Targeting Ion Channel Dysregulation in Colorectal Cancer: Identification of Clofilium as a Potent Inhibitor of Tumor Growth and Metastatic Potential

Background: Colorectal cancer (CRC) remains a leading cause of cancer mortality and a promising target for emerging “ionoceutical” therapies that modulate cancer channelopathies. Ion channels are key regulators of tumor bioelectric signaling, yet their biomarker and therapeutic potential in CRC are only beginning to emerge from preclinical and transcriptomic studies. Methods: Public RNA‑seq datasets from primary CRC samples and COLO 205 cells were compared with normal colon tissue to identify shared upregulation of ion channel genes. Guided by these findings, 19 ion‑modulating compounds targeting dysregulated channels were screened in COLO 205 reporter lines. Top hits were evaluated for effects on proliferation and cytotoxicity in COLO 205 (p53 wild‑type), HT‑29 (p53 mutant), and Caco‑2 (p53 null) cells, with cell‑cycle distribution analyzed in COLO 205. Activity, selectivity, and in vivo efficacy were assessed using 2D and 3D COLO 205 spheroid assays, heterotypic COLO 205–endothelial–fibroblast intravasation spheroids, non‑cancerous toxicity assays in normal colon epithelial, endothelial, and colon fibroblast cells, and in vivo COLO 205 xenografts. Mechanism was probed using immunoblotting and RNA‑seq. Results: Nine compounds inhibited CRC cell proliferation primarily through cytostatic mechanisms (G0/G1 accumulation). Most agents were less active in 3D spheroids than in 2D; however, the potassium‑channel, Na+/K+-ATPase pump, and NMDA receptor antagonist clofilium had greatly enhanced efficacy, impaired migration and invasion, and showed the highest selectivity (IC50 = 3.317 µM in COLO 205; therapeutic index = 47 vs normal colon epithelial cells). Clofilium induced acute depolarization followed by sustained hyperpolarization, caused broad transcriptional reprogramming, ER stress, cell‑cycle arrest, and innate immunity pathway activation, with immunoblotting confirming inhibition of AKT, JNK, p38, and NF‑κB and induction of NDRG1, senescence, autophagy, and caspase‑3 cleavage. In heterotypic spheroids, clofilium reduced intravasation and exerted anti‑angiogenic effects, and in COLO 205 xenografts daily treatment reduced tumor volume by 37% with minimal systemic toxicity. Conclusions: Clofilium demonstrates potent, selective anti‑CRC activity and supports ion channel targeting as a viable “ionoceutical” strategy for CRC.