## 3 Results and Discussion
### The Derivative **2** Inhibits Tubulin Polymerization  
Microtubules represent an important target in cancer therapy since they play a key role in various cellular functions such as mitosis, cell signaling, and organelle trafficking [37]. Indeed, microtubule-targeting agents, such as vinca alkaloids and taxanes, are successfully used in the clinic for the treatment of different kinds of cancers, but drug resistance onset and high toxicity represent the major issues. Different literature data indicated that some natural compounds, including RSV and some derivatives, exhibit their anticancer activity through the disruption of the microtubule dynamics, interfering with tubulin, without showing the limitations of traditional microtubule-targeting agents [38; 39; 40]. In previous  
Figure 3: (**A**) A schematic view of the derivative **2** (drawn as cyan sticks) binding site located at the interface between actin (pink ribbons) and profilin (violet ribbons); (**B**) A detailed view of the interface between actin and Profilin allows to identify the different residues involved in the derivative **2** binding.  
work, we assessed the interesting anticancer activity of the RSV derivative **2** on two breast cancer cell lines, MCF-7 and MDA-MB-231, without showing any cytotoxicity on the healthy cells [10]. Thus, in order to confirm in silico studies, we evaluated its ability to interfere with tubulin using immunofluorescence studies on both breast cancer cells. Figure 4 shows that the treatment with the derivative **2** (25 \(\mu\)M) induced a total microtubule disorganization with tubulin filaments amassed inside the cell cytoplasm, creating the typical crystals, indicated by the white arrows (Figure 4, Panels B, derivative **2**, MCF-7, and MDA-MB-231). Whereas in vehicle-treated cells, tubulin filaments resulted showed that they were well assembled and regularly distributed in the cytoplasm (Panels B, CTRL, MCF-7, and MDA-MB-231). A parallel experiment has been conducted on 3T3-L1 cells, demonstrating that the derivative **2** had no effect on the microtubules of the normal cell (Figure 4, Panels B, derivative **2**, 3T3-L1). Finally, the effect of RSV on the cytoskeleton of all the used cell lines was also assessed. The results demonstrated that MCF-7, MDA-MB-231 and 3T3-L1 cells exposed to RSV showed a regularly organized tubulin cytoskeleton (Figure 4, Panels B, RSV, MCF-7, MDA-MB-231, and 3T3-L1).  
In order to assess if the derivative **2** act as stabilizing- or destabilizing-microtubules agents, we also examined its effect on the tubulin polymerization reaction using a specific in vitro assay. We used three reference molecules, two tubulin polymerization inhibitors, Vinblastine and Nocodazole, and a stabilizing agent, Paclitaxel. The turbidity variation linked to the tubulin polymer formation was recorded each 30 s at 350 nm for 3600 s. In Figure 5, it is possible to observe that in the control reaction the tubulin polymerization rapidly occurred, in a time-dependent manner, reaching the plateau phase after 17 min with a maximal optical density (OD350) of 0.49. Paclitaxel caused a more rapid tubulin heterodimer assembly than in the control reaction. The steady-state was reached after less than 15 min with an OD350 of 0.54. The OD350 at 90 min was about 0.41 and 0.44 for control and Paclitaxel, respectively. Whereas, using Vinblastine or Nocodazole, the polymerization reaction was impeded (final OD350 of 0.19 and 0.28, respectively). The derivative **2** (used at the concentration of 10 \(\upmu\)M) was also able to inhibit the tubulin polymerization with an effectiveness similar to Nocodazole, but to a lesser extent with respect to Vinblastine, indeed the polymerization curve reached the plateau after 30 min at an OD350 1.4-fold reduced than the control reaction (OD350 = 0.35), with a final OD350 value of about 0.32. Thus, the derivative **2** resulted in a promising in vitro inhibitor of tubulin polymerization.