Cytotoxic Mechanism of Sphaerodactylomelol, an Uncommon Bromoditerpene Isolated from Sphaerococcus coronopifolius

Abstract

Marine natural products have exhibited uncommon chemical structures with relevant antitumor properties highlighting their potential to inspire the development of new anticancer agents. The goal of this work was to study the antitumor activities of the brominated diterpene sphaerodactylomelol, a rare example of the dactylomelane family. Cytotoxicity (10-100 µM; 24 h) was evaluated on tumor cells (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, SK-ML-28) and the effects estimated by MTT assay. Hydrogen peroxide (H₂O₂) levels and apoptosis biomarkers (membrane translocation of phosphatidylserine, depolarization of mitochondrial membrane potential, Caspase-9 activity, and DNA condensation and/or fragmentation) were studied in the breast adenocarcinoma cellular model (MCF-7) and its genotoxicity on mouse fibroblasts (L929). Sphaerodactylomelol displayed an IC₅₀ range between 33.04 and 89.41 µM without selective activity for a specific tumor tissue. The cells' viability decrease was accompanied by an increase on H₂O₂ production, a depolarization of mitochondrial membrane potential and an increase of Caspase-9 activity and DNA fragmentation. However, the DNA damage studies in L929 non-malignant cell line suggested that this compound is not genotoxic for normal fibroblasts. Overall, the results suggest that the cytotoxicity of sphaerodactylomelol seems to be mediated by an increase of H₂O₂ levels and downstream apoptosis.

Keywords: DNA damage; MCF-7 cells; apoptosis; biological activities; breast cancer; marine natural products; oxidative stress; red algae.

Figure 1

Chemical structure of sphaerodactylomelol.

Figure 2

Production of H₂O₂ by MCF-7 cells treated with (A) sphaerodactylomelol (48 µM) and (B) H₂O₂ (200 µM) for 1, 3, and 6 h. MCF-7 H₂O₂ levels following 1, 3, and 6 h treatment with sphaerodactylomelol and H₂O₂ (C). Values represent mean ± SEM of at least three independent experiments carried out in triplicate. Symbols represent significant differences (p < 0.05) when compared to: * control.

Figure 3

Mitochondrial membrane potential (MMP) of MCF-7 cells after treatment with sphaerodactylomelol (48 µM) and FCCP (2.5 µM) plus oligomycin A (1 µg/mL) for 15, 30, and 60 min. The results were expressed as the ratio between the monomers/aggregates of JC-1. Values represent the mean ± SEM of three or four independent experiments. Symbols represent significant differences (p < 0.05) when compared to: * control.

Figure 4

Sphaerodactylomelol effects on MCF-7 cells apoptosis biomarkers (A) externalization of phosphatidylserine, (B) Caspase-9 activity, and (C) DNA condensation and fragmentation. Values represent the mean ± SEM of three or four independent experiments. Symbols represent significant differences (p < 0.05) when compared to: * control. Images of 4′,6-diamidino-2-phenylindole (DAPI) stained cells were obtained using inverted fluorescence microscope at ×400. Arrows indicate alterations in DNA as comparing with control. The images are representative of one well of each situation tested.

Figure 5

Frequency of L929 fibroblasts DNA change following exposure to sphaerodactylomelol (48 µM) and ethyl methanesulfonate (EMS) for 3 h. Values represent the mean ± SEM of three independent experiments. Damage index: Σ (comet class: 1, 2, 3, 4). 0, nucleus without DNA damage. Symbols represent significant differences (p < 0.05) when compared to: * control; # EMS.