Is the Way to Fight Cancer Paved with Gold? Metal-Based Carbene Complexes with Multiple and Fascinating Biological Features

Abstract

Herein, we report the synthesis and the multiple anti-tumor properties of new gold and silver carbene complexes. The chemical modifications, grounded on our previous studies, led us to identify a good lead complex, gold-based, whose biological features are very exciting and promising in the anti-cancer research and could be further developed. Indeed, the bis-[4,5-dichloro-(N-methyl-N'(2-hydroxy-2-phenyl)ethyl-imidazole-2-ylidene)gold(I)]⁺[dichloro-gold]^- (AuL7) complex possesses the ability to interfere with at least three important and different intracellular targets, namely the human topoisomerases I and II and tubulin, which are able to modulate metabolic processes not directly correlated each other. We proved that the modifications of the ligands structure in AuL7, with respect to another already published complex, i.e., bis-[4,5-dichloro-(N-methyl-N'(cyclopentane-2ol)-imidazole-2-ylidine)gold(I)]⁺[dichloro-gold]^- (AuL4), produce a different behavior toward tubulin-polymerization process, since AuL7 is a tubulin-polymerization inhibitor and AuL4 a stabilizer, with the final same result of hampering the tumor growth. Taken together, our outcomes designate AuL7 as a promising compound for the development of multi-targeted anti-cancer therapies.

Keywords: carbene complexes; docking studies; human topoisomerases I and II; tubulin; women cancers.

Figure 1

NHC ligands and Ag and Au respective complexes.

Figure 2

New synthetized Ag and Au complexes.

Scheme 1

Synthesis of N-[4-(hydroxymethyl)phenyl]-N’-[(2-hydroxy-2-phenyl)ethyl]-imidazolium bromide (L5).

Scheme 2

Synthesis of [N,N’-[(2-hydroxy-2-phenyl)ethyl]-imidazolium iodide (L6).

Scheme 3

Synthesis of N-[4-(hydroxymethyl)phenyl]-N’-[(2-hydroxy-2-phenyl)ethyl]-imidazole-2-ylidene]silver(I)bromide] (AgL5).

Scheme 4

Synthesis of N-[4-(hydroxymethyl)phenyl]-N’-(2-hydroxy-2-phenyl)ethyl]-imidazole-2-ylidene]-gold(I)bromide](AuL5).

Scheme 5

Synthesis of [N,N’-(2-hydroxy-2-phenyl)ethyl]-imidazole-2-ylidene]-silver(I)]⁺[di-iodide-silver]⁻ (AgL6).

Scheme 6

Synthesis of bis[N,N’-(2-hydroxy-2-phenyl)ethyl]-imidazole-2-ylidene]-gold(I)]⁺[dichloro-gold]⁻ (AuL6).

Figure 3

Graphical representation of AuL4 and AuL7 binding to the tubulin α:β interface. (a) Ribbon representation of the protein dimeric interface (tanned ribbons), bound ligands are reported as sticks. Residues involved in the binding are properly labeled. (b) Tubulin dimeric interface in complex with AuL7 (protein is represented in tanned ribbons, amino acids involved in ligand binding are properly labeled, AuL7 is drawn as purple sticks). (c) Close up of AuL4 (orange sticks) binding.

Figure 3

Graphical representation of AuL4 and AuL7 binding to the tubulin α:β interface. (a) Ribbon representation of the protein dimeric interface (tanned ribbons), bound ligands are reported as sticks. Residues involved in the binding are properly labeled. (b) Tubulin dimeric interface in complex with AuL7 (protein is represented in tanned ribbons, amino acids involved in ligand binding are properly labeled, AuL7 is drawn as purple sticks). (c) Close up of AuL4 (orange sticks) binding.

Figure 4

Tubulin-polymerization assay. The effect of compounds AuL4 and AuL7 on in vitro tubulin polymerization was evaluated. Two positive control molecules, Paclitaxel and Vinblastine, were used as tubulin-stabilizing and tubulin-destabilizing agents, respectively. DMSO was used as a negative control. Assembly of tubulin into microtubules was determined by measuring the turbidity at 350 nm for 3600 s at 37 °C. All the compounds were used at a concentration of 10 μM.

Figure 5

Immunofluorescence studies. MDA-MB-231 cells were treated with AuL7, Vinblastine, Paclitaxel (used at their IC₅₀ values) or with a vehicle (CTRL) for 24 h. After treatment, the cells were methanol fixed, incubated with primary and secondary antibodies, stained with 4′,6-diamidino-2-phenylindole (DAPI) and observed and imaged under the inverted fluorescence microscope at 63× magnification (see Materials and methods). Vehicle-treated cells (CTRL) exhibited a normal arrangement and organization of the cytoskeleton. Microtubules of MDA-MB-231 cells treated with vinblastine and paclitaxel showed an irregular arrangement and organization: particularly, tubulin crystal formation (white arrows, panel B, vinblastine) and tubulin bundles and thicken fibers (white arrows, panel B, paclitaxel) can be noted for vinblastine and paclitaxel respectively. Treatment with AuL7 shows a vinblastine-like mode of action (white arrows, panels B, AuL7). Panels A: DAPI, excitation/emission wavelength 350 nm/460 nm; panels B: β-tubulin (Alexa Fluor^® 488) excitation/emission wavelength 490 nm/515 nm; panels C show a merge. Representative fields are shown.

Figure 6

Immunofluorescence studies. HeLa cells were treated with AuL4, Vinblastine, Paclitaxel (used at their IC₅₀ values) or with a vehicle (CTRL) for 24 h. After treatment, the cells were methanol fixed, incubated with primary and secondary antibodies, stained with DAPI and observed and imaged under the inverted fluorescence microscope at 20× magnification (see Materials and methods). Vehicle-treated cells (CTRL) exhibited a normal arrangement and organization of the cytoskeleton. Microtubules of HeLa cells treated with vinblastine and paclitaxel showed an irregular arrangement and organization: particularly, tubulin crystal formation (white arrows, panel B, vinblastine) and tubulin bundles and thicken fibers (white arrows, panel B, paclitaxel) can be noted for vinblastine and paclitaxel respectively. Treatment with AuL4 shows a paclitaxel-like mode of action (white arrows, panels B, AuL4). Panels A: DAPI, excitation/emission wavelength 350 nm/460 nm; panels B: β-tubulin (Alexa Fluor^® 488) excitation/emission wavelength 490 nm/515 nm; panels C show a merge. Representative fields are shown.

Figure 7

hTopoI assay. Supercoiled DNA was incubated without or with human Topoisomerase I in the absence or presence of the test compounds at 1 μM: lane 1, plasmid pHOT1, lane 2, vehicle (DMSO), lane 3, AuL7 1 μM and lane 4, AuL4 1 μM.

Figure 8

hTopo II inhibition assay. kinetoplast DNA (kDNA) was incubated with human topoisomerase II in the absence (lane 3, vehicle DMSO) or presence of compound AuL7 and AuL4 at1μM (lane 4 and 5 respectively). Lane 1 and 2, decatenated and linear kDNA, respectively.

Figure 9

MDA-MB-231 cells were treated with AuL7 at a concentration corresponding to its IC₅₀ value or with the vehicle (CTRL) for 24 h. Processed cells were observed under an inverted fluorescence microscope at 20× magnification. The green nuclear fluorescence, in AuL7-treated cells, indicates apoptotic death. Panels A: DAPI (CTRL and AuL7) excitation/emission wavelength 350 nm/460 nm. Panels B: CF™488A (CTRL and AuL7) excitation/emission wavelength 490 nm/515 nm. Panels C: a merge of panels A and B. Fields are representative of three separate experiments.

Figure 10

ROS induction by AuL7 treatment in MDA-MB-231 cells. (a) ROS production was evaluated by using DCF-DA in MDA-MB-231 cells treated with Menadione (Men, used as positive control), AuL7, or vehicle. Images were acquired at 20× magnification. (A): Nuclear stain with DAPI (excitation/emission wavelength 350 nm/460 nm) in treated (Men or AuL7) and untreated cells (vehicle, CTRL); (B): DCF (excitation/emission wavelength 490 nm/515 nm for CFQ488A) in treated (Men or AuL7) and untreated cells (vehicle, CTRL); (C): overlay channel; (b) Fluorescence quantification. *p < 0.001, AuL7 vs. Men. Fields are representative of three separate experiments.

Figure 11

Mitochondria staining and cytochrome c translocation in MDA-MB-231 cells. In vehicle-treated cells, cytochrome c (panel B, CTRL) is localized within intact mitochondria (panel C, CTRL), as visible in panel D (CTRL) where the overlay channel is shown. Treatment with AuL7 at a concentration corresponding to its IC₅₀ value for 24 h induces cytochrome c release in the cytosol (panel B, AuL7, white arrow) and loss of mitochondria integrity (panel C, AuL7, white arrows). (A) nuclear stain with DAPI (excitation/emission wavelength 350 nm/460 nm); (B) Alexa Fluor CF 488 (excitation/emission wavelength 490 nm/515 nm); (C) MitoTracker Deep Red FM probe (excitation/emission wavelength 644 nm/665 nm); (D) Overlay channels. Images were acquired at 63x magnification and representative fields are shown.

Figure 12

Caspases activity. Activation of caspases 3/7 and 9 due to the treatment of MDA-MB-231 cells with the compound AuL7 at a concentration equal to 5 μM for 24 h. Columns mean, bars standard deviations (SD), * p < 0.001. Results are representative of three separate experiments.

Figure 13

Cycle assay. (a) DNA content histogram of MDA-MB-231 cells treated with only DMSO; (b) DNA content histogram of MDA-MB-231 cells treated with AuL7 at the concentration corresponding to its IC₅₀ value for 72 h.