Efeito apoptótico do fenilpropanoide benzoato de (E)-4-(1-epoxi-7,8-propenil) fenila (CV2) em linhagem de célula de câncer de mama humano (MCF-7) e toxicidade em modelo de peixe zebra

Abstract

Despite considerable advances in diagnosis and treatment, cancer remains the second leading cause of death worldwide. Chemotherapy is one of the most widely used approaches in disease management; however, despite its effectiveness in suppressing tumor growth, it often causes significant side effects, motivating the search for safer and more effective alternatives. In this context, compounds derived from natural products stand out, including phenylpropanoids, which have been reported as potential therapeutic agents for cancer. (E)-4-(1-epoxy-7,8-propenyl) phenyl benzoate (CV2) is a novel phenylpropanoid isolated from the roots of Croton velutinus (Euphorbiaceae). Preliminary studies have demonstrated its potent anti-inflammatory, trypanocidal, and cytotoxic effects on human tumor cell lines. This study aimed to investigate the antitumor and toxicological effects of CV2. The cytotoxicity of CV2 was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay in human tumor cells (MCF-7, MDA-MB-231, SK-MEL-28, HeLa, and HCT-116) and non-tumor human cells (MCF-10A and PBMC). Additionally, toxicity was assessed in Danio rerio (zebrafish) embryos. The results showed that CV2 induced greater cytotoxicity in the human breast adenocarcinoma cell line (MCF-7), with a half-maximal inhibitory concentration (IC₅₀) of 4.63 ± 0.16 μM. Cytotoxicity was also analyzed in PBMCs (IC₅₀ = 1.75 ± 0.06 μM) and in the non-tumor human breast cell line (MCF-10A, IC₅₀ = 44.09 ± 1.00 μM), demonstrating nine times greater selectivity for the MCF-7 tumor cell line (SI, selectivity index: 9.69). Cell cycle analysis revealed that CV2 increased the percentage of cells in the sub-G1 phase (5 μM: 31.77 ± 4.93%; 10 μM: 11.29 ± 0.12%; p<0.05 for both). Furthermore, Annexin V-FITC/PI staining showed an increase in the percentage of apoptotic cells (5 μM: 50.17 ± 2.52%; 10 μM: 44.50 ± 2.37%; p<0.05 for both). CV2 (5 μM) also stimulated the production of reactive oxygen species (ROS). Pretreatment with N-acetyl-L-cysteine (NAC) prevented CV2 cytotoxicity (5 μM: 27.09 ± 1.02%; 10 μM: 13.90 ± 1.00%; p<0.05 for both, in the presence of NAC). Assays using specific antibody labeling indicated that CV2 (5 μM) increased the percentage of cells labeled for ERK1/2, JNK1/2, p38 MAPK, PKB/AKT, and NF-κB proteins. Moreover, pretreatment with JNK and p38 MAPK inhibitors prevented CV2-induced cytotoxicity. In the zebrafish model, the lethal concentration that killed half of the embryos was determined (LC₅₀ = 3.07 μM), and no non-lethal effects were observed in the embryos. In summary, CV2 demonstrated an in vitro antitumor effect by inducing apoptosis, increasing oxidative stress, and modulating intracellular signaling pathways, including MAPKs, PKB/AKT, and NF-κB. Additionally, it exhibited embryotoxicity in the zebrafish model, highlighting it as a promising antitumor compound for future therapeutic investigations.