O-Desmethyltramadol Enhanced Anti-Cancer Efficacy over Tramadol Through Non-μ-Opioid Receptor and Differential Cellular Contexts of Human Breast Cancer Cells

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, <i>O</i>-desmethyltramadol, has greater μ-opioid receptor affinity and stronger pharmacological activity. Hence, we sought to examine whether the cytotoxic effect of <i>O</i>-desmethyltramadol was better than tramadol on breast cancer cells. Our results showed that <i>O</i>-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 <i>O</i>-desmethyltramadol, indicating a non-opioid receptor-mediated mechanism. Compared with antitumor activity of tramadol mediated through ER stress, we confirmed that <i>O</i>-desmethyltramadol induced ER stress proteins, including the p-eIF2α/eIF2α ratio, ATF4, and CHOP. In MDA-MB-231 cells, <i>O</i>-desmethyltramadol treatment elevated mRNA expression levels of <i>ATF4</i>, <i>CHAC1</i>, and <i>DDIT3</i> by approximately 2-fold. In MCF-7 cells, the induction was even more pronounced, with <i>ATF4</i> increased 1.7-fold, <i>CHAC1</i> 12-fold, and <i>DDIT3</i> 9-fold. Beyond the opioid receptor-mediated pathway, we further analyzed the differential functions of <i>O</i>-desmethyltramadol than tramadol using the RNA-seq analysis. The pathway enrichment analyses revealed that <i>O</i>-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 <i>O</i>-desmethyltramadol's role in interferon response and tumor microenvironment modulation. Four upregulated genes and five downregulated genes were modulated by <i>O</i>-desmethyltramadol in MDA-MB-231 and MCF-7 cells. Overall, our findings indicated that <i>O</i>-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 <i>O</i>-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.