Novel Au Carbene Complexes as Promising Multi-Target Agents in Breast Cancer Treatment

Abstract

Over the past decade, metal complexes based on N-heterocyclic carbenes (NHCs) have attracted great attention due to their wide and exciting applications in material sciences and medicinal chemistry. In particular, the gold-based complexes are the focus of research efforts for the development of new anticancer compounds. Literature data and recent results, obtained by our research group, reported the design, the synthesis and the good anticancer activity of some silver and gold complexes with NHC ligands. In particular, some of these complexes were active towards some breast cancer cell lines. Considering this evidence, here we report some new Au-NHC complexes prepared in order to improve solubility and biological activity. Among them, the compounds 1 and 6 showed an interesting anticancer activity towards the breast cancer MDA-MB-231 and MCF-7 cell lines, respectively. In addition, in vitro and in silico studies demonstrated that they were able to inhibit the activity of the human topoisomerases I and II and the actin polymerization reaction. Moreover, a downregulation of vimentin expression and a reduced translocation of NF-kB into the nucleus was observed. The interference with these vital cell structures induced breast cancer cells' death by triggering the extrinsic apoptotic pathway.

Keywords: N-heterocyclic carbenes; actin; breast cancer cells; docking studies; human topoisomerases; multi-target agents.

Figure 1

New synthesized Au N-heterocyclic (NHC) carbene complexes.

Scheme 1

NHC-Au(I) complexes.

Figure 2

hTopo I and hTopo II assays. (A) hTopo I relaxation assay. hTopo I was exposed to DMSO (CTRL) or compounds 1 and 6 at the concentration of 1 μM, then the products of hTopo I reaction were visualized on agarose gel; (B) hTopo II decatenation assay. hTopo II was exposed to DMSO (CTRL) or compounds 1 and 6 at the concentration 1 μM. The hTopo II reaction products were visualized on agarose gel. pHOT1, supercoiled plasmid DNA; kDNA, kinetoplast DNA; CTRL, control (DMSO); 1 and 6, tested compounds.

Figure 3

Actin immunofluorescence studies. MDA-MB-231 cells were exposed to compound 1 (used at its IC₅₀ value), with 0.1 μM Latrunculin A (LA) or with a vehicle (CTRL) for 24 h. Then the cells were further processed, observed and imaged under the inverted fluorescence microscope at 40x magnification. CTRL cells showed a regular arrangement and organization of the actin cytoskeleton. MDA-MB-231 cells exposed to LA and, as well as those treated with compound 1, exerted an irregular arrangement and organization of the actin system. Panels (A): nuclear stain with DAPI (λ_ex/λ_em = 350/460 nm); Panels (B): β-actin (Alexa Fluor^® 568; λ_ex/λ_em = 644/665 nm); Panels (C): show a merge. Representative fields are shown.

Figure 4

Actin immunofluorescence studies. MCF-7 cells were exposed to compound 6 (used at its IC₅₀ value), with 0.1 μM Latrunculin A (LA) or with a vehicle (CTRL) for 24 h. Then the cells were further processed, observed and imaged under the inverted fluorescence microscope at 40× magnification. CTRL cells showed a regular arrangement and organization of the actin cytoskeleton. MCF-7 cells exposed to LA and, as well as those treated with compound 6, showed an irregular arrangement and organization of the actin system. Panels (A): nuclear stain with DAPI (λ_ex/λ_em = 350/460 nm); Panels (B): β-actin (Alexa Fluor^® 568; λ_ex/λ_em = 644/665 nm); Panels (C): show a merge. Representative fields are shown.

Figure 5

In vitro actin polymerization/depolymerization assay. The effect of compounds 1 and 6 (5 μM) on in vitro actin polymerization and depolymerization was examined. DMSO was used as a negative control. Actin-targeting agents, latrunculin A (LA) and cytochalasin B (CB), both at the concentration of 5 μM, were used as controls. (A) In vitro actin polymerization assay: compounds 1 and 6 were incubated with the labeled rabbit muscle actin in order to verify their ability to inhibit the protein polymerization; (B) In vitro actin depolymerization assay: after actin polymerization, the compounds 1 or 6 were added to the reaction mixture, in order to determine their ability to act as depolymerizing agents. For both the assays, the assembly of actin filaments was determined by measuring the fluorescence (Ex/Em: 365/410 nm) in kinetic mode for 1 h at room temperature using a microplate reader. The graphics are representative of three different assays.

Figure 5

In vitro actin polymerization/depolymerization assay. The effect of compounds 1 and 6 (5 μM) on in vitro actin polymerization and depolymerization was examined. DMSO was used as a negative control. Actin-targeting agents, latrunculin A (LA) and cytochalasin B (CB), both at the concentration of 5 μM, were used as controls. (A) In vitro actin polymerization assay: compounds 1 and 6 were incubated with the labeled rabbit muscle actin in order to verify their ability to inhibit the protein polymerization; (B) In vitro actin depolymerization assay: after actin polymerization, the compounds 1 or 6 were added to the reaction mixture, in order to determine their ability to act as depolymerizing agents. For both the assays, the assembly of actin filaments was determined by measuring the fluorescence (Ex/Em: 365/410 nm) in kinetic mode for 1 h at room temperature using a microplate reader. The graphics are representative of three different assays.

Figure 6

Evaluation of vimentin expression levels. (a) Immunofluorescence studies: MDA-MB-231 and MCF-7 cells were treated with compounds 1 and 6, respectively, (used at their IC₅₀ values) or with a vehicle (CTRL) for 24 h. After treatment, the cells were further processed, observed and imaged under the inverted fluorescence microscope at 40× magnification (for more details see Materials and Methods). Compound 1 induced a downregulation of the vimentin expression with respect to the CTRL cells in MDA-MB-231 cells. Panels A: nuclear stain with DAPI (λ_ex/λ_em = 350/460 nm); Panels B: vimentin (Alexa Fluor^® 568; λ_ex/λ_em = 644/665 nm); Panels C: show a merge. Images are representative of three separate experiments; (b) Fluorescence quantification carried out using ImageJ; p < 0.001; (c) Western blots analysis: MDA-MB-231 and MCF-7 cells were treated with compounds 1 and 6, respectively, (used at their IC₅₀ values) or with a vehicle (CTRL) for 24 h and then the total protein content was extracted and processed as reported in the Experimental Section. GAPDH: loading normalization. Images are representative of three separate experiments; (d) Western blots quantification carried out using ImageJ; p < 0.001.

Figure 7

Binding modes of compounds 1 and 6 with hTopo I and hTopo II. The proteins are depicted as tanned and olive-green ribbons, respectively. Compound 1 is drawn as light blue sticks, while compound 6 as green sticks. As reference, the violet bubble indicates the position of compound 1 with respect to compound 6 in binding hTopo II.

Figure 7

Binding modes of compounds 1 and 6 with hTopo I and hTopo II. The proteins are depicted as tanned and olive-green ribbons, respectively. Compound 1 is drawn as light blue sticks, while compound 6 as green sticks. As reference, the violet bubble indicates the position of compound 1 with respect to compound 6 in binding hTopo II.

Figure 8

Ribbon representation of the binding modes of compounds 1, 6 and Latrunculin B (Lab) to Actin. The proteins are drawn as pink ribbons. Compound 1 is drawn as light blue sticks, while compound 6 as green sticks. The binding pose of Latrunculin B (Lab), as determined by X-ray crystallography, is reported as gold sticks.

Figure 9

TUNEL assay. MDA-MB-231 and MCF-7 breast cancer cells were treated with compounds 1 and 6, respectively, at the concentration equal to their IC₅₀ or with vehicle (CTRL) for 24 h. Then they were exposed to the TdT enzyme and visualized under a fluorescence microscope (20× magnification). The green fluorescence indicates nuclei of cells undergoing apoptosis. Panels (A), DAPI λ_ex/em 350 nm/460 nm. Panels (B), CF^TM488 A λ_ex/em 490 nm/515 nm. Panels C show the overlay channel.

Figure 10

Caspases’ activity. Increase of caspases 3/7 and 8 activity levels, reported as percentage over the vehicle-treated cells used as control, following treatment of MDA-MB-231 cells with compound 1 (a) and MCF-7 cells with compound 6 (b). Both the compounds were used at their IC₅₀ values and incubated for 24 h. The data are representative of three independent experiments. *** p < 0.001 and n.s. not significant; treated vs. CTRL.

Figure 11

Mitochondria staining and cytochrome c detection in MDA-MB-231 cells. In the CTRL cells as well as in the cells treated with compound 1 at its IC50 value for 24 h, cytochrome c is localized within mitochondria. Panels (A), DAPI λ_ex/em = 350/460 nm; Panels (B), Alexa Fluor CF 488 λ_ex/em = 490/515 nm; Panels (C), MitoTracker Deep Red FM probe λ_ex/em = 644/665 nm; Panels (D), overlay channels. Images were acquired at 40× magnification and representative fields are shown.

Figure 12

Mitochondria staining and cytochrome c detection in MCF-7 cells. In the CTRL cells as well as in the cells treated with compound 6 at its IC50 value for 24 h, cytochrome c is localized within mitochondria. Panels (A), DAPI λ_ex/em = 350/460 nm; Panels (B), Alexa Fluor CF 488 λ_ex/em = 490/515 nm; Panels (C), MitoTracker Deep Red FM probe λ_ex/em = 644/665 nm; Panels (D), overlay channels. Images were acquired at 40× magnification and representative fields are shown.

Figure 13

NF-κB activation in MDA-MB-231 and MCF-7 cells after treatment with compounds 1 and 6, respectively. In the vehicle-treated cells, NF-κB is localized within the cytoplasm and nucleus, while the treatment of the breast cancer cells with compounds 1 and 6 for 24 h (used at their IC₅₀ values) reduced the NF-κB presence in the nucleus. Panels A, nuclear stain with DAPI (λ_ex/em = 350/460 nm); Panels B, Alexa Fluor CF 488 (λ_ex/em = 490/515 nm); Panels C, overlay channels. Images were acquired at 40× magnification and representative fields are shown.