Cu(II) complex that synergistically potentiates cytotoxicity and an antitumor immune response by targeting cellular redox homeostasis

Abstract

Developing anticancer drugs with low side effects is an ongoing challenge. Immunogenic cell death (ICD) has received extensive attention as a potential synergistic modality for cancer immunotherapy. However, only a limited set of drugs or treatment modalities can trigger an ICD response and none of them have cytotoxic selectivity. This provides an incentive to explore strategies that might provide more effective ICD inducers free of adverse side effects. Here, we report a metal-based complex (Cu-1) that disrupts cellular redox homeostasis and effectively stimulates an antitumor immune response with high cytotoxic specificity. Upon entering tumor cells, this Cu(II) complex enhances the production of intracellular radical oxidative species while concurrently depleting glutathione (GSH). As the result of heightening cellular oxidative stress, Cu-1 gives rise to a relatively high cytotoxicity to cancer cells, whereas normal cells with low levels of GSH are relatively unaffected. The present Cu(II) complex initiates a potent ferroptosis-dependent ICD response and effectively inhibits in vivo tumor growth in an animal model (c57BL/6 mice challenged with colorectal cancer). This study presents a strategy to develop metal-based drugs that could synergistically potentiate cytotoxic selectivity and promote apoptosis-independent ICD responses through perturbations in redox homeostasis.

Keywords: disrupting redox homeostasis; ferroptosis; immunogenic cell death; metal complex.

Fig. 1.

Chemical structures of the metal complexes considered in this study; n = 1 for Cu, Pt, and Pd; n = 2 for Co.

Scheme 1.

Graphic summary of Cu-1-induced antitumor immunity.

Fig. 2.

Single X-ray crystal structure of Cu-1 shown at the 50% probability level. All hydrogen atoms have been removed for clarity. CCDC number: 2289499.

Fig. 3.

Effects on in vitro cytotoxicity and redox homeostasis. (A) Cytotoxicity of the indicated metal complexes incubated with cells for 48 h. (B) Degradation of methylene blue (MB) seen upon exposure to different concentrations of GSH. (C) Degradation of MB seen at different time points. (D) Degradation of MB seen upon incubation of Cu-1 with H₂O₂ for 1 h at pH = 7.4. Photographs of the MB solutions are shown in the Inset. (E) GSH/GSSG ratio in the indicated cell lines before and after treatment with Cu-1 at the ~IC₅₀ concentration determined for the indicated cell lines. (F) Cellular relative ROS stress level resulting from treatment with Cu-1. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.

Ferroptosis induced by Cu-1. (A) Effects of inhibition of ferroptosis, necroptosis, apoptosis, cuproptosis, and autophagy on CT-26 cell viability as seen in the presence of Cu-1 (1.5 μM,48 h). (B) GSH levels in CT-26 cells treated with the indicated concentrations Cu-1 for 12 h. (C) Lipid peroxidation levels in CT-26 cells treated with the indicated concentrations of Cu-1 for 12 h. (Scale bars: 20 μm.) (D) Effects of Cu-1 on key ferroptosis proteins in CT-26 cells after treatment for the indicated time intervals. (E) Effects of ferroptosis inhibition (using 5.0 μM Fer-1 as an inhibitor) on key ferroptosis proteins in CT-26 cell lines treated with Cu-1 for 48 h. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

Induction of ICD by Cu-1. (A) Confocal microscopy images of CT-26 cells displaying CRT localization 3 h posttreatment with 1.0 or 2.0 μM Cu-1. (Scale bars: 20 μm.) (B) Percentage of cells expressing surface-CRT posttreatment with Cu-1 or oxaliplatin, as determined by flow cytometry and the effect of the ferroptosis inhibitor, Fer-1 (2.0 μM), on surface expression of CRT. (C) ATP release in CT-26 cells 6 h posttreatment with the indicated concentrations of Cu-1 or oxaliplatin, and the effect of Fer-1 on Cu-1-stimulated ATP release. (D) Release of HMGB1 protein in CT-26 cells 6 h posttreatment with the indicated concentrations of Cu-1 or oxaliplatin, and the effect of Fer-1 on Cu-1-stimulated HMGB1 release. (E) Antitumor vaccination where CT-26 cells were initially treated with Cu-1 (2, 4, 10 μM), oxaliplatin (100 μM), or solvent control for 6 h before the cells were subcutaneously injected into the left flanks of C57BL/6 mice (n = 10), which were then rechallenged in the right flanks with untreated CT-26 cells after 7 d. The curves show the percentage tumor-free after the 30-d treatment. (F–H) Percentages of (F) activated CD8⁺ T cells, (G) CD4⁺T cells, and (H) Foxp3⁺ T cells in the total cells isolated from tumors of C57BL/6 mice treated with PBS, Cu-1, or oxaliplatin. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.

Tests of the therapeutic potential of Cu-1 (10 mg/kg) and oxaliplatin (7 mg/kg) in vivo. (A) Image of isolated tumors. (B) Relative tumor volume changes during therapy (n = 5). (C) Body weights of mice receiving treatment (n = 5 per group). (D) Weights of isolated tumors. (E−G) Serum biochemical analyses showing the function of the liver (E), heart (F), and kidney (G). (H) Hematoxylin−eosin staining of major organs from mice receiving treatments. (I) Schematic schedule (x axis) for an in vivo Cu content biodistribution assay (n = 3 per group). The untreated mice were set as day 0 with the Cu content in major organs of mice receiving Cu-1 being shown on the y axis. Data represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.