Cytotoxic Potential of the Coelomic Fluid Extracted from the Sea Cucumber Holothuria tubulosa against Triple-Negative MDA-MB231 Breast Cancer Cells

Abstract

Growing evidence has demonstrated that the extracts of different holothurian species exert beneficial effects on human health. Triple negative breast cancers (TNBC) are highly malignant tumors that present a poor prognosis due to the lack of effective targeted therapies. In the attempt to identify novel compounds that might counteract TNBC cell growth, we studied the effect of the exposure of the TNBC cell line MDA-MB231 to total and filtered aqueous extracts of the coelomic fluid obtained from the sea cucumber Holoturia tubulosa, a widespread species in the Mediterranean Sea. In particular, we examined cell viability and proliferative behaviour, cell cycle distribution, apoptosis, autophagy, and mitochondrial metabolic/cell redox state. The results obtained indicate that both total and fractionated extracts are potent inhibitors of TNBC cell viability and growth, acting through both an impairment of cell cycle progression and mitochondrial transmembrane potential and a stimulation of cellular autophagy, as demonstrated by the increase of the acidic vesicular organelles and of the intracellular protein markers beclin-1, and total LC3 and LC3-II upon early exposure to the preparations. Identification of the water-soluble bioactive component(s) present in the extract merit further investigation aiming to develop novel prevention and/or treatment agents efficacious against highly metastatic breast carcinomas.

Keywords: Holothuria tubulosa; autophagy; breast cancer; cell cycle; cell viability; coelomic fluid; mitochondrial function.

Figure 1

A specimen of Holoturia tubulosa sea cucumber.

Figure 2

Dose response effect of total extracts and 10K fractions on the viability and growth of MDA-MB231 cells after either 24 or 48 h of exposure. Error bars correspond to the standard error of the mean (s.e.m.) of three independent measurements. * p < 0.05.

Figure 3

Cell cycle distribution of MDA-MB-231 cells exposed to total extracts and 10K fractions for 24 (A) and 48 h (B), compared to control conditions. Error bars correspond to the standard error of the mean (s.e.m.) of three independent measurements. * p < 0.05.

Figure 4

Histograms showing the percentage of apoptotic (annexin V+/propidium iodide−) vs. non-apoptotic MDA-MB-231 cells cultured in control conditions or exposed to either total extracts or 10K fractions for 24 and 48 h. Error bars correspond to the standard error of the mean (s.e.m.) of three independent measurements. * p < 0.05.

Figure 5

Representative flow cytometric analyses of the transmembrane mitochondrial potential (MMP) in control (A,E), total extract- (C,G), and 10K fraction-treated (D,H) MDA-MB231 cells for 24 (A–D) and 48 h (E–H). Dot plots (B) and (F) show the parallel positive controls of valinomycin-treated cells. The values indicated in the bottom quadrants in each frame quantitate the percentage of low red-emitting cells that underwent dissipation of MMP. The results are expressed as mean ± s.e.m. of triplicate assays.

Figure 6

Representative plots showing reactive oxygen species (ROS) accumulation in gated alive MDA-MB231 cells exposed to total extracts (A,C) and 10K fractions (B,D) for 24 (A,C) and 48 h (B,D), compared to controls and H₂O₂-treated cells as positive controls. The x-axis reports the intensity of green fluorescence emitted by the dye 2’,7’-dichlorodihydrofluorescein, which is proportional to the amount of cytosolic ROS.

Figure 7

Representative flow cytometric analyses of control (A,D), total extract- (B,E), and 10K fraction-treated (C,F) MDA-MB231 cells stained with acridine orange for evaluation of acidic vesicular organelle (AVO) accumulation after 24 (A–C) and 48 h (D,F) of exposure. The percentage indicated in the top quadrants refers to the high red fluorescence-emitting events. The results are expressed as mean ± s.e.m. of triplicate assays.

Figure 8

Histograms showing the intensity of the immunofluorescence of beclin-1 (A), LC3-II (B), and total LC3 (C) in control and treated MDA-MB-231 cells after 24 and 48 h of exposure. Negative controls lack the incubation with the primary antibody. Error bars correspond to the standard error of the mean (s.e.m.) of three independent measurements. * p < 0.05.