Dinuclear Cu²⁺-complexes disrupt cellular pathways and rewire the breast cancer proteome

Cancer's global burden highlights the urgent need for more effective therapies. Metal-based drugs, particularly Cu²-complexes, offer promising alternatives due to copper's diverse biological functions. This study investigates the antitumor potential of two novel dinuclear Cu²-complexes, [Cu₂(μ-CH₃COO)(L)(OH₂)]·2H₂O (R9) and [Cu₂(μ-OH)(HL)(OH₂)]ClO₄·2H₂O (R10), in MCF-7 breast cancer cells. Both compounds exhibited greater cytotoxicity than cisplatin, with IC₅₀ values of 1.01 ± 0.09 μM (R9) and 1.27 ± 0.14 μM (R10), while showing selectivity toward cancer cells, as indicated by higher IC₅₀ values in healthy MCF10A cells. Treated MCF-7 cells showed increased granularity, mitochondrial membrane depolarization, and elevated reactive oxygen species. At IC₅₀ concentrations, cell cycle analysis revealed Sub-G1 accumulation and DNA fragmentation (TUNEL assay), indicating apoptosis via intrinsic pathways, supported by caspase 9 activation. Label-free proteomics revealed distinct mechanisms for R10 compared to cisplatin. In R10-treated cells, key downregulated pathways included glycolysis, the TCA cycle, oxidative phosphorylation, PI3K-Akt signaling, and the ubiquitin-proteasome system. Apoptosis-related proteins such as structural proteins (ACTB, ACTG1, SPTAN1, TUBA4A), mitochondrial apoptotic factors (AIFM1), nuclear envelope components (LMNA), and stress-response regulators (JUN, EIF2S1) were dysregulated. Proteomics data is available via ProteomeXchange with identifier PXD064464. These findings support the potential of Cu²-complexes as effective antitumor agents with mechanisms distinct from cisplatin, offering superior efficacy through apoptosis induction. SIGNIFICANCE: Breast cancer remains one of the leading causes of cancer-related mortality among women, highlighting the need for more effective and selective therapeutic agents. While platinum-based drugs are widely used, their limitations call for novel alternatives. In this study, we demonstrate that two newly synthesized dinuclear copper(II) complexes, R9 and R10, exhibit strong cytotoxicity against MCF-7 breast cancer cells with higher selectivity compared to cisplatin. Through functional and proteomic analyses, we show that these compounds induce intrinsic apoptosis, disrupt cytoskeletal integrity, and modulate key signaling pathways such as PI3K-Akt and RAS-ERK. Our proteomic results reveal distinct molecular signatures for R10, underscoring its unique mechanism of action. Therefore, copper-based complexes represent promising candidates for breast cancer treatment, and proteomics provides critical insight into their therapeutic potential.