Autophagy-dependent cell death and immune responses induced by heteroleptic copper(II), manganese(II), and silver(I) complexes containing dicarboxylate and 1,10-phenanthroline ligands: novel insights from in vitro and in vivo models

Autophagy is a process that breaks down unwanted cellular components to maintain homeostasis. While it is defined as a ‘self-protection’ process, a disruption of autophagic mechanisms can lead to cellular death. Autophagy is interconnected with many other cellular processes including innate and adaptive immunity and can therefore have therapeutic potential as it can be modulated to control immune and other cellular responses. We therefore postulated the potential of our Cu(II), Mn(II) and Ag(I) complexes coordinated with 1,10-phenanthroline to interfere with the autophagy process and to induce an immune-response. Herein, we evaluated the ability of a series of heteroleptic complexes, [Cu₂(oda)(phen)₄](ClO₄)₂, [Cu(oda)(phen)₂], [Ag₂(oda)(phen)₃], [Ag₂(udda)(phen)₃], and {[Mn₂(oda)₃(phen)₄]²⁻[Mn₂(oda)(phen)₄(H₂O)₂]²⁺}(where:oda²⁻ = octanedioate; udda²⁻ = undecanedioate; and phen = 1,10-phenanthroline), to modulate autophagy in vitro using MCF-7 (breast cancer) and MCF-12A (non-tumorigenic breast) cell lines; alongside their in vivo immunomodulatory effects in the Galleria mellonella larva model. Differential autophagy induction was observed between the two cell types through quantitative flow cytometry and fluorescence microscopy of autophagosomes using GFP-LC3, with a pronounced upregulation in non-tumorigenic MCF-12A cells compared to cancerous MCF-7 cells, underscoring the context-dependent modes of action of these complexes. Among them, the Mn(II)-phen complex ({[Mn₂(oda)₃(phen)₄]²⁻[Mn₂(oda)(phen)₄(H₂O)₂]²⁺}) was identified as the most potent inducer of autophagy in MCF-7 cells (p < 0.01), highlighting its unique mechanistic interaction within this breast cancer cell line, compared to the Cu(II) and Ag(I) analogues. In vivo characterisation of the complexes in the larvae of G. mellonella revealed a high tolerance and broad therapeutic windows were established. Notably, the Ag(I)-phen complex [Ag₂(udda)(phen)₃] was determined as the most well-tolerated complex, with a 24-h LD₅₀ of 864.3 µg/mL, exemplifying the pronounced tolerance of the complexes in contrast to previously reported in vitro cytotoxicity at distinctly low micro-molar concentrations. Analysis of changes in the haemocyte density, an established marker of immune response, revealed significant immune activation in G. mellonella, particularly with the Ag(I) ([Ag₂(oda)(phen)₃] and [Ag₂(udda)(phen)₃]) and Mn(II) ({[Mn₂(oda)₃(phen)₄]²⁻[Mn₂(oda)(phen)₄(H₂O)₂]²⁺}) complexes (p < 0.001). This study offers novel insights into the interplay between autophagy and immune modulation building upon our recent in vitro apoptosis study with these complexes. Our work establishes a compelling link between autophagy and modulation of immune activation and provides further biological insights on the unique and metal-specific roles of these complexes in shaping cellular and systemic responses.