# Molecular Targets of Minor Cannabinoids in Breast Cancer: In Silico and In Vitro Studies
## 5 Conclusions  
Through in silico and in vitro assays, this study presents a comprehensive analysis of the potential therapeutic effects of the cannabinoids, described so far, for the treatment of hormone-dependent breast tumors. These compounds showed ability to modulate key targets responsible for tumor development, such as aromatase, ER, and AR, and the integrated activities implemented on them may together represent an advantage for treatment at different stages of the disease. In fact, the nine minor cannabinoids studied showed, in vitro, potential to act either as ER antagonists with inverse agonist properties, or as ER agonists. Moreover, despite the lack of significant aromatase inhibition, all cannabinoids may act as AR antagonists with inverse agonist action. Regarding their chemical characters, key features for ER antagonism like the chromene ring or detrimental substituents for aromatase inhibition, such as the carboxylic acid, were identified and can guide the selection of further investigations on minor cannabinoids. Moreover, by elucidating their molecular targets and mechanisms of action, this study opens up a prospective application of these compounds in other cancers and clinical conditions. From the best of our knowledge, this is the first study exploring the molecular targets of minor cannabinoids and, together with previous studies, it reinforces the importance and therapeutic potential of cannabinoids in breast cancer, paving the way for novel and alternative therapeutic approaches and highlighting the medicinal potential of _Cannabis_. **Supplementary Materials:** The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/ph17091245/s1](https://www.mdpi.com/article/10.3390/ph17091245/s1)]([https://www.mdpi.com/article/10.3390/ph17091245/s1](https://www.mdpi.com/article/10.3390/ph17091245/s1)), Figure S1. Chemical structures of the cannabinoids described in Table 2. Figure S3. Chemical structures of the cannabinoids described in Table 3. Figure S4. Molecular dynamics (MD) of ER\(\alpha\) complexed with CBC (A), CBCA (B), CBD (C), CBDA (D), CBDB (E), CBDM (F), CBDV (G), CBG (H), CBGVA (I), and CBN (J). Results at the beginning (t = 0 ps, green) and at the end (t = 10,200 ps, blue) of the simulation (left), RMSD plot (center) and Potential Energy plot (right) during the time of simulation. Figure S5. Effects of the nine minor cannabinoids, CBG (A), CBDV (B), CBN (C), CBDB (D), CBDA (E), CBDM (F), CBGVA (H) and CBC (I), on VM7 LucE2 cell viability. Cells were treated with the cannabinoids (1, 5 and 10 uM) in the absence (ER\(\alpha\) agonism) or presence (ER\(\alpha\) antagonism) of testosterone (T; 1 nM) or estradiol (E2; 91.8 pM) for 24h. Results are presented as mean \(\pm\) SEM of at least three independent experiments performed in triplicate. Statistically sig-nificant differences between control (cells not exposed to cannabinoids), set as 1, and cells not treated with T or E2 are expressed as * (\(p<0.05\)), ** (\(p<0.01\)) and *** (\(p<0.001\)), while the differences between control and cells treated with cannabinoids in the presence of T are expressed as # (\(p<0.05\)), #(\(p<0.01\)) and *** (\(p<0.001\)). Figure S6. Effects of the nine minor cannabinoids, CBG (A), CBDV (B), CBN (C), CBDB (D), CBDA (E), CBDM (F), CBCA (G), CBGVA (H) and CBC (I), on CHO-K1 cells viability. Cells were treated with the cannabinoids (1, 5 and 10 uM) in the absence (AR agonism) or presence (AR n-tagonism) of R1881 (0.1 nM) for 24h. Results are presented as mean \(\pm\) SEM of at least three inde-pendent experiments performed in triplicate. Statistically significant differences between control (cells not exposed to cannabinoids), set as 1, and cells not treated with R1881 are expressed as * (\(p<0.05\)), ** (\(p<0.01\)) and *** (\(p<0.001\)), and *** (\(p<0.001\)), while the differences between control and cells treated with cannabinoids in the presence of R1881 are expressed as $$$$ (\(p<0.001\)). **Author Contributions:** C.F.A.