# Simple Thalidomide Analogs in Melanoma: Synthesis and Biological Activity
## 3 Results and Discussion
### Biological Results
#### 3.2.2 Effect of Compound **2k** on Microtubule Dynamics and Cell Death by Apoptosis
concentration of 10 \(\upmu\)M. In this assay, we measured the turbidity at 350 nm for 3600 s at 37 \({}^{\circ}\)C to evaluate the tubulin assembly. The obtained results, shown in Figure 3, demonstrated that, in the CTRL reaction, tubulin heterodimers polymerize in a time-dependent manner, reaching a plateau after about 15 min with a final optical density value at 350 nm (OD350) of about 0.48. Instead, the exposure of tubulin to the microtubule-stabilizing agent, paclitaxel, caused an increase in tubulin polymerization, which reached a plateau after about 10 min with a final OD350 higher than CTRL (OD350 paclitaxel = 0.56). Contrarily, the microtubule-destabilizing agent vinblastine strongly blocked the tubulin polymerization and its curve reached the steady state only after 70 min with a very low final OD350 (0.19). Similarly, the exposure of tubulin to the compound **2k** exerted an important inhibitory activity on the protein polymerization, even if in a lesser extent if compared with vinblastine. Indeed, the **2k** curve reaches the plateau in about 25 min with a turbidity value 1.5-fold less than the control reaction (OD350 **2k** = 0.32). Taken together, these results suggest that compound **2k** perturbs microtubule dynamics due to its capability to inhibit tubulin polymerization. Due to the great involvement of microtubules in cellular growth, they represent a good target for cancer treatment. Indeed, the disorganization of microtubules can be responsible of cell cycle arrest, subsequently triggering the programmed cell death of tumoral cells [22, 23, 40, 41, 42]. In order to investigate if compound **2k** induced apoptosis, we performed a TUNEL assay on A2058 cells, exposed for 24 h to this compound, used at its IC\({}_{50}\) value. The obtained results indicate that compound **2k** induced apoptosis in these cells. Indeed, in the treated cells is notable a clear green nuclear fluorescence due to the formation of fragmented DNA, substrate of the terminal deoxynucleotidyl transferase (TdT) enzyme (Figure 4, Panel B, compound **2k**). Contrarily, green fluorescence is totally absent in the nuclei of the CTRL cells (vehicle-treated cells), indicating no DNA breakage (Figure 4, Panel B, CTRL). Figure 3: Tubulin polymerization assay. The in vitro tubulin polymerization was evaluated after tubulin incubation with compound **2k** used at the concentration of 10 \(\upmu\)M. Vinblastine and paclitaxel (10 \(\upmu\)M) were used as positive controls, while vehicle (DMSO) was used as a negative control. Tubulin polymerization was measured by determining the turbidity at 350 nm for 3600 s at 37 \({}^{\circ}\)C.  
The **2k**-induced apoptosis of melanoma A2058 cells was accompanied by Poly(ADPribose) polymerase-1 (PARP-1) cleavage. During the apoptosis process, cells exhibit several biochemical modifications, including the degradation of many proteins by caspases [43]. Among them, PARP-1 is converted from the 116-kDa form to a fragment of 89 kDa. PARP-1 is an eukaryotic protein that plays a vital role in DNA repair, replication, and differentiation, and is involved in the activation of cellular defense mechanisms against DNA damage [44]. During apoptotic death, caspases cause PARP-1 cleavage and inactivation, making it no longer able to repair DNA damage [45]. Thus, considering the importance of PARP-1 cleavage as a hallmark of apoptosis, we analyzed the status of this protein in A2058 cells treated with compound **2k** (used at its IC\({}_{50}\) value) at different times (48 h and 72 h), performing a Western blot analysis. Treatment with compound **2k** induced proteolytic cleavage of PARP-1.