Anti-leukemic effect of menthol, a peppermint compound, on induction of apoptosis and autophagy

Background: Menthol, a natural compound in peppermint leaves, has several biological activities, including antioxidant, anti-inflammatory, antiviral, antibacterial and anticancer properties. This study revealed the anti-leukemic effects and its underlying mechanisms of the menthol related apoptosis signaling pathway and autophagy in both NB4 and Molt-4 leukemic cell lines. Methods: Both leukemic cells were treated with menthol in various concentration. Cell viability was assessed using MTT assay, whereas apoptosis and autophagy were analyzed by flow cytometry using Annexin V-FITC/PI and anti-LC3/FITC antibodies staining, respectively. Apoptotic and autophagic related gene and protein expression were detected using RT-qPCR and western blot analysis, respectively. Moreover, STITCH database was used to predicts the interaction between menthol and proposed proteins. Results: Menthol significantly decreased cell viability in NB4 and Molt-4 cell lines in dose dependent manner. In combination of menthol and daunorubicin, synergistic cytotoxic effects were observed in leukemic cells. However, there was a minimal effect found on normal, peripheral blood mononuclear cells (PBMCs). Moreover, menthol significantly induced apoptosis induction via upregulation of caspase-3, BAX, p53 and downregulation of MDM2 mRNA expression. Autophagy was also induced by menthol through upregulating ATG3 and downregulating mTOR mRNA expression. For protein expression, menthol significantly increased caspase-3 whereas decreased mTOR in both leukemic cells. Conclusions. These results suggest that menthol exhibits cytotoxic activities by inhibition of cell proliferation, induction of apoptosis and autophagy through activating the caspase cascade, altering BAX and p53/MDM2, and regulating autophagy via the ATG3/mTOR signaling pathway.