# N-Heterocyclic Carbene-Gold(I) Complexes Targeting Actin Polymerization
## 3 Results
### AuL4 and AuL7 Interface with the Normal Intracellular Actin Organization  
The cell cytoskeleton is an important intracellular structure that is involved in several physiological processes, and is implied, as well, in tumor progression, most importantly in the epithelial-mesenchymal transition (EMT) process. The major components are tubulin and actin, which are targets of effective anticancer drugs. Some metal complexes have been proved to interfere with the cytoskeleton dynamics [15; 32; 33; 34; 35; 36], particularly inhibiting the tubulin polymerization but, more recently, they were discovered to play a different and specific role in regulating the actin system [37]. Since we have already proved that the two metal complexes exerted inhibitory activity against the tubulin polymerization [15], we wondered whether they could also interfere directly with the other major component of the cell cytoskeleton, _viz._ the actin, in the two cancer cell models used for the previous study. With this in mind, HeLa and MDA-MB-231 cells were treated for 24 h with compounds **AuL4** (at its IC\({}_{50}\) = 1.63 \(\pm\) 0.5 \(\upmu\)M) and **AuL7** (at its IC\({}_{50}\) = 2.1 \(\pm\) 0.7 \(\upmu\)M), respectively, together with the only vehicle (DMSO, negative control). As a reference molecule, we used latrunculin A (positive control), a well-known natural compound that targets the actin cytoskeleton, at a concentration of 1 \(\upmu\)M, under the same experimental conditions. After that, cancer cells were further processed and imaged under a fluorescent microscope, as described in the Material and Methods section. As visible in Figures 4 and 5, in the negative control (only DMSO), the actin filaments exhibited a normal organization in the cell cytoplasm, with differences relative to the normal HeLa and MDA-MB-231 cell morphology. Conversely, under LA exposure, both cancer cell lines showed a remarkable disorganization of the actin system. Indeed, in Figure 4, the actin bundles are very thick and bright, with respect to the vehicle-treated HeLa cells, and one can notice that the actin system is more compact, not fairly distributed, and some dot-like structures appeared (see white arrows). A similar arrangement was seen in **AuL4**-treated HeLa cells; indeed, the cell size is reduced and the actin is thickened around the cell nuclei, with respect to the vehicle-treated cells (negative control). The MDA-MB-231 cells under **AuL7** treatment lost their normal morphology, as well, but assumed different shapes; indeed, in Figure 5, some cells appear round, others with a thread-like structure or abnormally enlarged cytoplasm (see white arrows). Moreover, the actin filaments look very bright and arranged in dot-like structures or unevenly distributed within the cell cytoplasm. To summarize, we can affirm that both compounds interfere with the regular organization of the actin system in the cancer cells under investigation. Figure 4: Immunofluorescente analysis. HeLa cells were treated with DMSO (CTRL, negative control) 0.1 \(\upmu\)M Latrunculin A (LA, positive control) or **AuL4** (at its IC\({}_{50}\) value) for 24 h. After treatment, the cells were further processed (see Materials and Methods section) and imaged under inverted fluorescence microscope at 20\(\times\) magnification. In the CTRL cells, the actin filaments exhibited a normal organization in the cells cytoplasm. HeLa cells treated with LA and **AuL4**, showed an irregular arrangement and organization of the actin cytoskeleton: the actin system appear more compact, not fairly distributed and some dot-like structures appeared (see white arrows). (**Panels a**): nuclear stain with DAPI (\(\lambda\)ex/\(\lambda\)em = 350/460 nm); (**Panels b**): \(\beta\)-actin (Alexa Fluor® 568; \(\lambda\)ex/\(\lambda\)em = 644/665 nm); (**Panels c**): show the merge. Representative fields are shown.