Biphasic effect of the dietary phytochemical linalool on angiogenesis and metastasis

Cytotoxic chemotherapy dominates the field of cancer treatment. Consequently, anticancer phytochemicals are largely screened on the basis of their cytotoxicity towards cancer cells which are achieved at higher doses, leading to various toxic side effects. Some phytochemicals also showed pro-carcinogenic effects at certain doses. The concept of hormesis has taught us to look into biphasic responses of phytochemicals in a more systematic way. Interestingly, the monoterpenoid alcohol, linalool, also has been reported to display both anti-oxidant and pro-oxidant properties, which prompted us to explore a probable biphasic effect on cancer cells. Cytotoxicity of various concentrations of linalool (0.1-4 mM) was tested on B16F10 murine melanoma cell line, and two sub-lethal concentrations (0.4 and 0.8 mM) were selected for further experiments. 0.4 mM linalool inhibited angiogenesis and metastasis, while 0.8 mM increased them. Similarly, B16F10 cell migration, invasion, and epithelial-mesenchymal transition markers also showed inhibition and induction with lower and higher linalool concentrations, respectively. Chorioallantoic membrane assay, scratch wound assay, and Boyden's chamber were used to analyze angiogenesis and metastasis. Expression of molecular markers such as vascular endothelial growth factor (VEGF) and its receptor phosphorylated VEGF receptor II (p-VEGFRII or p-Flk-1), Hypoxia-inducible factor-1 α (HIF-1α), E-cadherin, and vimentin were detected using Western blot, ELISA, PCR, qPCR, and immunofluorescence. Finally, ChIP assay was performed to evaluate HIF-1α association with VEGF promoter. Interestingly, measurement of intracellular reactive oxygen species at the selected concentrations of linalool using DCFDA in a flow cytometer showed that the phytochemical induced significant amount of ROS at 0.8 mM. This work sheds light on bimodal dose-response relationship exhibited by dietary phytochemicals like linalool, and it should be taken into consideration to elicit a desirable therapeutic effect.