Ca2+-independent cytotoxicity of menthol in the A549 lung cancer cell line

Menthol evokes cooling sensations by activating the transient receptor potential melastatin 8 (TRPM8) channel, resulting in relaxing, anti-inflammatory, and analgesic effects when administered through inhalation and topical application. Although the toxicity of menthol is relatively low, the mechanism underlying menthol-induced cytotoxic effects remains unclear. Thus, this study aimed to investigate the cytotoxic effects of menthol in the A549 lung cancer cell line. Menthol induced increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in distinct modes depending on its concentration. A relatively low concentration (0.3 mM) of menthol activated transient receptor potential ankyrin 1 (TRPA1) despite the expression of TRPM8 in A549 cells. A higher concentration (3 mM) of menthol nonspecifically induced Ca²⁺ release from intracellular stores. Menthol inhibited Ca²⁺-ATPase in the organelle membrane. At 3 mM, menthol elicited necrotic cell death accompanied by morphological changes within 60 min. This cytotoxicity was not prevented by HC-030031 (a TRPA1 blocker) or BAPTA-AM (an intracellular Ca²⁺ chelator). Furthermore, the analysis of the cytotoxicity of monoterpene analogs of menthol revealed structure-related activity in menthol-induced cytotoxicity. These findings indicate that menthol-induced cytotoxic effects are concentration-dependent and may provide valuable insights into novel therapeutic strategies for lung cancer.