The Marine Dinoflagellate Alexandrium minutum Activates a Mitophagic Pathway in Human Lung Cancer Cells

Abstract

Marine dinoflagellates are a valuable source of bioactive molecules. Many species produce cytotoxic compounds and some of these compounds have also been investigated for their anticancer potential. Here, we report the first investigation of the toxic dinoflagellate Alexandrium minutum as source of water-soluble compounds with antiproliferative activity against human lung cancer cells. A multi-step enrichment of the phenol⁻water extract yielded a bioactive fraction with specific antiproliferative effect (IC₅₀ = 0.4 µg·mL^-1) against the human lung adenocarcinoma cells (A549 cell line). Preliminary characterization of this material suggested the presence of glycoprotein with molecular weight above 20 kDa. Interestingly, this fraction did not exhibit any cytotoxicity against human normal lung fibroblasts (WI38). Differential gene expression analysis in A549 cancer cells suggested that the active fraction induces specific cell death, triggered by mitochondrial autophagy (mitophagy). In agreement with the cell viability results, gene expression data also showed that no mitophagic event was activated in normal cells WI38.

Keywords: Alexandrium minutum; glycoprotein; marine antiproliferative compounds; mitophagy.

Figure 1

(A) 2D-Diffusion Ordered Spectroscopy (DOSY) spectra recorded in D₂O at 600 MHz of Fraction 1B; and (B) Electrophoresis gel of Fractions 3B (active sample) and 4B (deglycosylated Fraction 3B sample).

Figure 2

Effect of Fractions 3A (<10 KDa) and 3B (>10 KDa) on cell viability of human lung adenocarcinoma cells of (A) A549 and human normal lung fibroblasts and (B) WI38. Values are reported as mean ± S.D. compared to controls (100% viability) of three independent experiments. Concentrations tested were 0.1, 1 and 10 µg·mL⁻¹ for 48 h.

Figure 3

Effect of Fraction 3B on the expression levels of target genes in: human lung adenocarcinoma cells (A549) (A); and human normal lung fibroblasts (WI38) (B). All experiments were performed with RNA extracted from three different biological replicates and error bars represent ±S.D. Statistical analyses on the results obtained in (A,B); (C) One-way ANOVA); (D) Sidak; and (E) student’s t-test.