Efeito antitumoral de 22β-hidroxitingenona obtida de Salacia impressifolia (Miers) A.C. (Celastraceae) contra células de melanoma humano

Abstract

The search for new therapeutic options to melanoma remains one of the focus of scientific research around this type of cancer, including sources from natural products, such as 22β-hydroxytingenone, a quinonemethide triterpenes obtained from Salacia impressiofolia. The present thesis aimed to investigate the effect of the substance 22β-hydroxytingenone against melanoma cells. Initially, the effect of 22β-hydroxytingenone on properties related to tumor progression, such as proliferation, migration, cellular invasion and mechanisms of reprogramming of energy metabolism, through the enzymatic activity of lactate dehydrogenase (LDHA), was evaluated. Subsequently, the mechanism of cell death induced by 22-HTG was investigated through the analysis of morphological changes, following cell cycle, annexin V-FITC/propidium iodide assays (Annexin/PI), mitochondrial membrane potential, production of reactive oxygen species (ROS) by flow cytometry, and fluorescence microscopy with acridine orange/ ethidium bromide assay (AO/BE) staining. RT-qPCR was performed to evaluate the expression of BRAF, NRAS, and NRAS genes and docking mocelular was executed. The anti-invasiveness potential of 22-HTG was evaluated in a three-dimensional (3D) model of reconstructed human skin. With the results obtained, it was possible to identify the cytotoxic effect of 22-HTG against melanoma cells, with IC50 value 4.35, 3.72, and 3.29 μM after 24, 48, and 72 h of incubation, respectively. After, was defined the time of 24 h and the concentrations of 1,0, 2,0 and 2,5 µM were defined for the other tests, in which a reduction in cell viability, inhibition of new colony formation, reduction of LDHA gene expression, migration and invasion of melanoma cells, and decreased activity of metaloproteinases (MMP-2 and MMP-9), and LDHA. Regarding the investigation of cell death, treatment with 22-HTG caused morphological changes, as cell shrinkage, chromatin condensation, and nuclear fragmentation. Further investigation showed an increase in AO/BE-labelled cells and apoptosis was confirmed with Anexin/IP-labeling. Exposure to 22-HTG led to reduced mitochondrial membrane potential, which is not involved by increased oxidative stress. Using the 3D model of reconstructed human skin, 22-HTG reduced the ability of melanoma cells to invade the dermis, highlighting antimetastatic potential. Expression of BRAF, NRAS, and KRAS, important biomarkers in melanoma development, was reduced by 22-HTG treatment and was strong binding affinity with BRAF and NRAS in molecular docking, which may help explain the observed cellular events. According to the data from the thesis, it was possible to conclude that 22-HTG has anti-tumor effect on melanoma cells, with induction of apoptosis and reduction of events such as proliferation, migration, invasion, proteolytic action of MMPs. This study provide new insights for future work on investigating the utilization of 22-HTG in malignant melanoma treatment.