Antitumor effect and molecular mechanism of fucoidan in NSCLC

Abstract

Background: Fucoidan, a water-soluble polysaccharide, exerts anticoagulant and antiviral functions. It was recently reported that fucoidan also exerts an antitumor function. Lung cancer is one of the most common cancers in the world. The aim of this study was to investigate anti-tumor,apoptosis and anti-metastasis effects of fucoidan in both cell-based assays and mouse xenograft model, as well as to clarify possible role of m-TOR pathway in the protection.

Methods: In vitro: Different concentrations of fucoidan were given to act on non-small cell lung cancer (NSCLC) cell lines A549 and H1650. The effects of fucoidan on cell proliferation were observed by detecting cyclin expression levels, CCK8 and EDU experiments and cloning experiments. The apoptotic level was detected by flow cytometry and the apoptotic protein level was detected by Westernblot. By detecting the expression of adhesion molecules, the expression of matrix metalloproteinase (MMP) family, and Transwell cell invasion and migration experiment, the effect of fucoidan on cell adhesion, invasion and migration was observed. Meanwhile the effect of fucoidan on angiogenesis was observed by detecting the expression of vascular endothelial growth factor (VEGF). In vivo experiment: An animal model of NSCLC cell mouse subcutaneous xenograft tumor was established to analyze the correlation between the consumption of fucoidan and the size and volume of xenograft tumor through gross observation. Through immunohistochemical staining and immunofluorescence double staining, ki67 and cell adhesion molecules (E-cadherin, N-cadherin and CD31) and VEGF-A in the tumor were detected, and the correlation between the amount of fucoidan and the above indexes was analyzed.

Results: Fucoidan inhibited the proliferation and angiogenesis of NSCLC cells via the mTOR pathway and promoted their apoptosis by increasing the Bax/Bcl-2 ratio. Not only that, fucoidan inhibited NSCLC cell invasion via epithelial-mesenchymal transformation (EMT). The mice fed fucoidan exhibited significant reductions in tumor volumes and weights. These indicators (Ki67, VEGF-A,N-cadherin) were decreased and E-cadherin expression was up-regulated in A549 mice that treated with fucoidan. The results showed that fucoidan inhibited tumor proliferation in vivo by affecting the expression of related proteins.

Conclusion: Fucoidan conveys antitumor effects and our results represent an ideal therapeutic agent for NSCLC.

Keywords: Fucoidan; Non-small cell lung cancer (NSCLC).

Fig. 1

Fucoidan inhibited the proliferation of NSCLC cells. a The CCK8 assay was used to evaluate proliferation. Ctrl vs 10 mg**p < 0.01,ctrl vs 16 mg **p < 0.01.b A cloning formation experiment was used to visually detect cell proliferation. Ctrl vs 10 mg**p < 0.01,ctrl vs 16 mg **p < 0.01.c. Proliferation under fucoidan treatment was examined using EdU incorporation assays. Ctrl vs 10 mg**p < 0.01,ctrl vs 16 mg **p < 0.01

Fig. 2

Fucoidan inhibited the proliferation of cells via mTOR pathway. a The expression of cell cycle-associated molecules (cyclin D1) and VEGF were examined by western blot analyses.*p < 0.05 b. Expression levels of p-mTOR, p-P70S6K, p-4EBP1 and p-S6K were measured in A549 and H1650 cells treated with fucoidan using Western blot analysis.*p < 0.05

Fig. 3

Fucoidan inhibits invasion and EMT in NSCLC cells. a. Transwell assays were used to determine the invasive ability of NSCLC cells. Representative fields showing invasive cells following fucoidan treatment on the membrane are presented. We counted the cells separately. **p < 0.01.b. Western blot analyses were used to detect the expression of MMP-9, E-cadherin, N-cadherin and vimentin.*p < 0.05

Fig. 4

Fucoidan induces apoptosis in A549 and H1650 NSCLC cells. a. Annexin V-PE/7-AAD double-staining and flow cytometry revealed that fucoidan effectively induced apoptosis in A549 and H1650 cells. LL (low left), LR (low right), UR (upper right), and UL (upper left) denote viable (live), early apoptotic, late apoptotic and necrotic cells, respectively. b. Western blot analysis of Bcl-2 and Bax expression and the Bax/Bcl-2 ratio in cells treated with fucoidan or untreated. ** P < 0.01

Fig. 5

Fucoidan suppresses tumor growth in A549 cell xenograft mice in vivo. a. Representative images of subcutaneous A549 cell xenografts after surgical removal are shown. b. Tumor volumes and weights were measured. Tumor volume growth curves in mice from the fucoidan-treated and control groups.*p < 0.05. c. The tumor samples were homogenized, and immunohistochemical and western blot analyses of tumor samples were performed to detect the Ki-67 index. d. Immunofluorescence staining revealed the expression of VEGF-A and CD31 in tumor tissue. e. Immunofluorescence staining revealed the expression of N-cadherin and E-cadherin in tumor tissue