Antitumor Effects of Fucoidan Via Apoptotic and Autophagic Induction on HSC-3 Oral Squamous CellCarcinoma

Abstract

Objective: Many studies suggested that fucoidan has anticancer potential. The objective of the present study was to determine the cytotoxic effects and mechanism of cell death induced by fucoidan extracted from Fucus vesiculosus on HSC-3 oral squamous cell carcinoma.

Methods: HSC-3 cells were treated with 0, 100, 200, and 400 μg/mL of fucoidan. Cell viability was measured using MTT assay. Apoptosis and cell cycle were measured with a flow cytometry-based assay. Chromatin condensation and nuclear fragmentation were determined using Hoechst 33342 staining. Mitochondrial membrane potential (ΔΨm) was determined using the JC-1 kit. The apoptotic, anti-apoptotic, and autophagic markers study were done by western blot analysis.

Results: the viable cell number of treated HSC-3 cells was decreased. Moreover, treated cells were arrested in the G0/G1 phase. Annexin V/PI staining revealed that fucoidan could induce apoptosis in HSC-3 cells. Western blot analysis suggested the up-regulation of apoptotic markers including cleaved caspase-3, cleaved PARP, Bax, and autophagic markers including LC3-II and Beclin-1 but down-regulation of anti-apoptotic markers, Bcl-2. Fucoidan could disturb ΔΨm and induce chromatin condensation with nuclear fragmentation.

Conclusion: fucoidan has potential in anticancer properties against HSC-3 cells manifested by the induction of apoptosis, cell cycle arrest, and autophagy.<br />.

Keywords: Apoptosis; Autophagy; Cell cycle; Fucoidan; HSC-3 oral squamous cell carcinoma.

Figure 1

(A) Percent of cell viability of HSC-3 cell line after treatment with various concentrations of fucoidan for 24 h, after that, the IC50 value of fucoidan was calculated. (B) Cytotoxic effect of various concentrations of fucoidan on HSC-3 cells at different time points. Histogram is presented in mean ± SD.*p<0.05, ** p < 0.01, ***p < 0.001

Figure 2

Apoptotic Effects of Fucoidan in HSC-3 Cells were Observed by Using Flow Cytometry. (A-D) represent the results of cells incubated with 0, 100, 200, and 400 μg/ml of fucoidan, respectively. The numbers of AnnexinV-FITC and propidium iodide positive cells are shown in the scattered plot. (E) The percentage of cell populations in early and late apoptotic stages per total cells. *p < 0.05, ** p < 0.01, ***p < 0.001

Figure 3

Detection of Nuclear Damages in Treated HSC-3 Cells (A-D) represents the results of cells stained with Hoechst33342 and further observed under a fluorescence microscope after incubated with 0, 100, 200, and 400 μg/ml of fucoidan, respectively. Arrowheads display chromatin condensation and nuclear fragmentation. Scale bar = 50 μm

Figure 4

Effects of Fucoidan on Mitochondrial Membrane Potential and Apoptosis in Cultured HSC-3 Cells. (A) The red to green fluorescence intensity of JC-1 ratio was significantly decreased in the concentration-dependent manner (* p < 0.05, ** p < 0.01, ***p < 0.001) compared with the control group. (B-E) The observation under the fluorescence microscope revealed the decreased number of HSC-3 cells with red fluorescence in a concentration-dependent manner from 0-400 μg/ml of fucoidan. Scale bar = 50 μm

Figure 5

Cell Cycle Analyses of HSC-3 Cells Treated with Fucoidan were Observed by Using Flow Cytometry. The results suggested the distributions in various phases of the cell cycle of HSC-3 cells treated with various concentrations of fucoidan (A): control group; (B), (C) and (D): Fucoidan-treated groups at 100, 200, and 400 μg/ml, respectively. (E) Percentage of each phase are presented as mean ± SD.* p < 0.05

Figure 6

Western Blot Analysis of the Expression Levels of Apoptotic Related Proteins in HSC-3 Cells Treated with 0, 100, 200, and 400 μg/ml of Fucoidan for 48 h. (A) western blot analysis of Bax, Bcl-2, cleaved caspase-3, and cleaved PARP protein expressions. Relative expression levels of (B) Bax, (C) Bcl-2, (D) The ratio of Bax and Bcl-2, (E) cleaved caspase-3, and (F) cleaved PARP were analyzed and compared with the control group. * p< 0.05, ** p < 0.01, *** p < 0.001

Figure 7

Morphological Changes of Treated HSC-3 Cells were Observed Using a Phase-Contrast Microscope. (A) In control cells, a small number of vacuoles and granules (arrowheads) in the cytoplasm were observed. However, numerous vacuoles and granules were observed in the cytoplasm and cell surface of treated cells (B-D), and the quantities of those changes were increased in a concentration-dependent manner. (E) The levels of LC3-II and Beclin-1 expression were determined by using western blot. Relative expression levels of LC3-II (F) and Beclin-1 (G) were analyzed and compared with the control group. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar = 50 μm