Damnacanthal exerts anti-cancer effects in breast cancer cells via NAG-1 upregulation, CRM1 downregulation, and induction of autophagy

Breast cancer continues to be a major cause of cancer-related deaths worldwide, emphasizing the urgent need for innovative treatments. This study investigates the anti-cancer potential of damnacanthal in breast cancer cell lines BT-20 and MCF-7, highlighting its regulatory effects on key biomarkers NAG-1 (Nonsteroidal Anti-inflammatory Drug-Activated Gene-1) and CRM1 (Chromosome Region Maintenance 1). NAG-1 is recognized for its pro-apoptotic and anti-tumorigenic roles, while CRM1 is associated with oncogenic activity. We found that damnacanthal treatment significantly increased NAG-1 expression and decreased CRM1 expression in a dose- and time-dependent manner. Functional assays demonstrated that damnacanthal inhibited cell proliferation, reduced colony formation, and decreased tumor spheroid size. Mechanistically, luciferase reporter assays revealed that damnacanthal activates the −133 to +41 region of the NAG-1 promoter through C/EBP-β. Real-time PCR and Western blotting analyses confirmed that NAG-1 is upregulated transcriptionally, whereas CRM1 is downregulated post-translationally via enhanced protein degradation, as evidenced by cycloheximide chase assays. Molecular docking suggested direct binding of damnacanthal to CRM1, potentially explaining its reduced stability. Additionally, damnacanthal induced autophagy by upregulating NAG-1, as indicated by increased LC3-II expression and autophagic flux, further confirmed by immunocytochemistry. These findings suggest that damnacanthal exerts potent anti-cancer effects by modulating NAG-1 and CRM1 expressions, inhibiting tumor cell growth, and inducing autophagy. This highlights its therapeutic potential as a novel agent for breast cancer treatment, warranting further clinical exploration.