Characterization and anti-tumor activity of saponin-rich fractions of South Korean sea cucumbers (Apostichopus japonicus)

Abstract

In this study, the saponin-rich fractions of five individual (two Red and three Black) sea cucumbers (Apostichopus japonicus) in South Korea were investigated for their antiproliferative effect against HL-60, B16F10, MCF-7, and Hep3B tumor cell lines. The red sea cucumber saponin-rich fraction (SSC) from Jeju Island (JRe) decreased the growth of HL-60 with an IC₅₀ value of 23.55 ± 3.40 μg/mL, which represented the strongest anticancer activity among the extracts. Further, SSC downregulated B-cell lymphoma extra-large (Bcl-xL), while upregulating, to different degrees, Bcl-2-associated X protein (Bax), caspase-9, caspase-3, PARP cleavage, and apoptotic bodies in cancer cells. Evidence for SSC inducing apoptosis via the mitochondria-mediated pathway was found. The contents of SSCs were determined using ultra high-performance liquid chromatography coupled with a quadrupole orbitrap mass spectrometry to comparatively evaluate the regional influence. In West Sea, the total SSC content of A. japonicus was 15.5 mg/g, representing the highest content, while A. japonicus in the South Sea yielded the lowest content at 8 mg/g. The major saponin constituent in SSC was identified as Holotoxin A₁, which may the anti-tumor compound in A. japonicus.

Keywords: Anticancer activity; Apostichopus japonicus; Holotoxin A1; Saponin.

Fig. 1

Collection and extraction of sea cucumbers Apostichopus japonicus. a Harvesting regions, approximate sizes, and weights of A. japonicus. b Preparation of saponin-rich fraction from A. japonicus (SSC)

Fig. 2

Representative total ion current chromatograms (TICs) of SSCs obtained from a JRe and b WBa, and the fragmentation pattern of the [M-H]⁻ ion of Holotoxin A₁ (m/z 1391.6) c in UHPLC/Q-Orbitrap MS

Fig. 3

Percentage of viable cells as a measure of cell proliferation after treatment with different concentrations of five SSCs. a Vero cells, b HL-60, c B16F10, d Hep3B and e MCF-7 cells. Experiments were performed in triplicate (n = 3) and the data are expressed as mean ± SE. *p < 0.05, **p < 0.001

Fig. 4

Effects of JRe and WBa on the induction of apoptosis in HL-60 cells. a Flow cytometry analysis of JRe and WBa on the nuclear morphology in HL-60 cancer cell. JRe and WBa were treated with 12.5, 25, 50 μg/mL concentrations to HL-60 cells for 24 h. Cells were stained with 7-AAD and Annexin V. b Fluorescent microscopic evaluation of the effects of JRe and WBa on the nuclear morphology in HL-60 cancer cell. SSC treatment was performed using 12.5, 25 and 50 μg/mL sample concentrations. Observations were made 24 h after treatment by the acridine orange/ethidium bromide double-staining method. c Western blot analyses of JRe induced HL-60 cells for measuring the expression of Bax, Bcl-xL, cleaved PARP, caspase-9, and cleaved caspase-3. Experiments were performed in triplicate (n = 3) and the data are expressed as mean ± SE. *p < 0.05, **p < 0.001. Results are reproducible based on three independent determinations

Fig. 4

Effects of JRe and WBa on the induction of apoptosis in HL-60 cells. a Flow cytometry analysis of JRe and WBa on the nuclear morphology in HL-60 cancer cell. JRe and WBa were treated with 12.5, 25, 50 μg/mL concentrations to HL-60 cells for 24 h. Cells were stained with 7-AAD and Annexin V. b Fluorescent microscopic evaluation of the effects of JRe and WBa on the nuclear morphology in HL-60 cancer cell. SSC treatment was performed using 12.5, 25 and 50 μg/mL sample concentrations. Observations were made 24 h after treatment by the acridine orange/ethidium bromide double-staining method. c Western blot analyses of JRe induced HL-60 cells for measuring the expression of Bax, Bcl-xL, cleaved PARP, caspase-9, and cleaved caspase-3. Experiments were performed in triplicate (n = 3) and the data are expressed as mean ± SE. *p < 0.05, **p < 0.001. Results are reproducible based on three independent determinations