Derivado do carvacrol com propriedades antitumorais via modulação de canais de sódio dependentes de voltagem

Abstract

In cancer pathology, ion channels play an important role in the regulation of cell proliferation, tumor initiation, and progression. Cancers with high expression levels of functional voltage-gated ion channels tend to have a higher metastatic capacity, like neuroblastomas. Natural products, plant derivatives, have stood out in the field of medicinal chemistry because of their intrinsic medicinal properties. An example is carvacrol which, in addition to modular ion channels, has antitumor activity. The natural product carvacrol was used as a base for the drug design of the semi-synthetic carvacrol derivatives compounds. This study proposed to investigate the anticancer potential of carvacrol derivatives, which has previously shown an inhibition effect on voltage-gated sodium channels subtype 1.7 (Nav1.7). The effect of the twelve compounds Derivatives of Carvacrol (DC) was evaluated through the Patch Clamp technique in which Nav1.7 currents (INav1.7) were obtained in CHO-hNav1.7 cells. Derivative of Carvacrol 6 (DC6) with better effect, presenting IC50 of 39.26 μM for blocking Nav1.7. The toxicity of DC6 was evaluated in nauplii of Artemia Salinas, and the behavioral toxicological evaluation was performed in Swiss mice using the Irwin test, the compound has shown low toxicity in both experimental models. The cytotoxic effect of DC6 was evaluated by the MTT reduction method in the neuroblastoma cell lines, SH-SY5Y, Neuro-2A, IMR-32M, and SH-EP, presenting IC50 values for the 24 h times of 23.36 μM, 26.29 μM, 0.35 μM, and 50 μM respectively. In normal cell lines, HUVEC and L929 the IC50 of the compound were higher than 100 μM in both cell types. In sub-toxic concentrations, DC6 presented antiproliferative activity in neuroblastomas. Cell migration assay was performed to verify the effect of DC6 on SH-SY5Y cell motility, in which the cell migration rate was reduced within 48 h. To understand the correlation between Nav1.7 blockade and the reduction of cell migration presented by DC6, migration in CHO-hNav1.7 cells was evaluated in the presence of DC6 and 300 nM of tetrodotoxin (TTX). The cell migration rate after 72h of incubation with 300 nM of TTX was 42.79% and for 8 μM of DC6, it was 34.59%. Suggesting that the decrease in cell migration observed with DC6 treatment occurs mainly via Nav1.7 blockade. To evaluate the mechanism of cell death, the induction of apoptosis test by flow cytometry in neuroblastomas was performed, in which DC6 promoted the induction of cell death in a concentration-dependent manner, via late apoptosis and necrosis. Morphological analysis by fluorescence microscopy was performed on the SH-SY5Y cell line, and after 24 h of incubation with DC6, expressive disorganization of the cytoskeletal filaments was observed, more specifically of the

actin filaments, showing an effect dependent on the concentration of DC6. In a three- dimensional model of in vitro metastasis, DC6 inhibited the formation of cellular

spheroids in the neuroblastoma cell line SH-SY5Y. Thus it has been shown that DC6 has a double effect when it comes to tumor modulation, as it presents, in sub-toxic concentrations, the ability to induce cell death and reduce the migration of tumor cells via Nav1.7 modulation, as well as showing low toxicity in non-carcinogenic in vivo and in vitro models.