ANALYSIS OF THE ANTITUMOR EFFECTS OF AROMADENDRENE SESQUITERPENE ON MURINE AND HUMAN MELANOMA CELLS AND EVALUATION OF THE PROTECTIVE EFFECT IN VIVO

Abstract

Melanoma is skin cancer that originates from mutations in melanocytes, cells present in the basal layer of the epidermis. Although it has a low incidence, this type of cancer has a high mortality rate due to its metastatic potential. Cell death is a fundamental process for the development and maintenance of cells. However, tumor cells may be resistant to this mechanism. The most studied types of cell death are apoptosis, necroptosis, necrosis, and autophagy. Several studies have shown the antitumor activity of natural compounds that induce cell death by apoptosis through the accumulation of reactive oxygen species. Aromadendrene is a sesquiterpene that has shown great promise for the treatment of some types of cancer. The main objective of this project is to evaluate the antitumor activity of aromadendren, derived from Eucalyptus globulus, in cells of murine melanoma B16F10-Nex2 and human melanoma A375. To evaluate the cytotoxic effect of aromadendrene, as well as the mechanism of action of this terpene in the presence of death inhibitors and reactive oxygen species inhibitors, cell viability assays will be performed with the exclusion dye Trypan Blue. Fluorescence microscopy assays on cells treated with the terpene and labeled with the fluorophores DHE (dihydroxyetide), TUNEL and PI (propidium iodide) will be performed to analyze increased levels of the superoxide anion, DNA fragmentation and plasma membrane permeabilization, respectively. To investigate the mechanism of action of aromadendren in the process associated with the formation of melanoma metastasis, as well as its ability to interfere with cell proliferation, colonization and invasion, cell migration and invasion assays will be performed, as well as clonogenic assays. The Western Blotting assay will be done to investigate the alteration in the expression of proteins related to the death process in cells treated as aromadendren. Terpene-induced morphological modifications will be evaluated by transmission electron microscopy. The in vivo protective effect of aromadendren will be evaluated using the metastatic model in C57BL/6 mice.