Abstract
Redox homeostasis is crucial for cancer cell survival and resistance to therapy. The transcription factor NRF2, a master regulator of antioxidant and metabolic genes, is often upregulated in tumors to mitigate oxidative stress. Although NRF2 stability is canonically governed by KEAP1-CUL3-proteasome degradation, emerging evidence implicates lysosomal and autophagic pathways in non-canonical NRF2 turnover. The mechanisms by which these alternative pathways are engaged during chronic oxidative signaling remain unclear. We investigated whether sustained activation of the redox-sensitive ion channel TRPA1 by cannabidiol (CBD) disrupts redox homeostasis and promotes NRF2 degradation in colorectal cancer models. Using five independent CRC cell lines (RKO, HCT116, HT29, SW480, and MC38), we assessed reactive oxygen species (ROS), mitochondrial function, autophagy, and NRF2 protein dynamics through biochemical assays, lysosomal fractionation, and imaging. Xenograft models were used for in vivo validation. Chronic TRPA1 activation induced a biphasic ROS response, characterized by an early increase linked to mitochondrial Ca(2+) influx and a delayed ROS surge associated with mitochondrial dysfunction. This oxidative trajectory initially stabilized but subsequently led to its degradation after 24 h via a KEAP1-independent, autophagy-lysosome pathway. Proteasome inhibition failed to rescue NRF2, whereas bafilomycin A1 restored its levels and blocked co-localization with lysosomal markers (e.g., LAMP2A). Importantly, CBD-induced TRPA1 activation sensitized CRC cells to oxaliplatin, triggering apoptotic-not senescent-cell death. These effects were dose-dependent and consistent across all tested cell lines. Our findings reveal a non-canonical bioelectric-lysosomal axis that links TRPA1 activity to NRF2 destabilization in colorectal cancer. This work expands the understanding of NRF2 proteostasis under sustained oxidative stress and highlights TRPA1 as a tractable redox-modulating target for overcoming chemoresistance.