Cytotoxic effects of menthol in lung cancer cell line, A549

Abstract

Menthol, a cooling monoterpene derived from a peppermint, is used as an essential oil for various usages. It also functions as an agonist of TRPM8 and TRPA1, which are nociceptors related to various diseases including a lung injury. However, the cytotoxic action of menthol is not fully understood. In the present study, we investigated the effect of menthol on the viability of a lung cancer epithelial cell line, A549. The cell viability was measured by using a WST-8 reagent and a trypan blue dye exclusion test. The apoptotic cells were detected by DNA fragmentation and annexin V binding. The expression of TRPM8 and TRPA1 were detected by using RT-PCR and western blotting. The intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured by using Ca²⁺-imaging system with fura-2. The activity of Ca²⁺-ATPase was determined by using a colorimetric assay based on the molybdenum blue method. The expression of TRPM8 and TRPA1 was detected at both levels of mRNAs and proteins in A549. Menthol (0.3 mM) elicited the increase of [Ca²⁺]_i which was inhibited by HC-030031, a TRPA1 antagonist but not AMTB, a TRPM8 antagonist. A high concentration of menthol (3 mM) induced [Ca²⁺]_i increases in all cells. These responses were not attenuated by the removal of extracellular Ca²⁺ and the treatment with HC-030031, but inhibited by thapsigargin, a SERCA inhibitor. The activity of Ca²⁺-ATPase was attenuated by menthol. Menthol (3 mM) induced non-apoptotic cell death within 1 hrs, which was not attenuated by BAPTA-AM, an intracellular Ca²⁺ chelator. Apoptosis occurred at 12 hrs after the application of menthol. These results suggest that menthol induces the cytotoxicity at relative higher concentration in A549. Although menthol evoked [Ca²⁺]_i increases dependent on intracellular stores, its cytotoxic effect is not related to the activation of TRPM8 and TRPA1 and the increase of [Ca²⁺]_i.