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Fucoidan inhibition of lung cancer in vivo and in vitro : role of the Smurf2-dependent ubiquitin proteasome pathway in TGFβ receptor degradation.

Hsu HY, Lin TY, Wu YC, Tsao SM, Hwang PA, Shih YW, Hsu J - Oncotarget (2014)

Bottom Line: Herein, using lung cancer cells, we found that fucoidan effectively reduces TGFRI and TGFRII protein levels in vivo and in vitro.To elucidate how fucoidan decreases TGFR proteins in lung cancer cells, we found that fucoidan enhances the ubiquitination proteasome pathway (UPP)-mediated degradation of TGFRs in A549 and CL1-5 cells.Mechanistically, fucoidan promotes Smurf2 and Smad7 to conjugate TGFRs, resulting in TGF degradation; however, Smurf2-shRNA abolishes fucoidan-enhanced UPP-mediated TGFR degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan. The Genomics Research Center, Academia Sinica, Taipei, Taiwan. Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, Taiwan. Contributed equally to this work.

ABSTRACT
Fucoidan, a polysaccharide extracted from brown seaweeds, reduces tumor cell proliferation. In this study, we demonstrate that fucoidan reduces tumor size in LLC1-xenograft male C57BL/6 mice. Moreover, we found that LLC1-bearing mice continuously fed fucoidan showed greater antitumor activity than mice with discontinuous feeding. Fucoidan inhibited the in vitro growth of lung cancer cells. Transforming growth factor β (TGFβ) receptors (TGFRs) play important roles in the regulation of proliferation and progression, and high TGFRI expression in lung cancer specimens is associated with a worse prognosis. Herein, using lung cancer cells, we found that fucoidan effectively reduces TGFRI and TGFRII protein levels in vivo and in vitro. Moreover, fucoidan reduces TGFR downstream signaling events, including those in Smad2/3 and non-Smad pathways: Akt, Erk1/2, and FAK phosphorylation. Furthermore, fucoidan suppresses lung cancer cell mobility upon TGFβ stimulation. To elucidate how fucoidan decreases TGFR proteins in lung cancer cells, we found that fucoidan enhances the ubiquitination proteasome pathway (UPP)-mediated degradation of TGFRs in A549 and CL1-5 cells. Mechanistically, fucoidan promotes Smurf2 and Smad7 to conjugate TGFRs, resulting in TGF degradation; however, Smurf2-shRNA abolishes fucoidan-enhanced UPP-mediated TGFR degradation. Our study is the first to identify a novel mechanism for the antitumor activity of fucoidan, namely decreasing tumor growth by modulating the TGFR/Smad7/Smurf2-dependent axis, leading to TGFR protein degradation and inhibition of lung cancer cell progression in vitro and in vivo. Our current findings indicate that fucoidan is a potential therapeutic agent or dietary supplementation for lung cancer, acting via the Smurf2-dependent ubiquitin degradation of TGFβ receptors.

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Fucoidan inhibits proliferation and decreases TGFR-mediated Smad and non-Smad pathway activity in lung cancer cellsFucoidan inhibits the proliferation of lung cancer cells (A) A549, CL1-5, and LLC1. The cells were treated with various doses of fucoidan (0–300 μg/ml) for 24, 48, and 72 h; the viability of the cells was determined using the MTT assay. Each group of fucoidan-treated samples was normalized against each untreated control. The data are representative of three separate experiments and are presented as the mean ± SD; error bars indicate SD. Significant differences are shown (*P < 0.05 and **P < 0.01, compared with the control group). (B) CL1-5 cells were treated with fucoidan (100 or 200 μg/ml) for 1 h. (C) CL1-5 cells were pre-treated with fucoidan (200 μg/ml) for 2 h and then treated with TGFβ (10 ng/ml) for 1 h. The treatments were followed by western blotting analyses of whole cell lysates to detect the expression of p-Smad2/3 (Ser423/425), p-Akt (Ser473), p-Erk1/2 (Thr202/Tyr 204). Smad2/3, Akt and Erk were used as internal controls for loading.
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Figure 3: Fucoidan inhibits proliferation and decreases TGFR-mediated Smad and non-Smad pathway activity in lung cancer cellsFucoidan inhibits the proliferation of lung cancer cells (A) A549, CL1-5, and LLC1. The cells were treated with various doses of fucoidan (0–300 μg/ml) for 24, 48, and 72 h; the viability of the cells was determined using the MTT assay. Each group of fucoidan-treated samples was normalized against each untreated control. The data are representative of three separate experiments and are presented as the mean ± SD; error bars indicate SD. Significant differences are shown (*P < 0.05 and **P < 0.01, compared with the control group). (B) CL1-5 cells were treated with fucoidan (100 or 200 μg/ml) for 1 h. (C) CL1-5 cells were pre-treated with fucoidan (200 μg/ml) for 2 h and then treated with TGFβ (10 ng/ml) for 1 h. The treatments were followed by western blotting analyses of whole cell lysates to detect the expression of p-Smad2/3 (Ser423/425), p-Akt (Ser473), p-Erk1/2 (Thr202/Tyr 204). Smad2/3, Akt and Erk were used as internal controls for loading.

Mentions: Because we demonstrated that fucoidan suppresses the tumorigenesis of an LLC1-xenograft mouse model in vivo, we next examined the effect of fucoidan on human NSCLC cells, A549 and CL1-5, and on mouse LLC1 cells in vitro. We used the MTT assay to examine the effect of fucoidan on the growth pattern of these lung cancer cells at 24, 48 and 72 h and found 40–60% inhibition of cell viability after 72 h of fucoidan treatment (at 100 and 200 μg/ml) in these cells compared with the control (Fig. 3A). We further examined the synergistic effect of fucoidan combined with cisplatin and found synergistic cytotoxic effects on LLC1 (Supplementary Fig. 3).


Fucoidan inhibition of lung cancer in vivo and in vitro : role of the Smurf2-dependent ubiquitin proteasome pathway in TGFβ receptor degradation.

Hsu HY, Lin TY, Wu YC, Tsao SM, Hwang PA, Shih YW, Hsu J - Oncotarget (2014)

Fucoidan inhibits proliferation and decreases TGFR-mediated Smad and non-Smad pathway activity in lung cancer cellsFucoidan inhibits the proliferation of lung cancer cells (A) A549, CL1-5, and LLC1. The cells were treated with various doses of fucoidan (0–300 μg/ml) for 24, 48, and 72 h; the viability of the cells was determined using the MTT assay. Each group of fucoidan-treated samples was normalized against each untreated control. The data are representative of three separate experiments and are presented as the mean ± SD; error bars indicate SD. Significant differences are shown (*P < 0.05 and **P < 0.01, compared with the control group). (B) CL1-5 cells were treated with fucoidan (100 or 200 μg/ml) for 1 h. (C) CL1-5 cells were pre-treated with fucoidan (200 μg/ml) for 2 h and then treated with TGFβ (10 ng/ml) for 1 h. The treatments were followed by western blotting analyses of whole cell lysates to detect the expression of p-Smad2/3 (Ser423/425), p-Akt (Ser473), p-Erk1/2 (Thr202/Tyr 204). Smad2/3, Akt and Erk were used as internal controls for loading.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4202167&req=5

Figure 3: Fucoidan inhibits proliferation and decreases TGFR-mediated Smad and non-Smad pathway activity in lung cancer cellsFucoidan inhibits the proliferation of lung cancer cells (A) A549, CL1-5, and LLC1. The cells were treated with various doses of fucoidan (0–300 μg/ml) for 24, 48, and 72 h; the viability of the cells was determined using the MTT assay. Each group of fucoidan-treated samples was normalized against each untreated control. The data are representative of three separate experiments and are presented as the mean ± SD; error bars indicate SD. Significant differences are shown (*P < 0.05 and **P < 0.01, compared with the control group). (B) CL1-5 cells were treated with fucoidan (100 or 200 μg/ml) for 1 h. (C) CL1-5 cells were pre-treated with fucoidan (200 μg/ml) for 2 h and then treated with TGFβ (10 ng/ml) for 1 h. The treatments were followed by western blotting analyses of whole cell lysates to detect the expression of p-Smad2/3 (Ser423/425), p-Akt (Ser473), p-Erk1/2 (Thr202/Tyr 204). Smad2/3, Akt and Erk were used as internal controls for loading.
Mentions: Because we demonstrated that fucoidan suppresses the tumorigenesis of an LLC1-xenograft mouse model in vivo, we next examined the effect of fucoidan on human NSCLC cells, A549 and CL1-5, and on mouse LLC1 cells in vitro. We used the MTT assay to examine the effect of fucoidan on the growth pattern of these lung cancer cells at 24, 48 and 72 h and found 40–60% inhibition of cell viability after 72 h of fucoidan treatment (at 100 and 200 μg/ml) in these cells compared with the control (Fig. 3A). We further examined the synergistic effect of fucoidan combined with cisplatin and found synergistic cytotoxic effects on LLC1 (Supplementary Fig. 3).

Bottom Line: Herein, using lung cancer cells, we found that fucoidan effectively reduces TGFRI and TGFRII protein levels in vivo and in vitro.To elucidate how fucoidan decreases TGFR proteins in lung cancer cells, we found that fucoidan enhances the ubiquitination proteasome pathway (UPP)-mediated degradation of TGFRs in A549 and CL1-5 cells.Mechanistically, fucoidan promotes Smurf2 and Smad7 to conjugate TGFRs, resulting in TGF degradation; however, Smurf2-shRNA abolishes fucoidan-enhanced UPP-mediated TGFR degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan. The Genomics Research Center, Academia Sinica, Taipei, Taiwan. Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, Taiwan. Contributed equally to this work.

ABSTRACT
Fucoidan, a polysaccharide extracted from brown seaweeds, reduces tumor cell proliferation. In this study, we demonstrate that fucoidan reduces tumor size in LLC1-xenograft male C57BL/6 mice. Moreover, we found that LLC1-bearing mice continuously fed fucoidan showed greater antitumor activity than mice with discontinuous feeding. Fucoidan inhibited the in vitro growth of lung cancer cells. Transforming growth factor β (TGFβ) receptors (TGFRs) play important roles in the regulation of proliferation and progression, and high TGFRI expression in lung cancer specimens is associated with a worse prognosis. Herein, using lung cancer cells, we found that fucoidan effectively reduces TGFRI and TGFRII protein levels in vivo and in vitro. Moreover, fucoidan reduces TGFR downstream signaling events, including those in Smad2/3 and non-Smad pathways: Akt, Erk1/2, and FAK phosphorylation. Furthermore, fucoidan suppresses lung cancer cell mobility upon TGFβ stimulation. To elucidate how fucoidan decreases TGFR proteins in lung cancer cells, we found that fucoidan enhances the ubiquitination proteasome pathway (UPP)-mediated degradation of TGFRs in A549 and CL1-5 cells. Mechanistically, fucoidan promotes Smurf2 and Smad7 to conjugate TGFRs, resulting in TGF degradation; however, Smurf2-shRNA abolishes fucoidan-enhanced UPP-mediated TGFR degradation. Our study is the first to identify a novel mechanism for the antitumor activity of fucoidan, namely decreasing tumor growth by modulating the TGFR/Smad7/Smurf2-dependent axis, leading to TGFR protein degradation and inhibition of lung cancer cell progression in vitro and in vivo. Our current findings indicate that fucoidan is a potential therapeutic agent or dietary supplementation for lung cancer, acting via the Smurf2-dependent ubiquitin degradation of TGFβ receptors.

Show MeSH
Related in: MedlinePlus