Background
Despite the success of endocrine therapy, resistance development demands the discovery of novel therapeutic strategies for estrogen receptor-positive (ER+) breast cancer (BC). Several studies revealed that cannabinoids exert crucial anti-cancer effects in these tumors. In fact, we showed that Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) decrease ER+ BC cell viability, modulating aromatase and ERs. Considering this and the fact that Cannabis has several minor phytocannabinoids whose therapeutic effects are still unknown, the actions of cannabigerol (CBG), cannabinol (CBN) and cannabichromene (CBC) in sensitive and resistant ER+ BC models were explored.

Methods
The effects of CBG, CBN, and CBC on the viability of sensitive and resistant ER+ BC cells were evaluated for 3 and 6 days in MCF-7aro and LTEDaro cells, respectively. In MCF-7aro cells, apoptosis was assessed by caspase-7 activity and PARP levels, via luminescent and Western blot (WB) assays, respectively. Cell cycle progression was studied by flow cytometry and the effects on aromatase, ERα, and androgen receptor (AR) by WB and qPCR analysis.

Results
The three cannabinoids reduced MCF-7aro cell viability, promoting apoptosis and cell cycle arrest at G2/M phase, with CBN being the most effective. All the cannabinoids reduced aromatase and ERα expression, but only CBN and CBC decreased the transcription of estrogen-responsive genes. Only CBG reduced AR protein levels. Regarding the effects on LTEDaro cells, all the cannabinoids decreased cell viability and CBN, once again, induced the most pronounced effects.

Conclusions
This study reveals that minor phytocannabinoids modulate key targets responsible for ER+ BC progression, highlighting their importance as potential anti-cancer drugs with multi-target action for ER+ BC. Moreover, it suggests that cannabinoids may also be effective in cases where resistance to endocrine therapy was already developed, which may represent a breakthrough in the development of novel anti-cancer therapies.