N-Heterocyclic Carbene (NHC) Silver Complexes as Versatile Chemotherapeutic Agents Targeting Human Topoisomerases and Actin

Abstract

In recent years, the number of people suffering from cancer has risen rapidly and the World Health Organization and U.S. and European governments have identified this pathology as a priority issue. It is known that most bioactive anticancer molecules do not target a single protein but exert pleiotropic effects, simultaneously affecting multiple pathways. In our study, we designed and synthesized a new series of silver N-heterocyclic carbene (NHC) complexes [(NHC)₂ Ag]⁺ [AgX₂ ]^- (X=iodide or acetate). The new complexes were active against two human breast cancer cell lines, MCF-7 and MDA-MB-231. These compounds showed multiple target actions as anticancer, by inhibiting in vitro the activity of the human topoisomerases I and II and interfering with the cytoskeleton dynamic, as also confirmed by in silico studies. Moreover, the antimicrobial activity of these silver complexes was studied against Gram-positive/negative bacteria. These dual properties provide a two-tiered approach, making these compounds of interest to be further deepened for the development of new chemotherapeutic agents.

Keywords: actin; breast cancer; docking studies; human topoisomerases; silver N-heterocyclic carbine complexes.

Scheme 1

Structures of silver N‐heterocyclic carbene (NHC) complexes.

Scheme 2

Synthesis of NHC silver(I) acetate 2, 4, 5, and 6 complexes.

Figure 1

Minimum energy structures of 1–6. Distance is in Å. Part of hydrogens of NHC skeleton have been omitted for clarity.

Figure 2

a) In vitro actin polymerization assay. The effect of compounds 2 and 6 (5 μM) on in vitro actin polymerization was evaluated, incubating them with the labeled rabbit muscle actin. In vitro actin polymerization assay: compounds 1 and 6. b) In vitro actin depolymerization assay. Compounds 2 or 6 were added to the reaction mixture after actin polymerization, in order to determine their ability to interfere with the depolymerization process. For both the assays, DMSO was used as a negative control, while actin‐targeting agents, LA and CB (5 μM), were used as reference molecules. The assembly of actin filaments was determined by measuring the fluorescence (λ_ex/em: 365/410 nm) in kinetic mode for 1 h at room temperature using a microplate reader. The graphics are representative of three separated tests.

Figure 3

Immunofluorescence analysis. MDA‐MB‐231 cells were exposed for 24 h to DMSO (CTRL), to LA or compound 2, both used at the concentration of 5 μM. The cells were further processed and imaged under an inverted fluorescence microscope at 20× magnification (see experimental section). The treatment with compound 2 induced the formation of prominent actin cords at the level of the MDA‐MB‐231 cell periphery. Panels A: nuclear stain with DAPI (λex/λem=350/460 nm); Panels B: β‐actin (Alexa Fluor® 568; λex/λem=644/665 nm); Panels C: show the overlay channel. Representative fields of three independent experiments are shown.

Figure 4

Immunofluorescence analysis. MCF‐7 cells were exposed for 24 h to DMSO (CTRL), to LA or compound 6, both used at the concentration of 5 μM. The cells were further processed (see experimental section) and imaged under an inverted fluorescence microscope at 20× magnification. The treatment with compound 6, similarly to LA, induced the formation of cytoplasm with a thread‐like structure or abnormally enlarged, with very bright actin filaments arranged in dot‐like structures. Panels A: nuclear stain with DAPI (λex/λem=350/460 nm); Panels B: β‐actin (Alexa Fluor® 568; λex/λem=644/665 nm); Panels C: show the overlay channel. Representative fields of three independent experiments are shown.

Figure 5

Human Topoisomerases I relaxation assays. Supercoiled DNA was used as substrate and incubated with hTopoI in the absence or presence of the tested compounds. Lane 1, pHOT1 DNA; lane 2, CTRL (DMSO); lane 3, compound 2 1 μM; lane 4, compound 6 1 μM; lane 5, a marker of relaxed DNA.

Figure 6

Human Topoisomerase II decatenation assay. Kinetoplast DNA (kDNA) was used as substrate and incubated with hTopoII, in the absence or presence of the tested compounds. (a) Lane 1, linear kDNA, lane 2, decatenated kDNA, lane 3, kDNA; lane 4, CTRL (DMSO); lane 5, compound 2 1 μM; lane 6, compound 6 1 μM. (b) Lane 1, linear kDNA, lane 2, decatenated kDNA, lane 3, kDNA; lane 4, CTRL (DMSO); lane 5, compound 2 10 μM; lane 6, compound 6 10 μM.

Figure 7

Schematic representation of compound 2 (Panel A, blue sticks), 6 (Panel B, yellow sticks), and Latrunculin B (Panel C, orange sticks) binding modes to Actin (Pink ribbons).

Figure 8

Panels A and B show the binding modes of compounds 2 (pink) and 6 (violet) to human Topoisomerase I (sandy ribbons). Panels C and D illustrate the two compounds (same colors) poses within human Topoisomerase II (green ribbons).

Figure 9

TUNEL assay. MDA‐MB‐231 cells treated with compound 2 (a) or MCF‐7 cells treated with compound 6 (b), both at concentrations equal to their IC₅₀ o with vehicle (CTRL) for 24 h. The presence of the green nuclear fluorescence indicates apoptotic death. Panels A: DAPI (CTRL, compounds 2 and 6) excitation/emission wavelength 350 nm/460 nm. Panels B: CF™488 A (CTRL, compounds 2 and 6) excitation/emission wavelength 490 nm/515 nm. Panels C: a merge of panels A and B. Fields are representative of three independent experiments.