Phormidolide D, a novel cytotoxic cyanobacterial macrolide: Assessing its effects on the extracellular matrix, apoptosis, and angiogenesis.

Marine cyanobacteria are a rich source of structurally diverse secondary metabolites and have inspired at least four anticancer drugs currently on the market, highlighting their potential for the discovery of new therapeutic candidates. This study aimed to discover and elucidate the structure of a novel halogenated polyketide from marine cyanobacteria and to characterize its cytotoxic and mechanistic profile. Phormidolide D (1) was discovered through bioactivity-guided isolation, and its structure was elucidated using NMR and HR-MS/MS analyses. Cytotoxicity was evaluated in SH-SY5Y, HepG2, HCT 116, and hCMEC/D3 cell lines. Its biological activity was further characterized in 2D and 3D HCT 116 models by assessing spheroid morphology, extracellular matrix-related mRNA expression, combination effects with staurosporine, cell cycle distribution and apoptosis. In vivo anti-angiogenic activity was evaluated in transgenic zebrafish (Tg(fli1a:EGFP) embryos. Phormidolide D (1) exhibited potent cytotoxicity against the human colon carcinoma cell line HCT 116 in both monolayer and spheroid cultures (IC50 = 0.83 μM and 30.80 μM, respectively), whereas phormidolide A (2) was inactive. Phormidolide D (1) affected the 3D spheroid architecture, as demonstrated by fluorescence imaging. Its effects on the extracellular matrix were further observed by increased matrix metalloproteinase 1 mRNA expression and reduced collagen type I transcripts. Co-treatment of a phormidolide rich fraction with sub-effective concentrations of staurosporine reduced spheroid viability, with Bliss analysis confirming a synergistic interaction. Treatment with phormidolide D (1) increased sub-G1 cell population and early apoptotic cells. Furthermore, transgenic zebrafish embryos exposed to phormidolide D (1) had reduced vascularization, suggesting further studies for anti-angiogenic potential. These findings highlight phormidolide D (1), a structurally unique marine natural product distinguished by an unprecedented bromomethylene ketone, as a promising lead for anticancer drug development.