Lunatina-1: Desvendando o seu mecanismo citotóxico em células de câncer de mama triplo negativo

Abstract

Lunatin-1 is a peptide isolated from the venom of the Peruvian scorpion Hadruroides lunatus that induces apoptosis in in vitro experiments on the human promyelocytic leukemia cell line HL-60 and causes cell death in human cancer cells MCF-7 and MDA-MB-231 through unknown mechanisms, thus far. Therefore, this study aimed to investigate the mechanism of cell death induced by Lunatin-1 in MDA-MB-231 cells, a basal-like breast cancer cell line, wich represents a triple-negative breast cancer with a high recurrence rate. A propidium iodide (PI) uptake kinetics assay measured by a Cytation 5 microplate reader showed that Lunatin-1 induces cell membrane permeability, evidenced by positive PI staining from the first minute of treatment (p < 0.05). Furthermore, Lunatin-1 was able to reduce Red CellMask staining, a plasma membrane-binding probe, after 5 minutes of treatment, indicating that Lunatin-1 targets the cell membrane of MDA-MB-231 cells. Scanning electron microscopy data demonstrated that Lunatin-1 induced damage to the cell membrane, with a reduction in microvilli from the first minutes of treatment and an increase in pore-like structures after 10 minutes. The quantification of microvilli was performed using Cell Profiler and CellPose software, which confirmed a significant reduction (p < 0.05) in microvilli after 10 minutes of treatment with Lunatin-1. The quantification of pore-like structures was performed using Fiji/ImageJ software, confirming a significant increase (p < 0.05) in the number of these structures, with no increase in their diameter: control (0.5% DMSO) = 41.8 ± 2.1 nm vs. Lunatin-1 (25 µM) = 42.3 ± 1.2 nm (p > 0.05). The results demonstrated that Lunatin-1 causes death in the MDA-MB-231 breast cancer cell line through mechanisms related to different forms of cell membrane disruption, being classified as a membrane-disrupting peptide (MDP). Lunatin-1 could be used as a prototype for conjugation with tumor marker proteins or tumor homing peptides to improve its selectivity for tumor cells, as it also induced cytotoxicity against the non-tumoral cell line HEK-293.