1.1 Copper homeostasis in tumor cells

Copper is a cofactor for essential enzymes required by the human body and can maintain their homeostasis by acting across concentration gradients^[1].

Normally, intracellular copper concentrations are kept at extremely low levels, and this is also true in cancer cells. The normal function of copper ions relies on the interaction of different types of proteins: copper is transported through the blood system and transferred to the cell surface. Cu²⁺ will be catalytically reduced to Cu⁺ by the STEAP protein on the membrane surface, which has stronger cytotoxicity^[2][3].

Copper ions entering the cell pass through the outer mitochondrial membrane and inner mitochondrial membrane through COX17 and SLC25A3 in sequence, and enter the mitochondrial matrix. Of course, the copper ions still in the cytoplasm will also combine with the copper ion chelators GSH and MT to neutralize the cytotoxicity of copper, or be carried to SOD1 by the copper ion chaperone CCS to regulate the balance of intracellular reactive oxygen species. Various links are involved in the cell. Regulation of copper homeostasis^[2].

1.2 Copper homeostasis disorder

Dysregulation of copper homeostasis can lead to cellular metabolic disorders. When copper ions are excessively accumulated due to ionophores or transporters, on the one hand, FDX1 reduces Cu²⁺ to more toxic Cu⁺, inhibits the synthesis of iron-sulfur cluster proteins (Fe-S Cluster) related to mitochondrial respiration, and causes proteotoxic stress. reaction, ultimately leading to cell death^[1].

On the other hand, FDX1, as an upstream regulator of protein lipoylation modification, is involved in regulating the lipoylation of DLAT^[1][3]. Cu²⁺ can directly bind and induce the heteromerization of DLAT. This increase in insoluble DLAT leads to cellular proteotoxic stress and induces cell death^[3].

The battle against cancer treatment has always been difficult, and the study of cuproptosis provides a new way to kill tumors. So how do we trigger the weapon of cuproptosis to be used by us?

In general, cuproptosis can be triggered by increasing the intracellular concentration of free copper ions and from copper absorption, output, and storage. The transfer of copper ions into and out of cells is controlled by the copper ion transporters SLC31A1 and ATP7B. accessible^[2]:

(1) Overexpress SLC31A1 and absorb more copper ions;

(2) Knock down ATP7B, reduce copper efflux, and regulate intracellular copper ion concentration;

(3) Use copper ionophores such as Elesclomol and Disulfiram to directly transport extracellular Cu²⁺ into cells;

Deplete the endogenous intracellular copper chelator glutathione (GSH) by using butthionine sulfenimide (BSO) to avoid GSH chelating free copper ions.

Thus, excess Cu²⁺ or its more toxic reduced form Cu²⁺ is imported into cells and further leads to DLAT oligomerization by binding to lipoylated DLAT^[2]. At the same time, Cu also induces the reduction of Fe-S stability or the inactivation of Npl4-p97, resulting in cell death with a dedicated copper-induced mechanism^[2][4].

Of course, indispensable for the study of cuproptosis is the detection of relevant indicators! It mainly includes:

(1) Morphological observations, such as plasma membrane rupture, mitochondrial rupture, etc.^[2][5];

(2) Detection of related markers, such as Fe-S cluster proteins FDX1 and LIAS, which are markers of cuproptosis, the reduction of fatty acylation of DLAT and DLST and the increase of HSP70 levels, resulting in proteotoxic stress and ultimately cell death^[1];

(3) Detection of metabolic indicators, such as copper ion accumulation, α-ketoglutarate accumulation, and succinic acid reduction.

3.1 Morphological detection

When studying the relationship between copper accumulation and retinal developmental malformations and diseases, Guang Zhao et al. measured the morphological characteristics of embryos with excess copper (Figure 4 E). TEM analysis showed that the endoplasmic reticulum and mitochondrial structures of copper-treated embryonic retinal cells were disrupted. Compared with the control group, in copper-treated retinal cells, the inner mitochondrial membrane was reduced and large vacuoles were produced (E1-E3, red), and the endoplasmic reticulum formed a loose structure (E4-E6, green)^[5].

3.2 Marker detection

Studies have found that the copper ionophore Elesclomol induces cuproptosis in cardiomyocytes, which is characterized by a decrease in Fe-S cluster proteins and a decrease in mitochondrial enzyme fatty acylation^[6]. In “Exploring the potential impact of cuproptosis on AGEs-induced cardiomyocyte dysfunction in diabetic cardiomyopathy” published by Int J Mol Sci^[6], the author used Western Blot to detect the landmark changes after ES-Cu induced copper apoptosis in myocytes.

The results showed that the expression of various mitochondrial Fe-S cluster proteins, such as FDX1, LIAS, ACO2, ETFDH and NDUFV1, was down-regulated (Figure 5A); the fatty acylation of DLAT and DLST proteins was reduced (Figure 5B). In addition, the authors performed Western blot analysis of Fe-S cluster proteins in the myocardial tissue of diabetic (db/db) mice (Figure 5C-D) and found that the Fe-S cluster proteins FDX1, LIAS, and NDUFS8 in the hearts of db/db mice and ACO2 are lost, and HSP70 abundance increases^[6].

3.3 Metabolic index detection

The function of FDX1 depends on the accumulation of pyruvate and α-ketoglutarate, avoiding the consumption of succinate, thereby ensuring the TCA cycle of PDH and α-ketoglutarate dehydrogenase, leading to protein fatty acylation^[1]. Tian et al. designed a nanoadjuvant CS/MTO-Cu@AMI, which contains MTO , Cu²⁺ and exosome secretion inhibitor AMI (Figure 6A)^[7].

To verify cuproptosis, the authors analyzed the expression of HSP 70 and LIAS and detected changes in key metabolites in the Krebs cycle. The results showed that CS/MTO-Cu@AMI treatment significantly promoted the expression of HSP 70 and down-regulated the expression of LIAS (not shown in the figure). In addition, compared with the control group, the pyruvate and α-ketoglutarate contents in the tricarboxylic acid cycle in the CS/MTO-Cu@AMI group increased by 40%, while the succinic acid content decreased by 45% (Figure 6B-D), strongly supporting that CS/MTO-Cu@AMI can effectively induce cuproptosis^[7].

Nano-adjuvant CS/MTO-Cu@AMI: Cu²⁺ effectively triggers mitochondrial dysfunction induced by cuproptosis, activates PD-L1 protein degradation mediated by the AMPK pathway, and deprives macrophages and exosomes of the energy supply released. Amplifies oxidative stress, inactivates intracellular bacteria, thereby effectively sensitizing chemotherapy and activating systemic anti-tumor immunity in vitro and in vivo.

Copper is homeostatically regulated in tumor cells. The induced crproptosis will effectively inhibit tumor development. Crproptosis can be triggered by increasing copper ions concentration in cells, via modualting the absorption, output and storage of copper. In addition, relevant detection indicators and methods for copper death have been listed.

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