Fucoidan inhibition of lung cancer in vivo andin vitro: role of the Smurf2-dependent ubiquitin proteasome pathway in TGFβ receptor degradation
Hsien-Yeh Hsu1,2,3,*, Tung-Yi Lin1,3,*, Yu-Chung Wu4,5, Shu-Ming Tsao1, Pai-An Hwang6, Yu-Wei Shih1 and Jason Hsu7
1 Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan
2 The Genomics Research Center, Academia Sinica, Taipei, Taiwan
3 Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, Taiwan
4 Division of Thoracic Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan
5 School of Medicine, National Yang-Ming University, Taipei, Taiwan
6 Seafood Technology Division, Fisheries Research Institute, Council of Agriculture, Keelung, Taiwan
7 Fordham University, New York, NY, USA
* These authors contributed equally to this work
Correspondence to: Hsien-Yeh Hsu, email: hsienyeh@gmail.com
Keywords: Fucoidan, lung cancer, transforming growth factor β receptors (TGFRs), Smad ubiquitination regulatory factor 2 (Smurf2), ubiquitination proteasome pathway (UPP)
Received: June 09, 2014 Accepted: August 05, 2014 Published: August 06, 2014
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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 levelsin vivo andin 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 TGFR 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 offucoidan, 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.
www.impactjournals.com/oncotarget/ Oncotarget, Advance Publications 2014
台湾小分子褐藻糖胶抑制肺癌在体内和体外的增殖:
依赖Smurf2的泛素蛋白分解酶通路在降解转化生長因子β受体的作用
许先业(Hsien-Yeh Hsu)
国立阳明大学医学生物技术暨检验学系、研究所教授
台湾褐藻醣胶发展学会理事长
摘要：
台湾小分子褐藻糖胶是一种从褐藻中所萃取的多糖体，过去研究中已经证实具有抗肿瘤细胞生 长的功用。目前对于台湾小分子褐藻糖胶研究，我们利用肺癌细胞(LLC1)异种移植雄性小 鼠(C57BL/6) 模式，成功证明了台湾小分子褐藻糖胶可以降低老鼠的肺癌肿瘤大小。
更进一步，我们发现，将 LLC1异种移植小鼠连续喂食台湾小分子褐藻糖胶比不连续喂食表现 出更强烈的抗肿瘤活性，证实小 鼠长期服用台湾小分子褐藻糖胶可以有效对抗肺癌增长。我们于活体外研究发现，台湾小分子褐藻糖胶能有效地抑制肺癌细胞生长。细胞中转化生长因 子β（TGFβ）受体（TGFRs）在癌细胞中调控肿瘤之生成与移动型为担任重要角色；同时，在临 床肺癌肿瘤样本中，已被证实TGFRI表达的病人则有预后较差的特性。我们于体外试验与肺癌老鼠 实验中，发现台湾小分子褐藻糖胶可以有效地减少TGFRI和TGFRII的表现量。
更进一步，台湾小分子褐藻糖胶减少TGFR下游讯息传递路径，包括Smad2/3的与非Smad途 径：Akt和Erk1/2，和FAK的磷酸化。另外，我们也证实台湾小分子褐藻糖胶有效地抑制TGFβ所诱 导的肺癌细胞移动行为。为了阐明肺癌细胞中台湾小分子褐藻糖胶如何减少TGFR表现量，我们利 用肺癌细胞(A549和CL1-5)发现，台湾小分子褐藻糖胶会增强TGFRs的泛素化蛋白分解酶体途 径（ubiquitination proteasome pathway, UPP），有效地促进TGFRs被降解。
在机制探讨中，台湾小分子褐藻糖胶会促进Smurf2和Smad7蛋白与TGFRs结合，导 致TGFR被快速降解;实验中，我们利用Smurf2-shRNA将细胞内Smurf2去除，反而阻碍台湾小分子 褐藻糖胶增强UPP调节的TGFR降解，成功证实台湾小分子褐藻糖胶诱导TGFR表现量的原因。我们的研究首次发现一种新的台湾小分子褐藻糖胶抗肿瘤机制，而降低肿瘤的生长，即是通过调 控TGFR/Smad7/Smurf2依赖之主轴，从而导致TGFR蛋白降解和抑制肺癌细胞在体外和体内的进 展。我们目前的研究结果显示出，台湾 小分子褐藻糖胶是一种对肺癌有潜力的治疗药物或膳食补 充剂，其主要机制是透过Smurf2依赖性的泛素化作用降解TGFβ受体。
台湾小分子褐藻糖胶抑制癌症肿瘤
RESULTS:
Fucoidan suppresses tumorigenesis and reduces transforming growth factor β (TGFβ) receptor (TGFR) protein levels in LLC1 cell-xenograft male C57BL6 mice in vivo
Greater efficacy in the reduction of tumor volume:
weight and inhibition of TGFRI/II protein expression in LLC1-bearing mice orally fed continuously with fucoidan
实验结果——减小肿瘤体积：
台湾小分子褐藻糖胶透过刺激、调控包括Smurf2以及Smad7这两个因子，诱导泛素介质蛋白质降解机制的功能，而能有效抑制肺癌细胞生长与扩散，并使得恶性肺癌肿瘤体积缩小。
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