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.本研究针对一类新型吡唑-吡啶铜配合物（pyrazole-pyridine copper complexes）开展了定量构效关系（quantitative structure−activity relationship）研究，明确了其促进铜蓄积的能力与细胞毒性之间的显著相关性。当配体亲脂性使得配合物能够快速穿透细胞膜时，细胞内金属蓄积可达到峰值。铜与配体的摄取动力学存在差异，且最终达到的细胞内平衡浓度亦不相同。上述结果支持如下模型：配体作为金属离子的离子载体（ionophore），以解离或结合状态的实体形式在细胞内外区室之间循环。当使用结构互不相关的双硫仑（disulfiram）、吡唑-吡啶铜配合物以及无机铜处理癌细胞时，均可重现相同的形态学与分子学变化，表明铜过载是引发细胞毒性效应的根源。铜基疗法可使敏感癌细胞走向副凋亡细胞死亡，该过程以内质网（endoplasmic reticulum, ER）应激与大量空泡化为典型特征，且无凋亡特征。经铜处理的濒死细胞未检测到半胱天冬酶（caspase）激活，这一现象是金属介导的半胱天冬酶-3（caspase-3）抑制的结果。因此，铜可同时作为内质网应激诱导剂与半胱天冬酶-3抑制剂，迫使细胞进入不依赖半胱天冬酶的副凋亡死亡途径。明确不同铜结合剂共有的作用机制，为将铜毒性开发为抗肿瘤工具提供了理论依据。