# Molecular Targets of Minor Cannabinoids in Breast Cancer: In Silico and In Vitro Studies
## 2 Results
### Effects of Cannabinoids in Estrogen Receptor (ER)  
For the evaluation of the effects of cannabinoids on ER activity, the VM7 Luc4E2 cell line was treated for 24 h with each phytocannabinoid (1-10 \(\upmu\)M) in the presence (ER antagonism) or absence (ER agonism) of E\({}_{2}\) (91.8 pM) or T (1 nM). Our results (Figure 7) demonstrated that, in the absence of E\({}_{2}\) or T, **CBN** (\(p<\) 0.05, \(p<\) 0.01, \(p<\) 0.001; Figure 7C), **CBDA** (\(p<\) 0.01; Figure 7E), **CBCA** (\(p<\) 0.05; \(p<\) 0.01; \(p<\) 0.001; Figure 7G), and **CBC** (\(p<\) 0.05; \(p<\) 0.01; Figure 7I) presented a negative effect for agonism, whereas **CBDV** (\(p<\) 0.001; Figure 7B), **CBDB** (\(p<\) 0.001; Figure 7D), and **CBDM** (\(p<\) 0.001; Figure 7F) presented a positive effect and thus exhibited ER agonist activity. Of note, **CBG** (Figure 7A) and **CBGVA** (Figure 7H) did not exert any significant effect on the activity of this receptor. A reduction in VM7 Luc4E2 cell viability for these conditions was observed for **CBDV** (\(p<\) 0.01; Supplementary Figure S5B), **CBN** (\(p<\) 0.001; Supplementary Figure S5C), **CBDB** (\(p<\) 0.01; \(p<\) 0.001; Supplementary Figure S5D), and **CBDM** (\(p<\) 0.05; \(p<\) 0.01; Supplementary Figure S5F). In the presence of E\({}_{2}\), for ER antagonism evaluation, a signifi \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Cannabinoid**} & \multirow{2}{*}{**Aromatase Inhibition (\%) \(\pm\) SEM**} & \multicolumn{2}{c}{**Binding Affinity–Aromatase (kcal/mol)**} \\ \cline{2-3}  & & \(8.9\pm 1.7\) & \(-6.0\) \\  & & \(3.0\pm 0.9\) & \(-6.4\) \\  & & \(83.2\pm 0.6\) & \(-8.7\) \\  & & \(3.4\pm 1.3\) & \(-5.7\) \\  & & \(6.4\pm 3.6\) & \(-6.9\) \\  & & \(4.5\pm 2.8\) & \(-7.3\) \\  & & \(12.0\pm 2.5\) & \(-7.6\) \\  & & \(3.6\pm 2.3\) & \(-6.1\) \\  & & \(7.8\pm 0.9\) & \(-5.8\) \\  & & \(3.8\pm 0.5\) & \(-5.9\) \\  & & \(98.8\pm 0.2\) & \(-8.2\) \\  & & \(99.4\pm 0.2\) & \(-7.3\) \\  & & \(98.4\pm 0.3\) & \(-8.2\) \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Percentages of anti-aromatase activity obtained for the cannabinoids under study. cant decrease in relation to the control value was only observed for 5 and 10 \(\upmu\)M of **CBN** (\(p<0.001\); Figure 7C), and for **CBDB** (\(p<0.001\); Figure 7D), and **CBC** (\(p<0.05\); Figure 7I), both at 10 \(\upmu\)M, suggesting that these compounds act as ER antagonists. For the remaining cannabinoids under study, no significant differences were observed (Figure 7). However, it is important to consider that at these conditions, contrary to **CBG** and **CBGVA**, a significant reduction (\(p<0.05\); \(p<0.01\); \(p<0.001\); Supplementary Figure S5) in cell viability was detected, which may influence the transactivation results. When the antagonism was addressed in the presence of T instead of E\({}_{2}\), different behaviors were observed. Regarding **CBDV** (\(p<0.01\); \(p<0.001\); Figure 7B), **CBDB** (\(p<0.001\); Figure 7D), and **CBDM** (\(p<0.05\); \(p<0.01\); \(p<0.001\); Figure 7F), no effects were noted for the antagonism over T. On the other hand, **CBN** at 10 \(\upmu\)M (\(p<0.001\); Figure 7C), **CBDA** at 1 and 10 \(\upmu\)M (\(p<0.05\); \(p<0.001\); Figure 7E), and **CBC** at 10 \(\upmu\)M (\(p<0.05\); Figure 7I) acted as ER antagonists under these conditions. No significant effects were detected for **CBG** (Figure 7A), **CBCA** (Figure 7G), and **CBC** (Figure 7H). Nevertheless, **CBDV** (\(p<0.05\); \(p<0.001\); Supplementary Figure S5B), **CBN** (\(p<0.001\); Supplementary Figure S5C), **CBDB** (\(p<0.01\); \(p<0.001\); Supplementary Figure S5D), **CBDM** (\(p<0.05\); \(p<0.01\); \(p<0.001\); Supplementary Figure S5F), and **CBC** (\(p<0.05\); \(p<0.001\); Supplementary Figure S5I) reduced cell viability under these conditions. As previously reported by our group, **CBD** acts as an ER antagonist, with inverse agonist properties [44].