋亡性死亡。建立不同铜结合剂共有的作用机制，为利用铜毒性作为抗癌工具提供了理论依据。