# Antiproliferative Activity of Aminobenzylnaphthols Deriving from the Betti Reaction
## 3 Results and Discussion
### Membrane Integrity  
It is interesting to highlight that the Caco-2 and SH-SYSY cells showed a dose-dependent decrease in live cells with respect to the control after 24 h of exposure to molecule **1** and molecule **2** (200, 100, 50, and 25 uM). Linear trends of dose-dependent cytotoxic effects were observed. Concentrations of molecule **1** from 25 to 200 uM were associated with a reduction of about 80-56% and 83-64% in the number of live cells for Caco-2 and SH-SYSY, respectively. In the case of molecule **2**, a decrease in the number of live cells of about 79-52% and 77-35% for Caco-2 and SH-SYSY, respectively, was observed (Figure 4).  
After 72 h of exposure to **1** and **2** (200, 100, 50, and 25 uM) of Caco-2 and SH-SYSY, the results obtained with the MTT assay were confirmed (Figure 5).  
Concentrations of **1** ranging from 25 to 200 uM were associated with a significant reduction in the number of live cells of about 74-17% and 86-67% for Caco-2 and SH-SYSY, respectively, while the **2** treatments were associated with a signifi  
\begin{table}
\begin{tabular}{c c c c} \hline
**Cell Line** & **Cisplatin** & **1** & **2** \\ \hline Caco-2—24 h & 97 & 125 & 94 \\ Caco-2—72 h & 88 & 80 & 83 \\ SH-SYSY—24 h & 88 & 80 & 182 \\ SH-SYSY—72 h & 105 & 113 & 93 \\ \hline \end{tabular} Statistical analysis by ANOVA of cisplatin compared to compounds **1** and **2** for Caco-2 and SH-SYSY cell lines (\(p<0.05\)).  
\end{table}
Table 1: EC\({}_{50}\) values for cell viability of Caco-2 and SH-SYSY cell lines after treatments with cisplatin, molecule **1** and molecule **2** (200, 100, 50, and 25 uM).  
Figure 4: Cell viability of human Caco-2 adenocarcinoma and SH-SYSY neuroblastoma cells after 24 h of exposure to cisplatin, **1**, and **2** (200, 100, 50, and 25 uM), after exposure to Trypan blue for 2 min. The 100% control concerns the corresponding control sample.  
Colony-Forming Inhibition of Molecules **1** and **2** in Caco-2 Adenocarcinoma and SH-SYSY Neuroblastoma Cells  
Eventually, Caco-2 adenocarcinoma and SH-SYSY neuroblastoma cells were seeded and monitored to evaluate if molecules **1**, **2**, and cisplatin might inhibit colony-forming capacity and proliferation ability (colony size). The colonies were detected by Coomassieie blue staining as violet spots after 7- or 10-day treatments with the molecules under investigation.  
Pictures of randomly selected microscopic fields of each cell line are represented in Figures 8 and 9. Caco-2 and SH-SYSY controls had homogeneous and similar colony patterns. Decreasing concentrations of molecules **1**, **2**, and cisplatin (200, 100, 50, and 25 \(\upmu\)M) acted on colonies of Caco-2 and SH-SYSY with a dose-dependent reduction in size and colony number (\(p<0.05\)). In particular, the colony numbers of Caco-2 and SH-SYSY were reduced by 30% and 47%, respectively, when compared to the control at the highest treatment dose of 200 \(\upmu\)M of **1** (Figures 8 and 9). On the other hand, the same concentration of **2** caused a reduction of 50% and 60% in the colony numbers. In this respect, the colonies of SH-SYSY cells were more affected than those of Caco-2 cells (Figures 8 and 9).  
The first direction is the great ability of coordinating metal cations due to the combined action of the naphthod hydroxyl moieties with the amino residue. The coordinated cations could alter the molecular equilibrium [19], with a focus on the production of ROS (reactive oxygen species) that are connected in many chemotherapeutic actions.  
On the other hand, many papers [22; 25; 26; 29] seem to underline the ability of similar molecules to bind and alter the DNA or its action. This last mechanism is probably connected to the presence of heterocyclic moieties that are not present in our molecules.  
Thus, we are inclined to think that the anti-proliferative activity that we observed is connected to the first line of action that was reported.