1 INTRODUCTION
Cancer is one of the leading causes of human death worldwide, characterized by uncontrolled proliferation and locally invasive infiltration.1-3 Because of the evading immune recognition through genetic mutations, the balance between cell death and proliferation is broken, leading to the unrestricted cell proliferation and ultimately cancer.4,5 ​While advanced techniques have been applied to basic and clinical cancer research, most of them are arduous and unsatisfactory in performance, as existing approaches can barely kill cancer cells while sparing surrounding normal cells.6,7 Selectively inducing programmed cell death (PCD) in cancer cells is a promising option for cancer therapy, including apoptosis, autophagy, necrosis. Among them, apoptosis is considered the preferred alternative, but the therapeutic effects are still far from satisfactory due to the intrinsic resistance induced by tumor heterogeneity.8,9 Furthermore, acquired resistance results in high doses of medication, which brings severe side effects.10,11 Thus, development of novel cell death mode with more efficiency and less side effect is an urgent concern in the field of cancer therapy. Fortunately, recent studies have proposed several unidentified cell death forms with unique regulatory pathways, including ferroptosis, pyroptosis and cuproptosis, which can circumvent the limitations of classical cell death methods and open up new opportunities for the cancer treatment.12-15
Among these non-apoptotic forms of PCDs, cuproptosis has received much attention as the most emerging regulatory pathways of cell death. ​Interestingly, the connection between copper homeostasis and physical health was explored long before the term cuproptosis was established. The disequilibrium of copper homeostasis was repeatedly found to be associated with development of various diseases, such as Menkes disease, Wilson’s disease, neurodegenerative diseases, cardiovascular diseases, and cancer.16-20 Despite the apparent importance of copper for physiology and pathology, the underlying cellular mechanisms are still largely unknown, which prompts extensive exploration of copper in the treatment of various diseases. For example, the same morphological and molecular changes were observed after treating cancer cells with disulfiram (copper ionophore), pyrazole-pyridine copper complexes and inorganic copper, indicating that copper overload was the cause of cell death.21 Moreover, the killing effect of elesclomol (copper ionophore) was totally lost on coadministration of MDA-MB435 melanoma cells in the absence of serum (the source of copper), while it can be restored after adding copper instead of iron, manganese and zinc to the serum-free medium, suggesting a potential copper ion-regulated cell death mode.22 Afterwards, the anti-cancer effect of elesclomol was further corroborated through inducing a variety of cells, including melanoma cells, lung cancer cells, glioblastoma stem cells (GSCs) and gynecological tumor cells, to produce reactive oxygen species (ROS).23-26 However, the oxidative stress alone does not fully explain the mechanism of cell death, because the use of ROS scavenger N-acetylcysteine (NAC) can only partly reverse the elesclomol induced cancer cell death.27,28 Therefore, apart from oxidative stress, elesclomol should have additional mechanisms to regulate cancer cell death.
Gratifyingly, the “cuproptosis” proposed by Tsvetkov et al . provides a definitive explanation for the anti-cancer mechanism of elesclomol (Figure 1 ).29 Firstly, different metal ions including copper were carried by elesclomol to verify that only copper ions can mediate cancer cell death, which could be reversed by the copper ion chelating agents glutathione (GSH) and tetrathiomolybdate (TTM) but not by known inhibitors of various cell death pathways (ferrostatin-1, necrostatin-1, NAC), confirming that cell death induced by copper ions, namely cuproptosis, may be a new type of cell death mode different from traditional cell death such as apoptosis, ferroptosis, necrosis and autophagy. Then, mitochondrial respiration-dependent cells showed higher sensitivity to copper ions compared with glycolysis-dependent cells, suggesting that cuproptosis was related to mitochondrial metabolism. Further investigation revealed that the respiratory reserve capacity was significantly reduced after copper ion treatment, while basic respiration or ATP-related respiration remained stable, indicating that copper acted on the components of the tricarboxylic acid (TCA) cycle rather than electron transport chain (ETC). After that, seven TCA cycle genes were identified as relevant for cuproptosis mechanism, including FDX1 (a reductase reducing Cu2+ to Cu+), LIPT1, LIAS, DLD (three key enzymes of the lipoic acid pathway), DLAT, PDHA1, and PDHB (three components of the pyruvate dehydrogenase complex). Moreover, FDX1 and LIAS knockdown alleviated copper ionophore-triggered cytotoxicity, emphasizing the inherent association between TCA cycle and cuproptosis. Furthermore, the copper ions reduced by FDX1 bound to the lipoacyl group of DLAT to promote its lipoacylation and aggregation, thereby exerting cytotoxicity. In addition, FDX1 also leaded to the instability of Fe–S cluster protein, which exacerbated cell death. Notably, in vivoexperiments confirmed that cell death caused by copper homeostasis imbalance and cell death induced by copper ionophore belonged to the same cuproptosis mechanism. Altogether, cuproptosis is a copper-dependent form of novel cell death. The establishment of the concept not only illuminates the cytotoxic mechanism in copper ionophore, but also provides new insights for the treatment of various diseases including cancer.