# N-Heterocyclic Carbene-Gold(I) Complexes Targeting Actin Polymerization
## 4 Discussion  
Microtubules and actin filaments are cytoskeletal components that play pivotal roles in cell signaling, division, and motility, and regulate tumor relevant processes, for instance, morphological changes or cell migration [38]. For these reasons, they are interesting targets of an even higher number of anticancer drugs, such as the clinically used vinca alkaloids or taxanes that target microtubules, whereas compounds directed toward the actin have relatively lesser therapeutic applications, probably because of poor knowledge on their underlying mechanisms of action [39]. Under physiological conditions, the monomeric form of actin, called G-actin, polymerizes in a head-to-tail manner and constitutes the filamentous F-actin. The turnover of these forms is ensured by a tightly regulated equilibrium between G- and F-actin, but is substantially modified in cancer cells, together with changes in the filament-associated regulatory proteins, and is involved in the uncontrolled growth of tumor cells and, ultimately, in the increased ability to metastasize [40,41]. Generally, the migrating cancer cells undergo to shape rearrangements that depend on the formation of actin-based protrusions, cellular contractility, and new adhesions to surfaces [42]. For example, actin participates in the formation of specialized invasive and adhesive structures with proteolytic activity, called invadosomes [41]. Since we have already proved that the two NHC-gold complexes exerted inhibitory activity against the tubulin polymerization, we wondered whether they could also interfere directly with the other major component of the  
Figure 7: In vitro actin depolymerization assay. The effect of compounds **AuL4** and **AuL7** (5 \(\upmu\)M) on in vitro actin depolymerization was examined. DMSO was used as a negative control (CTRL). Latrunculin A (LA) and cytochalasin B (CB), were employed as reference molecules and used at the concentration of 5 \(\upmu\)M. **AuL4** and **AuL7**, added to the polymerized actin, do not accelerate the depolymerization reaction. The graphic was obtained measuring the fluorescence (Ex/Em: 365/410 nm) given by actin filaments disassembly in a kinetic mode for 1 h at room temperature. Data are representative of three independent experiments; standard deviations (SDs) are shown. cell cytoskeleton, _viz._ the actin. Indeed, some literature data reported that the actin system disorganization can be a consequence of the inhibition of the tubulin polymerization and not a direct regulation of the actin system [39; 43]. Amongst the most well-known molecules able to interfere directly with the intracellular actin, different types of cytochalasin and latrunculin were employed in many research studies, because of their easy membrane permeation and different types of behavior against the actin polymerization and depolymerization processes. In our assays, we used, as references molecules, cytochalasin B (CB), which inhibits the addition of monomers to the \"barbed\" end of the actin filaments [44], and latrunculin A (LA), which blocks the actin polymerization, and increases its depolymerization by trapping the actin monomers [45]. In our previous studies, we studied the anticancer properties of **AuL4** and **AuL7**, individuating some intracellular targets, as the human topoisomerases I and II, and the cytoskeleton tubulin, which decreased cancer cell growth by inducing the ROS-depending intrinsic apoptotic pathway. Encouraged by the outcomes, we continued studying other interactions of our compounds with other key-points belonging to the cell cytoskeleton. In particular, we investigated the possible implication of our complexes in regulating the actin dynamics in a direct way, and not as a consequence of the already observed interference with the tubulin polymerization reaction. Indeed, some microtubule poisons may promote the reorganization of the actin system, which is a consequent effect rather than a direct interaction.