An Organometallic Gold(I) Bis-N-Heterocyclic Carbene Complex with Multimodal Activity in Ovarian Cancer Cells

Abstract

The organometallic Au^I bis-N-heterocyclic carbene complex [Au(9-methylcaffeine-8-ylidene)₂ ]⁺ (AuTMX₂ ) was previously shown to selectively and potently stabilise telomeric DNA G-quadruplex (G4) structures. This study sheds light on the molecular reactivity and mode of action of AuTMX₂ in the cellular context using mass spectrometry-based methods, including shotgun proteomics in A2780 ovarian cancer cells. In contrast to other metal-based anticancer agents, this organogold compound is less prone to form coordinative bonds with biological nucleophiles and is expected to exert its drug effects mainly by non-covalent interactions. Global protein expression changes of treated cancer cells revealed a multimodal mode of action of AuTMX₂ by alterations in the nucleolus, telomeres, actin stress-fibres and stress-responses, which were further supported by pharmacological assays, fluorescence microscopy and cellular accumulation experiments. Proteomic data are available via ProteomeXchange with identifier PXD020560.

Keywords: G-quadruplexes; N-heterocyclic carbenes; cancer; gold complexes; proteomics; telomeres.

Figure 1

Chemical structures of AuTMX₂ and Au(BMI)₂, BMI=1‐butyl‐3‐methyl‐imidazole‐2‐ylidene. The latter was previously investigated using gel‐based proteomic methods in ref. [19a].

Figure 2

Cellular accumulation and distribution of AuTMX₂ in A2780 cancer cells by analysing ¹⁹⁷Au using ICP‐MS. (A) AuTMX₂ accumulates in cells more efficiently with increasing dose or longer incubation time. (B) AuTMX₂ distributes to one third in the nucleus (NE) and two thirds in the cytoplasmic (CYT) fraction irrespective of time when applied at 8 μm for 24 h. (C) Relative and (D) absolute amount of Au‐bound to DNA increases with applied dose after 24 h.

Figure 3

Response profiling by shotgun proteomics. The profile (A) and scatter (B) plots display the stability of the proteomic workflow, which contained six biological replicates per condition of AuTMX₂‐treated A2780 cancer cells. The scatter plot highlights the global LFQ‐correlation between replicate 1 and 2. Global protein expression changes were visualized by grouping significantly regulated proteins according to cellular compartment (C) and functional groups (D). The position of the protein group reflects the summed regulation in the cytoplasmic (CYT) and nuclear (NE) fractions.

Figure 4

Effect of AuTMX₂ on the cell cycle (A) of A2780 cancer cells after 24 h treatment and on their apoptosis induction (B) after 72 h treatment at the indicated concentrations measured by flow cytometry.

Figure 5

Effect of low dose treatments of AuTMX₂ on A2780 cancer cells after 7 d and 14 d treatment: (A) Duplication times and number of duplications, (B) cell cycle distribution assessed by flow cytometry after the end of week 1 (7 d) and week 2 (14 d), (C) cellular accumulation of ¹⁹⁷Au in trypsinized cells analysed by ICP‐MS and (D) absolute telomere lengths after 14 d. All experiments were performed in triplicate.

Figure 6

Fluorescence microscopy of nuclei (DAPI staining) and actin cytoskeleton (Alexa Fluor 488 phalloidin staining) of AuTMX₂‐treated A2780 cancer cells at different concentrations for 24 h vs. untreated cells (Con). Scalebars=20 μm.