Transient receptor potential melastatin 6 and transient receptor potential melastatin 6/7 antagonists suppress colon adenocarcinoma HT-29 cells

Abstract

BACKGROUND Magnesium (Mg2+) plays a fundamental role in numerous cellular processes, including enzymatic reactions, DNA replication, oxidative stress response, and cytoskeletal dynamics. In fact, dysregulation of Mg2+ homeostasis has been increasingly associated with the development and progression of cancer, particularly colorectal cancer (CRC). Transient receptor potential melastatin (TRPM) channels, especially TRPM6 and TRPM7, are essential regulators of epithelial Mg2+ influx. While TRPM7 promotes CRC progression, the role of TRPM6 and TRPM6/ 7 channels remains unclear. AIM To investigate the role of membrane-localized TRPM6 and TRPM6/7 channels in Mg2+ influx, spheroid (SP) formation, stemness, and migration. METHODS We used parental and SP-derived HT-29 cells at comparable passages as in vitro models. Mass spectrometry confirmed full-length sequences, phosphorylation, and methionine oxidation of TRPM6 and TRPM7. Mg2+ influx, total and free Mg2+ levels were measured by fluorescence imaging and biochemical assays. TRPM6 / TRPM7 expression and markers were analyzed by western blot. Func-tional assays, including secondary SP formation and wound healing, assessed stemness and migration. Cells were treated with Mg2+ transport inhibitors: Co(III)hexamine, 2-aminoethyl diphenylborinate (TRPM6/7 blocker), and Mesendogen (TRPM6 inhibitor). RESULTS The expression of membrane-bound TRPM6, TRPM7, and TRPM6/7 was significantly higher in SP cells than in parental cells. Mass spectrometric analysis confirmed the presence of full-length TRPM6 and TRPM7 with increased phosphorylation and oxidation in SP cells. Enhanced Mg2+ influx and total intracellular Mg2+ levels were observed in SP cells. Free ionized intracellular Mg2+ levels remained comparable across all experimental groups. Pharmacological inhibition of TRPM6 and TRPM6/7 significantly reduced Mg2+ influx, decreased total Mg2+ content, compromised CRC SP stability, abolished cancer stem-like properties, impaired cell migration, and downregulated pro-tumorigenic markers, including Nanog, cyclooxygenase-2, and matrix metalloproteinase-9. CONCLUSION Membrane-localized TRPM6 and TRPM6/7 channels regulate Mg2+ influx and promote CRC stemness, SP stability, and migration, highlighting their potential as therapeutic targets to inhibit CRC progression and metastasis.