Dose-Dependent Cytotoxic Effects of Menthol on Human Malignant Melanoma A-375 Cells: Correlation with TRPM8 Transcript Expression

Background: Transient receptor potential melastatin 8 (TRPM8), a principle membrane receptor involved in calcium ion influx and cell signal transduction, has been found to be up-regulated in some cancer types, including melanomas. Efficiency of menthol, an agonist of TRPM8, in killing melanoma cancer cells has been reported previously, but the mechanisms remain unclear. We here determined whether in vitro cytotoxic effects of menthol on A-375 human malignant melanoma cells might be related to TRPM8 transcript expression. Materials and Methods: The $PrestoBlue^{(R)}$ cell viability assay was used to assess the in vitro cytotoxic effect of menthol after 24h of treatment. RT-PCR was used to quantify TRPM8 transcript expression levels in normal and menthol-treated cells. Cell morphology was observed under inverted phase contrast light microscopy. Results: TRPM8 transcript expression was found at low levels in A-375 cells and down-regulated in a potentially dose-dependent manner by menthol. Menthol exerted in vitro cytotoxic effects on A-375 cells with an $IC_{50}$ value of 11.8 ${\mu}M$, which was at least as effective as 5-fluorouracil ($IC_{50}=120{\mu}M$), a commonly applied chemotherapeutic drug. Menthol showed no dose-dependent cytotoxicity on HeLa cells, a TRPM8 non-expressing cell line. Conclusions: The cytotoxic effects on A-375 cells caused by menthol might be related to reduction of the TRPM8 transcript level. This suggests that menthol might activate TRPM8 to increase cytosolic $Ca^{2+}$ levels, which leads to cytosolic $Ca^{2+}$ imbalance and triggers cell death.