Osteosarcoma (OS) has been demonstrated to be difficult to cure due to its potently malignant metastasis. Therefore, new therapeutic approaches blocking the metastatic potential of OS are urgently required to improve the outcomes for OS patients. In the present study, the anti-metastatic capacity of sea cucumber (
Fucoidan, a type of marine polysaccharide containing substantial percentages of L-fucose and sulfate ester groups, is an essential constituent of brown seaweed (
Osteosarcoma (OS) is the most primary malignant bone cancer commonly found in children and adolescents worldwide (
Sea cucumber is a widely used traditional Chinese medicine (TCM) with numerous healthy benefits (
Dry sea cucumber
The preparation of sea cucumber (
U2OS cells were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology. U2OS cells were routinely cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin, incubated in a 5% CO2 incubator at 37°C.
Cells (1×104) were seeded into each well of 96-well plates. After incubation at 37°C for 24 h, Cf-Fuc was administered to the cells at 50 and 100 µg/ml. After 24 h of incubation at 37°C, the medium was replaced with 100 µl fresh medium containing 0.5 mg/ml of MTT. Subsequently, the cells were incubated at 37°C for another 4 h, and 150 µl of DMSO was added after removal of the supernatant. The absorbance at 570 nm was determined using a microplate reader.
U2OS cells were detached by trypsin digestion and suspended in serum-free medium with or without Cf-Fuc (50 and 100 µg/ml) for 30 min. Then, the cells were seeded into a 96-well plate coated with fibronectin (10 µg/ml), and incubated for 0.5, 1, 2 or 4 h. After washing with PBS, the adherent cells were fixed with 4% paraformaldehyde at room temperature (RT) for 30 min, and stained with 0.5% crystal violet at RT for 15 min. The dye, extracted with 33% acetic acid after washing with PBS, was quantified using a microplate reader.
U2OS cells incubated with or without Cf-Fuc (50 and 100 µg/ml) at 37°C for 4 h were then placed in 24-well Transwell™ (Corning Costar, Corning, NY) filter inserts (8 µm pore diameter). DMEM containing 10% FBS as the chemoattractant was added in the lower well. After incubation for 12 h, the non-migrated U2OS cells inside the inserts were gently removed using cotton swab. The migrated cells were then fixed and stained with 0.1% crystal violet at RT for 15 min. Migrated cells were counted in five random fields under a light microscope at ×400 magnification.
U2OS cells were seeded into a CELLview cell culture dish (Greiner Bio-One) pre-coated with fibronectin (10 µg/ml), then incubated with or without Cf-Fuc (50 and 100 µg/ml) at 37°C for 4 h. U2OS cells were then placed in a 37°C heating chamber with CO2 supply. Images were captured at 5 min intervals with a CCD camera for 4 h. Image stacks were quantitatively analyzed and wind-rose plots of tracked migration paths of U2OS cells were plotted by NIH ImageJ software (version 1.30; National Institutes of Health).
Six-well plates were pre-coated with fibronectin (10 µg/ml) at 37°C overnight, and then U2OS cells were seeded into each well and incubated at 37°C for 2 h. Cells were further treated with or without Cf-Fuc (50 and 100 µg/ml) for 0.5, 1, 2 or 4 h. After being washed with PBS the cells were fixed and stained with rhodamine phalloidin (0.1% Triton X-100, 3.7% formaldehyde, and 2 µM rhodamine phalloidin in PBS) for 1 h. Cells were incubated with methanol to extract the dye and rhodamine fluorescence (excitation wavelength: 530 nm, emission wavelength: 590 nm) was further assessed with a fluorescence microplate reader (TECAN GENios; Tecan Austria GmbH). F-actin content at time 0 was considered as 1; F-actin content at 0.5, 1, 2 and 4 h are expressed as a relative fold change of mean fluorescence intensity at time 0.
Rac1 activation was determined according to a previous method (
Cells were harvested and lysed with RIPA lysis buffer. Cell lysates were collected and the concentration of proteins was quantified by BCA protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein (30 µg of total protein per lane) were subjected to 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 3% BSA for 2 h at room temperature and then incubated with indicated primary antibodies (1:1,000 dilution) at 4°C overnight. HRP-conjugated secondary antibody (Beyotime Institute of Biotechnology) was incubated at RT for another 1 h after washing with TBST three times. Protein bands were subsequently visualized using ECL Western blotting chemiluminescent detection reagent. The band intensities for quantitative analysis were measured by densitometric analysis using NIH ImageJ software (version 1.30; National Institutes of Health).
Data were expressed as the mean ± SD. Significant differences were determined by one-way ANOVA, followed by Bonferroni post hoc test using GraphPad Prism 6 (GraphPad Software, Inc.). P-values <0.05 were considered to indicate a statistically significant difference.
As revealed in
As revealed in
First, the inhibitory effect of Cf-Fuc on U2OS cell migration was evaluated by Transwell assay. Cf-Fuc caused strong inhibition on U2OS cell migration, leading to a significant decrease in the number of migrated cells compared with the control group (
Cytoskeleton remodeling is a critical event involved in cancer metastasis (
The effect of Cf-Fuc on cell adhesion signaling of U2OS cells was further examined. As revealed in
Small GTPase Rac1 plays a crucial role in the remodeling of the actin cytoskeleton. Whether Cf-Fuc could inhibit Rac1 activation in U2OS cells was subsequently examined. As revealed in
The Rac1/PAK1/LIMK1/cofilin signaling axis could regulate the assembly of the actin cytoskeleton and has been revealed to be involved in cancer metastasis (
Osteosarcoma (OS), a primary malignancy of bone, is prone to early metastasis (
Growing evidence reveals that polysaccharides from marine resources possess extensive health benefits, such as anti-metastatic ability (
As an essential cell adhesion molecule in OS cells, integrin plays important roles in mediating OS metastasis (
Cancer metastasis is associated with increased motility characteristics, such as an extensively cross-linked actin network to form pseudopods, which requires appropriate cytoskeleton remodeling (
In summary, Cf-Fuc significantly inhibited OS cell adhesion and migration, reduced F-actin formation, and downregulated adhesion signaling via suppression of the phosphorylation of FAK and paxillin. Cf-Fuc also impaired Rac1 activation and the PAK1/LIMK1/cofilin signaling axis for reorganization of the actin cytoskeleton, providing the potential anti-metastatic mechanism of Cf-Fuc.
Not applicable.
No funding was received.
The datasets used during the present study are available from the corresponding author upon reasonable request.
MZ, LC and YL designed and performed the experiments, analyzed the data and wrote the manuscript. MC and SZ also performed the experiments. DK designed, interpreted and funded the study, and also wrote the manuscript. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Cytotoxic effect of Cf-Fuc on U2OS cells. Data are expressed as the mean ± SD. *P<0.05 vs. the control group. Cf-Fuc,
Cf-Fuc inhibits U2OS cell adhesion. U2OS cells were seeded into 96-well plates pre-coated with fibronectin to assess adhesion at indicated time-points (0.5, 1, 2 and 4 h). The adherent cells were fixed and then stained with crystal violet. The dye was quantified using a microplate reader. Cf-Fuc,
Transwell assay. U2OS cells incubated with or without Cf-Fuc were placed in Transwell inserts. The migrated cells were then fixed and stained with crystal violet. Migrated cells were counted in five random fields. Representative images are presented and data are expressed as the mean ± SD. *P<0.05, **P<0.01 vs. the control group. Cf-Fuc,
Effect of Cf-Fuc on U2OS cell migration tracks demonstrated by wind-rose plots. Cf-Fuc,
Effect of Cf-Fuc on the velocity of U2OS cells. ***P<0.001 vs. the control group. Cf-Fuc,
Effect of Cf-Fuc on actin polymerization in U2OS cells. U2OS cells were seeded into 24-well plates pre-coated with fibronectin, and then treated with or without Cf-Fuc. F-actin was calculated by measuring the fluorescence of extracted rhodamine phalloidin with a fluorescence microplate reader. Bar graphs indicate the changes in F-actin content expressed as a relative fold change in the mean fluorescence intensity of the cells at time 0. Data are presented as the mean ± SD. Cf-Fuc,
Cf-Fuc inhibits the phosphorylation of FAK and paxillin. (A) U2OS cells were seeded into 6-well plates pre-coated with fibronectin and allowed to adhere for 4 h. The total and phosphorylated proteins were detected by western blotting. The blots for GAPDH demonstrated the equal loading. The quantitative analysis of (B) p-FAK/total FAK and (C) p-paxillin/total paxillin were determined by ImageJ software. Data are presented as the mean ± SD. **P<0.01, ***P<0.001 vs. the control. Cf-Fuc,
Effect of Cf-Fuc on Rac1 activation in U2OS cells. (A) Rac1 activation assay. (B) Quantitative analysis of Rac1 activation. Data are presented as the mean ± SD. ***P<0.001 vs. the control group. Cf-Fuc,
Cf-Fuc impacts the PAK1/LIMK1/cofilin signaling axis in U2OS cells. (A) The total and phosphorylated PAK1, LIMK1, and cofilin were determined by immunoblotting. The blots for GAPDH demonstrated the equal loading. (B) Quantitative analysis was determined by ImageJ. Data are presented as the mean ± SD. *P<0.05, **P<0.01, ***P<0.001 vs. the control group. Cf-Fuc,