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Cited by 167Seaweed-derived Bioactive CompoundsCell death mechanisms and regulationNatural product bioactivities and synthesis

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Fucoidan Extract Induces Apoptosis in MCF-7 Cells via a Mechanism Involving the ROS-Dependent JNK Activation and Mitochondria-Mediated Pathways

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Zhang, Zhongyuan, Teruya, Kiichiro, Eto, Hiroshi, Shirahata, Sanetaka·2011·PLoS ONE
DOI 10.1371/journal.pone.0027441PMC PMC3214060
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Abstract

BACKGROUND: Fucoidan extract (FE), an enzymatically digested compound with a low molecular weight, is extracted from brown seaweed. As a natural compound with various actions, FE is attractive, especially in Asian countries, for improving the therapeutic efficacy and safety of cancer treatment. The present study was carried out to investigate the anti-tumor properties of FE in human carcinoma cells and further examine the underlying mechanisms of its activities. METHODOLOGY/PRINCIPAL FINDING: FE inhibits the growth of MCF-7, MDA-MB-231, HeLa, and HT1080 cells. FE-mediated apoptosis in MCF-7 cancer cells is accompanied by DNA fragmentation, nuclear condensation, and phosphatidylserine exposure. FE induces mitochondrial membrane permeabilization (MMP) through loss of mitochondrial membrane potential (ΔΨm) and regulation of the expression of Bcl-2 family members. Release of apoptosis-inducing factor (AIF) and cytochrome c precedes MMP. AIF release causes DNA fragmentation, the final stage of apoptosis, via a caspase-independent mitochondrial pathway. Additionally, FE was found to induce phosphorylation of c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK) 1/2, and apoptosis was found to be attenuated by inhibition of JNK. Furthermore, FE-mediated apoptosis was found to involve the generation of reactive oxygen species (ROS), which are responsible for the decrease of ΔΨm and phosphorylation of JNK, p38, and ERK1/2 kinases. CONCLUSIONS/SIGNIFICANCE: These data suggest that FE activates a caspase-independent apoptotic pathway in MCF-7 cancer cells through activation of ROS-mediated MAP kinases and regulation of the Bcl-2 family protein-mediated mitochondrial pathway. They also provide evidence that FE deserves further investigation as a natural anticancer and cancer preventive agent.

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Figure 1 Effect of FE on the growth of cancer cell lines. Cell proliferation was analyzed using the MTT assay. Cells were treated with 82, 410, or 820 µg/mL FE or with PBS as the vehicle control for different time periods (n = 6). Results represent the means ± SD from three independent experiments.
Figure 1 Effect of FE on the growth of cancer cell lines. Cell proliferation was analyzed using the MTT assay. Cells were treated with 82, 410, or 820 µg/mL FE or with PBS as the vehicle control for different time periods (n = 6). Results represent the means ± SD from three independent experiments.CC BY · PubMed Central
Figure 2 FE induces apoptosis in MCF-7 cancer cells. (A and B) Sub-G1 cell population in control and FE-treated (820 µg/mL) MCF-7 cells was measured by flow cytometry as described in the methods section. The cell distribution was analyzed by flow cytometry analysis software. (C) Hoechst 33342 staining of MCF-7 cells treated with or without 820 µg/mL FE for 96 h. Each experiment shown is representative of 20 random fields observed. (D) Analysis of apoptotic cells by annexin/PI double staining using the IN Cell Analyzer 1000. MCF-7 cells were treated for different time periods with or without 820 µg/mL FE. Apoptosis was evaluated by determining the percentage of annexin V-positive cells. (E) Annexin/PI double staining of MCF-7 cells pretreated with 10 µM Z-VAD-fmk or 2 mM NAC. After a 1-h incubation, cells were exposed to 820 µg/mL FE for the indicated time periods. Cells grown in a medium containing 1 mg/mL L-fucose without FE served as a sugar control. Each experiment shown is representative of 20 random fields observed. All of the experiments were performed at least in triplicate. All results were obtained from three independent experiments. A significant difference between treatment and control groups is indicated by p<0.05(*) or p<0.01(**).
Figure 2 FE induces apoptosis in MCF-7 cancer cells. (A and B) Sub-G1 cell population in control and FE-treated (820 µg/mL) MCF-7 cells was measured by flow cytometry as described in the methods section. The cell distribution was analyzed by flow cytometry analysis software. (C) Hoechst 33342 staining of MCF-7 cells treated with or without 820 µg/mL FE for 96 h. Each experiment shown is representative of 20 random fields observed. (D) Analysis of apoptotic cells by annexin/PI double staining using the IN Cell Analyzer 1000. MCF-7 cells were treated for different time periods with or without 820 µg/mL FE. Apoptosis was evaluated by determining the percentage of annexin V-positive cells. (E) Annexin/PI double staining of MCF-7 cells pretreated with 10 µM Z-VAD-fmk or 2 mM NAC. After a 1-h incubation, cells were exposed to 820 µg/mL FE for the indicated time periods. Cells grown in a medium containing 1 mg/mL L-fucose without FE served as a sugar control. Each experiment shown is representative of 20 random fields observed. All of the experiments were performed at least in triplicate. All results were obtained from three independent experiments. A significant difference between treatment and control groups is indicated by p<0.05(*) or p<0.01(**).CC BY · PubMed Central
Figure 3 Caspase-independent apoptosis is induced by FE. (A) MCF-7 cells were treated with or without 820 µg/mL FE for different time periods. Cells treated for 6 h with 5 nM staurosporine (STS) served as a positive control. Cleavage of PARP, Bid, and caspase family members was detected using western blotting. (B) Effects of caspase inhibitors on apoptosis induced by FE. MCF-7 cells were pretreated with various caspase inhibitors at 10 µM for 1 h followed by incubation with 820 µg/mL FE for 48 h. The apoptotic cells were evaluated by annexin/PI double staining using the IN Cell Analyzer 1000. All results were obtained from 3 independent experiments.
Figure 3 Caspase-independent apoptosis is induced by FE. (A) MCF-7 cells were treated with or without 820 µg/mL FE for different time periods. Cells treated for 6 h with 5 nM staurosporine (STS) served as a positive control. Cleavage of PARP, Bid, and caspase family members was detected using western blotting. (B) Effects of caspase inhibitors on apoptosis induced by FE. MCF-7 cells were pretreated with various caspase inhibitors at 10 µM for 1 h followed by incubation with 820 µg/mL FE for 48 h. The apoptotic cells were evaluated by annexin/PI double staining using the IN Cell Analyzer 1000. All results were obtained from 3 independent experiments.CC BY · PubMed Central
Figure 4 FE induces loss of ΔΨm and regulation of Bcl-2 family proteins expression. (A and B) MCF-7 cells were treated with or without 820 µg/mL FE for the indicated time periods. Disruption of ΔΨm was observed by Rh123/PI double staining and analyzed by the IN Cell Analyzer 1000. The percentage of cells retaining Rh123 in each treatment group was compared with the untreated control. (C) MCF-7 cells were treated with or without 820 µg/mL FE for different time periods. Bcl-2, Bcl-xl, Bax, and Bad were determined by western blotting using specific antibodies. (D) A densitometric analysis was used to quantify the levels of Bax and Bcl-2 to evaluate the effect of FE on the Bax/Bcl-2 ratio. (E) Bcl-2 levels in MCF-7/Vec and MCF-7/Bcl-2 cells were detected by Western blotting. MCF-7/Vec and MCF-7/Bcl-2 cells were treated with or without 820 µg/mL FE for 48 h and analyzed by Annexin V/PI assay. All of the experiments were performed at least in triplicate. All results were obtained from three independent experiments. A significant difference between treatment and control groups is indicated by p<0.05(*) or p<0.01(**).
Figure 4 FE induces loss of ΔΨm and regulation of Bcl-2 family proteins expression. (A and B) MCF-7 cells were treated with or without 820 µg/mL FE for the indicated time periods. Disruption of ΔΨm was observed by Rh123/PI double staining and analyzed by the IN Cell Analyzer 1000. The percentage of cells retaining Rh123 in each treatment group was compared with the untreated control. (C) MCF-7 cells were treated with or without 820 µg/mL FE for different time periods. Bcl-2, Bcl-xl, Bax, and Bad were determined by western blotting using specific antibodies. (D) A densitometric analysis was used to quantify the levels of Bax and Bcl-2 to evaluate the effect of FE on the Bax/Bcl-2 ratio. (E) Bcl-2 levels in MCF-7/Vec and MCF-7/Bcl-2 cells were detected by Western blotting. MCF-7/Vec and MCF-7/Bcl-2 cells were treated with or without 820 µg/mL FE for 48 h and analyzed by Annexin V/PI assay. All of the experiments were performed at least in triplicate. All results were obtained from three independent experiments. A significant difference between treatment and control groups is indicated by p<0.05(*) or p<0.01(**).CC BY · PubMed Central
Figure 5 Translocation of mitochondrial apoptotic factors is induced by FE. (A) Time-dependent release of AIF from mitochondria into the cytosol was measured by western blotting analysis. (B) FE induces translocation of AIF from the mitochondria to the nucleus. MCF-7 cells were treated with 820 µg/mL FE for 96 h. After incubation with 100 nM MitoTracker Red (red fluorescence), the cells were fixed and permeabilized followed by immunostaining with an antibody specific for AIF (green fluorescence) and counterstaining with Hoechst 33342 (blue fluorescence) to visualize nuclear morphology. (C) Time-dependent release of cytochrome c from mitochondria into the cytosol was measured by western blotting analysis. Similar results were obtained from 3 independent experiments. Each image shown is representative of 20 random fields observed.
Figure 5 Translocation of mitochondrial apoptotic factors is induced by FE. (A) Time-dependent release of AIF from mitochondria into the cytosol was measured by western blotting analysis. (B) FE induces translocation of AIF from the mitochondria to the nucleus. MCF-7 cells were treated with 820 µg/mL FE for 96 h. After incubation with 100 nM MitoTracker Red (red fluorescence), the cells were fixed and permeabilized followed by immunostaining with an antibody specific for AIF (green fluorescence) and counterstaining with Hoechst 33342 (blue fluorescence) to visualize nuclear morphology. (C) Time-dependent release of cytochrome c from mitochondria into the cytosol was measured by western blotting analysis. Similar results were obtained from 3 independent experiments. Each image shown is representative of 20 random fields observed.CC BY · PubMed Central
Figure 6 Roles of MAPKs in FE-induced apoptosis. (A) MCF-7 cells were treated with 820 µg/mL FE for the indicated time periods. JNK, p38, and ERK1/2 MAPKs and their phosphorylated forms were determined by western blotting using specific antibodies. (B) Effects of MAPK inhibitors on apoptosis induced by FE. MCF-7 cells were pretreated with 10 µM SP600125, SB203580, or PD98059 for 1 h and then exposed to 820 µg/mL FE for 48 h (controls were not exposed to FE). After the treatment, annexin V-PI double staining was used to analyze apoptotic cell death using the IN Cell Analyzer 1000. All results were obtained from 3 independent experiments. Differences with p<0.05(*) or p<0.01(**) are considered statistically significant. (C) Cells were treated for 1 h with or without 10 µM SP600125 followed by 820 µg/mL FE for 6 h. Phosphorylated forms of JNK were determined by western blotting using a specific antibody.
Figure 6 Roles of MAPKs in FE-induced apoptosis. (A) MCF-7 cells were treated with 820 µg/mL FE for the indicated time periods. JNK, p38, and ERK1/2 MAPKs and their phosphorylated forms were determined by western blotting using specific antibodies. (B) Effects of MAPK inhibitors on apoptosis induced by FE. MCF-7 cells were pretreated with 10 µM SP600125, SB203580, or PD98059 for 1 h and then exposed to 820 µg/mL FE for 48 h (controls were not exposed to FE). After the treatment, annexin V-PI double staining was used to analyze apoptotic cell death using the IN Cell Analyzer 1000. All results were obtained from 3 independent experiments. Differences with p<0.05(*) or p<0.01(**) are considered statistically significant. (C) Cells were treated for 1 h with or without 10 µM SP600125 followed by 820 µg/mL FE for 6 h. Phosphorylated forms of JNK were determined by western blotting using a specific antibody.CC BY · PubMed Central
Figure 7 Effect of ROS on FE-induced apoptosis. (A and B) Cells were pretreated for 1 h with or without 2 mM NAC followed by 820 µg/mL FE for 15–360 min. The cells were labeled with 5 µM DCFH-DA, and the fluorescence was measured using the IN Cell Analyzer 1000 as explained in the methods section. (C) Cells were treated with 820 µg/mL FE for 48 h after incubation with 2 mM NAC for 1 h and then the apoptotic cells were analyzed using the IN Cell Analyzer 1000. (D) MCF-7 cells were treated with 2 mM NAC for 1 h before treatment with 820 µg/mL FE for 6 h. Phosphorylation of JNK, p38, and ERK1/2 MAPKs was detected by western blotting. (E) Cells were pretreated with 2 mM NAC and 10 µM SP600125 for 1 h and then incubated with 820 µg/mL FE for 48 h. Disruption of ΔΨm was detected by Rh123 staining by using the IN Cell Analyzer 1000. All results were obtained from 3 independent experiments. Differences with p<0.05(*) or p<0.01(**) are considered statistically significant.
Figure 7 Effect of ROS on FE-induced apoptosis. (A and B) Cells were pretreated for 1 h with or without 2 mM NAC followed by 820 µg/mL FE for 15–360 min. The cells were labeled with 5 µM DCFH-DA, and the fluorescence was measured using the IN Cell Analyzer 1000 as explained in the methods section. (C) Cells were treated with 820 µg/mL FE for 48 h after incubation with 2 mM NAC for 1 h and then the apoptotic cells were analyzed using the IN Cell Analyzer 1000. (D) MCF-7 cells were treated with 2 mM NAC for 1 h before treatment with 820 µg/mL FE for 6 h. Phosphorylation of JNK, p38, and ERK1/2 MAPKs was detected by western blotting. (E) Cells were pretreated with 2 mM NAC and 10 µM SP600125 for 1 h and then incubated with 820 µg/mL FE for 48 h. Disruption of ΔΨm was detected by Rh123 staining by using the IN Cell Analyzer 1000. All results were obtained from 3 independent experiments. Differences with p<0.05(*) or p<0.01(**) are considered statistically significant.CC BY · PubMed Central
Figure 8 Effect of FE on the cellular metabolism. (A) Cells were treated with glucose deprivation, 820 µg/mL FE, or 820 µg/mL FE in combination with glucose deprivation. The ATP concentration was analyzed with the Bioluminescence assay Kit. Reactions were carried out in triplicate and ATP concentrations were expressed as percentages of untreated controls. (B) Cells were treated with glucose deprivation, 820 µg/mL FE, or 820 µg/mL FE in combination with glucose deprivation. Annexin V-PI double staining was used to analyze apoptotic cell death using the IN Cell Analyzer 1000.
Figure 8 Effect of FE on the cellular metabolism. (A) Cells were treated with glucose deprivation, 820 µg/mL FE, or 820 µg/mL FE in combination with glucose deprivation. The ATP concentration was analyzed with the Bioluminescence assay Kit. Reactions were carried out in triplicate and ATP concentrations were expressed as percentages of untreated controls. (B) Cells were treated with glucose deprivation, 820 µg/mL FE, or 820 µg/mL FE in combination with glucose deprivation. Annexin V-PI double staining was used to analyze apoptotic cell death using the IN Cell Analyzer 1000.CC BY · PubMed Central
Figure 9 A model summarizing the mechanism of FE-induced apoptotic cell death in MCF-7 breast cancer cells.
Figure 9 A model summarizing the mechanism of FE-induced apoptotic cell death in MCF-7 breast cancer cells.CC BY · PubMed Central

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Metadata

Authors
Zhang, Z., Teruya, K., Eto, H., & Shirahata, S.
Affiliations
  • Kyushu University (JP)
  • Daiichi-Sankyo (Japan) (JP)
Corresponding author
Teruya, Kiichiro
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2011-11-11
Volume
6
Issue
11
Pages
e27441
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0027441
PMC
PMC3214060
OpenAlex
W2129739698
Cited by
167
References
59
Citations by year
2026: 5 · 2025: 8 · 2024: 8 · 2023: 14 · 2022: 12 · 2021: 10 · 2020: 9 · 2019: 15 · 2018: 13 · 2017: 10 · 2016: 12 · 2015: 13 · 2014: 16 · 2013: 14 · 2012: 8
FWCI
18.42
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Japan Society for the Promotion of Science (23580172)
Topics
  • Life Sciences › Agricultural and Biological Sciences › Aquatic Science › Seaweed-derived Bioactive Compounds
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
  • Physical Sciences › Chemistry › Organic Chemistry › Natural product bioactivities and synthesis
Keywords
fucoidan, apoptosis, MCF-7 cells, breast cancer, reactive oxygen species, JNK activation, MAPK signaling, mitochondrial membrane permeabilization, mitochondrial membrane potential, Bcl-2 family proteins, apoptosis-inducing factor, cytochrome c release, caspase-independent apoptosis, p38 MAPK, ERK1/2, anticancer natural compounds, brown seaweed extract
MeSH terms
Enzyme Activation, Humans, Mitochondria, Polysaccharides, Signal Transduction, Apoptosis, Reactive Oxygen Species, Cell Line, Tumor, JNK Mitogen-Activated Protein Kinases

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