Triple-negative breast cancer (TNBC) presents poor prognosis, early recurrence, limited therapeutic options, and high drug resistance, calling for the search of molecules that may serve as new drugs, drug adjuvants, or scaffolds for drug development. Preussin is a naturally derived compound that reduces cell viability and proliferation and promotes cell death and cell cycle arrest in 2D cultured cell lines. In this study, we investigated the effects of preussin isolated from the marine sponge-associated fungi Aspergillus candidus on the TNBC cell line MDA-MB-231, comparing 2D and 3D cell culture models. Three-dimensional cultures typically better simulate tumour behaviour in vivo and were generated here using ultra-low adhesion U bottom plates. Preussin at 25 µM or higher decreased cell viability in 2D (72 h exposure) and 3D cultured cells (96 h exposure), as assessed with MTT assay. These effects align with the observed decrease in cell proliferation (above 25 µM of preussin) in both culture models, evaluated with BrdU assay, as well as the increase in late apoptosis and necrosis at 35 µM for 2D culture and over 25 µM in 3D culture, as measured via flow cytometry using Annexin V-PI staining. In agreement with this, apoptosis and necrosis characteristics were observed in the cells' ultrastructure after exposure to preussin. With standard and enzymatic versions of the comet assay, we excluded the genotoxic effects of preussin at 25 or 35 µM after 2 h and 24 h of exposure. In an in vitro scratch assay, preussin decreased cell migration in the MDA-MB-231 cell line at a starting concentration of 5 µM. This study sheds light on the mechanisms underlying preussin's effects on a TNBC cell line, supporting its value as an antiproliferative, cytotoxic, and antimigratory agent in a dose-dependent manner in both culture models.

FCT funding UIDB/04423/2020 and UIDP/04423/2020.