**4. Discussion**

EOs and some of their components exert antitumor activities against numerous cancer models, such as colon [28], lung and liver [29], by affecting multiple pathways [30–33]. Di Martile et al. [34] summarized studies showing the properties of EOs to induce in vitro and in vivo cell death in melanoma models. They also indicated the use of EOs in clinical trials with the reduction of the side effects in cancer patients. Moreover, frankincense, pine needle and geranium EOs are also able to suppress tumor progression through the regulation of the AMPK/mTOR pathway in breast cancer [35]. Several EOs also have chemopreventive properties [34]. In eukaryotic cells, EOs can cause depolarization of mitochondrial membranes and decrease their fluidity. In this way, EOs are able to induce severe damage in the mitochondria, leading to cell death. Bhakkiyalakshmi et al. demonstrated that carvacrol, a phenolic monoterpenoid, is able to cause cell death as apoptosis. This process is associated with the production of free radicals that cause the rapid consumption of the intracellular pool of antioxidants [36]. Another study by Arunasree et al. evaluated the mechanism of action of carvacrol in the MDA-MB-231 cell line. This monoterpenoid induced cell death via cytochrome C release after mitochondria permeabilization [37]. Moreover, we also demonstrated the antitumoral activity of Tea Tree Oil (TTO), with an apoptotic effect on melanoma cell lines [38]. In general, the cytotoxic effects of EOs in toto, or those of some of their components, such as monoterpenoids [39], are due to their lipophilicity, which allows them to cross membranes and destroy them [40,41].

Herein, the damage caused by *V. macrophylla* EO to the cell membranes of SKBR3 cells can be attributed to its main component, citral, which is a mixture of the two monoterpenes, neral and geranial. Citral is generally recognized as safe (GRAS) by the FDA, and is commonly used as a flavoring agen<sup>t</sup> in the EU. On the other hand, this compound is endowed with antimicrotubule [42] and chemopreventive properties [43]. Several studies have shown that citral is able to cause cell death to tumor cells and severely damage cytoskeletal structures. On the other hand, other components occurring in the *V. macrophylla* EO, including both major and minor constituents, may be involved in the overall synergistic effect normally displayed by the EO [26]. Moreover, as demonstrated by other authors [44], our results show that *V. macrophylla* EO significantly changes the cytoskeletal organization of SKBR3 cancer cells, suppressing actin cytoskeletal rearrangemen<sup>t</sup> and destroying the cell membrane thanks in part to its monoterpenoid activities. Furthermore, this EO significantly decreased the proliferation and migration of human SKBR3 cells in a concentration and time-dependent manner.
