Abstract
Tramadol, a widely used analgesic, has recently been explored for its potential anti-cancer effects. However, the antitumor dosage of tramadol is over its current clinical application. Its primary metabolite, O-desmethyltramadol, has greater μ-opioid receptor affinity and stronger pharmacological activity. Hence, we sought to examine whether the cytotoxic effect of O-desmethyltramadol was better than tramadol on breast cancer cells. Our results showed that O-desmethyltramadol significantly reduced cell viability in breast cancer cells, with IC50 values of 64.2 μg/mL (MDA-MB-231) and 96.7 μg/mL (MCF-7), demonstrating over ten-fold greater potency than tramadol. The presence of a μ-opioid receptor antagonist Alvimopan did not alter the cytotoxic effects of tramadol and O-desmethyltramadol, indicating a non-opioid receptor-mediated mechanism. Compared with antitumor activity of tramadol mediated through ER stress, we confirmed that O-desmethyltramadol induced ER stress proteins, including the p-eIF2α/eIF2α ratio, ATF4, and CHOP. In MDA-MB-231 cells, O-desmethyltramadol treatment elevated mRNA expression levels of ATF4, CHAC1, and DDIT3 by approximately 2-fold. In MCF-7 cells, the induction was even more pronounced, with ATF4 increased 1.7-fold, CHAC1 12-fold, and DDIT3 9-fold. Beyond the opioid receptor-mediated pathway, we further analyzed the differential functions of O-desmethyltramadol than tramadol using the RNA-seq analysis. The pathway enrichment analyses revealed that O-desmethyltramadol influenced immune and inflammatory pathways, such as TNF and IL-6/JAK/STAT3 signaling in MDA-MB-231 cells, while in MCF-7 cells, it affected metabolic and transcriptional pathways, including mTOR and MAPK signaling. Gene Set Enrichment Analysis further highlighted O-desmethyltramadol's role in interferon response and tumor microenvironment modulation. Four upregulated genes and five downregulated genes were modulated by O-desmethyltramadol in MDA-MB-231 and MCF-7 cells. Overall, our findings indicated that O-desmethyltramadol exerted potent anti-cancer effects through multiple non-opioid mechanisms, with distinct response from tramadol depending on breast cancer subtype. These findings not only highlight the therapeutic potential of O-desmethyltramadol as a novel adjunct in breast cancer treatment, but also emphasize the need for further investigation into its safety and clinical applicability in oncology.