褐藻多糖硫酸酯对人肺腺癌细胞系A549、SPCA-1增殖的影响
摘要
目的：褐藻多糖硫酸酯(Fucoidan),又称为褐藻糖胶、岩藻聚糖硫酸酯、岩藻聚糖,是存在于所有褐藻中的细胞间多糖。它是一种水溶性杂多糖,除了主要成分L-岩藻糖和硫酸根外,还含有半乳糖、木糖、甘露糖、葡萄糖、糖醛酸、蛋白质等。本实验所用褐藻多糖硫酸酯由市售裙带菜(Undaria pinnatifida)中提取。研究表明,褐藻多糖硫酸酯具有多种生物学活性,包括较强的抗肿瘤活性,在体内、体外实验中,对多种肿瘤细胞均表现出明显的抑制增殖效应。具有广泛的研究和开发应用前景。大连地区,褐藻类裙带菜来源丰富、物美价廉、便于取材,具有明显的地域优势。目前,裙带菜仍然主要作为该地常见的食用藻类,有很大的开发利用和深加工的潜力和价值。
肺癌是现代社会中,随着环境压力的加剧,严重威胁人类健康和生命的主要疾病之一。传统的肿瘤治疗均不可避免地对机体正常组织产生毒副作用。因此,寻找安全有效的抗肿瘤药物成为肿瘤治疗和药物开发领域急待解决的一个方向,对促进人类健康具有重要意义。研究表明,褐藻多糖硫酸酯对正常细胞的影响很小,安全性好,可望在肿瘤治疗中发挥作用。本文旨在探讨裙带菜中褐藻多糖硫酸酯对人肺腺癌细胞系A549.SPCA-1体外增殖的影响。
方法：1.采用乙醇分级沉淀法、三氯乙酸除蛋白法从市售裙带菜干粉中提取褐藻多糖硫酸酯；硫酸-苯酚法检测所提样品中的糖含量。
2.MTT(四甲基偶氮唑盐)比色法检测褐藻多糖硫酸酯对A549及SPCA-1细胞增殖的抑制作用。
3.Hoechst33258染色检测经褐藻多糖硫酸酯处理后A549细胞是否发生凋亡的形态学改变；免疫细胞化学方法检测经处理后A549细胞中凋亡相关蛋白Caspase-3.Caspase-9及核转录因子NF-kappa B的表达情况,并分别提取各组细胞的总蛋白,通过ELISA法对上述蛋白进行定量；流式细胞术检测褐藻多糖硫酸酯对SPCA-1细胞周期及凋亡的影响。
结果：1.提取所得多糖样品的平均得率约为6.20%,样品中平均糖含量约为40.84%,Fucoidan的平均提取率约为2.53%。
2.Fucoidan对A549细胞处理组与对照组间及不同浓度组间吸光度值均有显著差异(P<0.05)。IC50反映当细胞存活率为50%时,对应的Fucoidan处理浓度。各处理时间组6h、12h、24h组IC 50分别为14.77 mg/ml, 8.46mg/ml,3.08mg/ml.
Fucoidan对SPCA-1细胞12h、24h、48h、72h处理组组与对照组间及不同浓度组间吸光度值均有显著差异(P<0.05)。各处理时间组,即12h、24h、48h、72h组IC50分别为19.18 mg/ml,9.58mg/ml,2.62mg/ml, 11.61mg/ml.
3.免疫细胞化学染色结果显示：Fucoidan呈时间剂量依赖方式上调调Caspase-3.Caspase-9蛋白的活性,伴随NF-kappa B转录因子的上调。
ELISA法结果显示：各处理组与对照组均有差异,P<0.05,各处理组间呈剂量依赖性上调Caspase-3.9,呈剂量和时间依赖性下调NF-kappaB。而5mg/ml组对Caspase-3上调的时间依赖关系不显著,2.5mg/ml组对Caspase-9上调的时间依赖性不显著。
流式细胞术对Fucoidan处理的SPCA-1细胞检测的结果显示：Fucoidan具有诱导细胞凋亡的效应,经10mg/ml Fucoidan处理1 2h后,凋亡率达37.78%,处理24h凋亡率为18.4%。Fucoidan对细胞周期的影响结果显示：与对照组相比G2／M期细胞比例下降,12h对照组为9.16%,5mg/ml组为6.8%,10mg/ml组未检出G2／M期细胞。24h对照组为7.37%,5mg/ml组为0.38%,10mg/ml组为4.7%。
结论：1.综合分析选择、改良方法,降低了Fucoidan对细胞作用的IC50值。
2.Fucoidan对人肺腺癌细胞A549.SPCA-1均有增殖抑制作用。
3.Fucoidan对A549细胞的增殖抑制作用与其上调Caspase-3,9诱导细胞凋亡和下调NF-kappa B抑制细胞生长的效应有关。
Fucoidan对SPCA-1细胞的增殖抑制作用也与诱导细胞凋亡有关,同时由于可降低G2／M期细胞的百分比提示可能与对细胞周期的阻滞有关。
Objective:Fucoidan, a kind of intercellular polysaccharide of brown algae, is a water-soluble polysaccharide mixture including L-fucose and sulfate as main composition, galactose, xylose, mannose, glucose, alduronicacid, and proteins et al. Fucoidan sample using in this study was extracted from Undaria pinnatifida produced in Dalian. Recent researches indicate that in vitro experiment, fucoidan presents a significant anti-proliferation activity on many kinds of tumor cells. Therefore, it has been expected to be valuable widely in study and application fields. In Dalian district, the resource of Undaria pinnatifida is rich with good quality and small cost, having advantages in study and exploitation. At present, Undaria pinnatifida is still as a kind of food stuff mainly, having big potentials in further processing and application.
Lung cancer has become one of the severe diseases in modern world with the development of modern industry and environmental degradation, threatening human health and life. Traditional cancer therapy is unavoidable to impair normal cells when resisting tumor cells. That now it's important to find new safer therapeutic methods and drugs. Studies indicate that fucoidan hardly produce effect on normal human cells, having advantage of safty and expecting to exert curing effect. This article aimed to investigate influence of fucoidan on two kinds of human lung adenocarcinoma cells:A549 and SPCA-1.
Method:1. Acquiring fucoidan sample by precipitating with the different concentrations of ethanol, moving protein impurities with TCA (trichloroacetic acid), detecting sugar content of the sample by sulphoacid-phenol method.
2. Using MTT colorimetry to quantify cell vitality of the two cell lines A549 and SPCA-1 after being treated with fucoidan,
3. Determining expression of apoptosis-associated proteins Caspase-3, Caspase-9 and nuclear transcription factor NF-kappa B in A549 cells of each group..
Extracting total cell protein of each group of A549 cells, then quatifying intracellular proteins above (Caspase-3, Caspase-9 and NF-kappa B) with ELISA(enzyme-linked immuno sorbent assay).
Using flow cytometry to detect influence of fucoidan on SPCA-1 cell cycle and apoptosis.
Results:1. Acquired fucoidan sample with acquisition rate of 6.20% at average. The average sugar content of the fucoidan sample is 40.84%, The fucoidan extraction ratio of the dry Undaria pinnatifida raw material is about 2.53%.
2. The difference of absorbency of A549 cells from the control group and the treatment group is significant, so as the difference between each treatment groups. IC50 values of fucoidan treatment for 6h,12h, and 24h are respectively 14.77mg/ml,8.46mg/ml and 3.08mg/ml. The inhibition rate has a positive relationship with treating time, also with treating concentration.
For SPCA-1 cell line, the positive relationship between inhibitionl rate and the treating time or concentration is also tenable. IC50 values are 19.18 mg/ml for 12h,9.58mg/ml for 24h,2.62mg/ml for 48h,11.61mg/ml for 72h.
3. Results of immunocytochemical staining indicate that fucoidan can up-regulate the activity of Caspase-3 and Caspase-9 in A549 cells by time and dose-dependent manner, accompanied with down-regulation of transcription factor NF-kappa B.
Results of ELISA display that there is significant difference (P<0.05) between each treatment group and control group. Between different treatment groups, fucoidan presents activity of up-regulating Caspase-3,9 by dose-dependent manner, down-regulating NF-kappa B by dose and time dependent manner. However, there's no time-dependent up-regulation of Caspase-3 within 5mg/ml group and no time-dependent up-regulation of Caspase-9 within 2.5mg/ml group.
Results of flow cetometry show that fucoidan can also induce apoptosis of SPCA-1 cells with the apoptosis rate of 37.78% for 12h treatment and 18.4% for 24h treatment. Fucoidan also has influence on cell cycle of SPCA-1 cells, the percentage of G2/M stage cells presents the trend of decline. For 12h treatment group, the percentage of G2/M stage cells in the control group is 9.16%, the 5mg/ml group 6.8%, and no detection of G2/M stage cells in 10mg/ml group. For 24h treatment group, the percentage of G2/M stage cells in the control group is 7.37%, the 5mg/ml group 0.38%, and the 10mg/ml group 4.7%.
Conclusions:1. The purer fucoidan sample has been acquired by improving extraction method.
2. Fucoidan can inhibit growth of human lung adenocarcinoma cell line A549 and SPCA-1 in vitro. Relatively, the A549 cells are more sensitive.
3. Antiproliferation ability of fucoidan may associated with its up-regulation effect of Caspase-3,9 and down-regulation of NF-kappa B.
Fucoidan influence growth of SPCA-1 probably by inducing apoptosis and interfering cell cycle to make G2/M stage cells decline.
肺癌是现代社会中,随着环境压力的加剧,严重威胁人类健康和生命的主要疾病之一。传统的肿瘤治疗均不可避免地对机体正常组织产生毒副作用。因此,寻找安全有效的抗肿瘤药物成为肿瘤治疗和药物开发领域急待解决的一个方向,对促进人类健康具有重要意义。研究表明,褐藻多糖硫酸酯对正常细胞的影响很小,安全性好,可望在肿瘤治疗中发挥作用。本文旨在探讨裙带菜中褐藻多糖硫酸酯对人肺腺癌细胞系A549.SPCA-1体外增殖的影响。
方法：1.采用乙醇分级沉淀法、三氯乙酸除蛋白法从市售裙带菜干粉中提取褐藻多糖硫酸酯；硫酸-苯酚法检测所提样品中的糖含量。
2.MTT(四甲基偶氮唑盐)比色法检测褐藻多糖硫酸酯对A549及SPCA-1细胞增殖的抑制作用。
3.Hoechst33258染色检测经褐藻多糖硫酸酯处理后A549细胞是否发生凋亡的形态学改变；免疫细胞化学方法检测经处理后A549细胞中凋亡相关蛋白Caspase-3.Caspase-9及核转录因子NF-kappa B的表达情况,并分别提取各组细胞的总蛋白,通过ELISA法对上述蛋白进行定量；流式细胞术检测褐藻多糖硫酸酯对SPCA-1细胞周期及凋亡的影响。
结果：1.提取所得多糖样品的平均得率约为6.20%,样品中平均糖含量约为40.84%,Fucoidan的平均提取率约为2.53%。
2.Fucoidan对A549细胞处理组与对照组间及不同浓度组间吸光度值均有显著差异(P<0.05)。IC50反映当细胞存活率为50%时,对应的Fucoidan处理浓度。各处理时间组6h、12h、24h组IC 50分别为14.77 mg/ml, 8.46mg/ml,3.08mg/ml.
Fucoidan对SPCA-1细胞12h、24h、48h、72h处理组组与对照组间及不同浓度组间吸光度值均有显著差异(P<0.05)。各处理时间组,即12h、24h、48h、72h组IC50分别为19.18 mg/ml,9.58mg/ml,2.62mg/ml, 11.61mg/ml.
3.免疫细胞化学染色结果显示：Fucoidan呈时间剂量依赖方式上调调Caspase-3.Caspase-9蛋白的活性,伴随NF-kappa B转录因子的上调。
ELISA法结果显示：各处理组与对照组均有差异,P<0.05,各处理组间呈剂量依赖性上调Caspase-3.9,呈剂量和时间依赖性下调NF-kappaB。而5mg/ml组对Caspase-3上调的时间依赖关系不显著,2.5mg/ml组对Caspase-9上调的时间依赖性不显著。
流式细胞术对Fucoidan处理的SPCA-1细胞检测的结果显示：Fucoidan具有诱导细胞凋亡的效应,经10mg/ml Fucoidan处理1 2h后,凋亡率达37.78%,处理24h凋亡率为18.4%。Fucoidan对细胞周期的影响结果显示：与对照组相比G2／M期细胞比例下降,12h对照组为9.16%,5mg/ml组为6.8%,10mg/ml组未检出G2／M期细胞。24h对照组为7.37%,5mg/ml组为0.38%,10mg/ml组为4.7%。
结论：1.综合分析选择、改良方法,降低了Fucoidan对细胞作用的IC50值。
2.Fucoidan对人肺腺癌细胞A549.SPCA-1均有增殖抑制作用。
3.Fucoidan对A549细胞的增殖抑制作用与其上调Caspase-3,9诱导细胞凋亡和下调NF-kappa B抑制细胞生长的效应有关。
Fucoidan对SPCA-1细胞的增殖抑制作用也与诱导细胞凋亡有关,同时由于可降低G2／M期细胞的百分比提示可能与对细胞周期的阻滞有关。
Objective:Fucoidan, a kind of intercellular polysaccharide of brown algae, is a water-soluble polysaccharide mixture including L-fucose and sulfate as main composition, galactose, xylose, mannose, glucose, alduronicacid, and proteins et al. Fucoidan sample using in this study was extracted from Undaria pinnatifida produced in Dalian. Recent researches indicate that in vitro experiment, fucoidan presents a significant anti-proliferation activity on many kinds of tumor cells. Therefore, it has been expected to be valuable widely in study and application fields. In Dalian district, the resource of Undaria pinnatifida is rich with good quality and small cost, having advantages in study and exploitation. At present, Undaria pinnatifida is still as a kind of food stuff mainly, having big potentials in further processing and application.
Lung cancer has become one of the severe diseases in modern world with the development of modern industry and environmental degradation, threatening human health and life. Traditional cancer therapy is unavoidable to impair normal cells when resisting tumor cells. That now it's important to find new safer therapeutic methods and drugs. Studies indicate that fucoidan hardly produce effect on normal human cells, having advantage of safty and expecting to exert curing effect. This article aimed to investigate influence of fucoidan on two kinds of human lung adenocarcinoma cells:A549 and SPCA-1.
Method:1. Acquiring fucoidan sample by precipitating with the different concentrations of ethanol, moving protein impurities with TCA (trichloroacetic acid), detecting sugar content of the sample by sulphoacid-phenol method.
2. Using MTT colorimetry to quantify cell vitality of the two cell lines A549 and SPCA-1 after being treated with fucoidan,
3. Determining expression of apoptosis-associated proteins Caspase-3, Caspase-9 and nuclear transcription factor NF-kappa B in A549 cells of each group..
Extracting total cell protein of each group of A549 cells, then quatifying intracellular proteins above (Caspase-3, Caspase-9 and NF-kappa B) with ELISA(enzyme-linked immuno sorbent assay).
Using flow cytometry to detect influence of fucoidan on SPCA-1 cell cycle and apoptosis.
Results:1. Acquired fucoidan sample with acquisition rate of 6.20% at average. The average sugar content of the fucoidan sample is 40.84%, The fucoidan extraction ratio of the dry Undaria pinnatifida raw material is about 2.53%.
2. The difference of absorbency of A549 cells from the control group and the treatment group is significant, so as the difference between each treatment groups. IC50 values of fucoidan treatment for 6h,12h, and 24h are respectively 14.77mg/ml,8.46mg/ml and 3.08mg/ml. The inhibition rate has a positive relationship with treating time, also with treating concentration.
For SPCA-1 cell line, the positive relationship between inhibitionl rate and the treating time or concentration is also tenable. IC50 values are 19.18 mg/ml for 12h,9.58mg/ml for 24h,2.62mg/ml for 48h,11.61mg/ml for 72h.
3. Results of immunocytochemical staining indicate that fucoidan can up-regulate the activity of Caspase-3 and Caspase-9 in A549 cells by time and dose-dependent manner, accompanied with down-regulation of transcription factor NF-kappa B.
Results of ELISA display that there is significant difference (P<0.05) between each treatment group and control group. Between different treatment groups, fucoidan presents activity of up-regulating Caspase-3,9 by dose-dependent manner, down-regulating NF-kappa B by dose and time dependent manner. However, there's no time-dependent up-regulation of Caspase-3 within 5mg/ml group and no time-dependent up-regulation of Caspase-9 within 2.5mg/ml group.
Results of flow cetometry show that fucoidan can also induce apoptosis of SPCA-1 cells with the apoptosis rate of 37.78% for 12h treatment and 18.4% for 24h treatment. Fucoidan also has influence on cell cycle of SPCA-1 cells, the percentage of G2/M stage cells presents the trend of decline. For 12h treatment group, the percentage of G2/M stage cells in the control group is 9.16%, the 5mg/ml group 6.8%, and no detection of G2/M stage cells in 10mg/ml group. For 24h treatment group, the percentage of G2/M stage cells in the control group is 7.37%, the 5mg/ml group 0.38%, and the 10mg/ml group 4.7%.
Conclusions:1. The purer fucoidan sample has been acquired by improving extraction method.
2. Fucoidan can inhibit growth of human lung adenocarcinoma cell line A549 and SPCA-1 in vitro. Relatively, the A549 cells are more sensitive.
3. Antiproliferation ability of fucoidan may associated with its up-regulation effect of Caspase-3,9 and down-regulation of NF-kappa B.
Fucoidan influence growth of SPCA-1 probably by inducing apoptosis and interfering cell cycle to make G2/M stage cells decline.
引文
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29. Yang C, Chung D, Shin IS, Lee H, Kim J, Lee Y, You S. Effects of molecular weight and hydrolysis conditions fucoidan on anticancer activity of fucoidans from sporophyll of Undaria pinnatifida, International journsl of biological macromolecules 2008 Dec; 43(5):433-7
30. Byon YY, Kim MH, Yoo ES, Hwang KK, Jee Y, Shin T, Joo HG. Radioprotective effects of fucoidan on bone marrow cells:improvement of the cell survival and immunoreactivity, Journal of veterinary science 2008 Dec; 9(4):359-65
31. Philchenkov A, Zavelevich M, Imbs T, Zvyagintseva T, Zaporozhets T. Sensitization of human malignant lymphoid cells to etoposide by fucoidan, a brown seaweed polysaccharide, Experimental oncology 2007 Sep; 29(3):181-5
1. Li B, Lu F, Wei X, Zhao R. Fucoidan:structure and bioactivity, Molecules 2008 Aug;13(8):1671-95
2. Yang C, Chung D, Shin IS, Lee H, Kim J, Lee Y, You S. Effects of molecular weight and hydrolysis conditions fucoidan on anticancer activity of fucoidans from sporophyll of Undaria pinnatifida, International journsl of biological macromolecules 2008 Dec; 43(5):433-7
3. Hu T, Liu D, Chen Y, Wu J, Wang S. Antioxidant activity of sulfated polysaccharide fractions extracted from Undaria pinnitafida in vitro, International journal of biological macromolecules 2010 Mar; 46(2):193-8
4. Chen JH, Lim JD, Sohn EH, Choi YS, Han ET. Growth-inhibitory effect of a fucoidan from brown seaweed Undaria pinnatifida on Plasmodium parasites, Parasitology research 2009 Jan; 104(2):245-50
5. Kolb N, Vallorani L, Milanovic N, Stocchi V. Evaluation of marine algae wakame (Undaria pinnatifida) and kombu (Laminaria digitata japonica) as food supplements, Food Technology and Biotechnology 2004 Jan; 42(1):57-61
6. Okai Y, Higashi-Okai K, Ishizaka S, Yamashita U. Enhancing effect of polysac-charides from an edible brown alga, Hijikia fusiforme (Hijiki), on release of tumor necrosis factor-a from macrophages of endotoxin-nonresponder C3H/HeJ mice. Nutrition and cancer 1997;27:74-79
7. Byon YY, Kim MH, Yoo ES, Hwang KK, Jee Y, Shin T, Joo HG. Radioprotective effects of fucoidan on bone marrow cells:improvement of the cell survival and immunoreactivity, Journal of veterinary science 2008 Dec; 9(4):359-65
8. 高梦祥,叶森。海带多糖的提取工艺研究,长江大学学报(自然科学版)2005 Feb；25(2)：73-7
9. Jimenez-Escrig A, Sanchez-Muniz FJ. Dietary fibre from edible seaweeds: Chemical structure, physicochemical properties and effects on cholesterol metabolism, Nutrition Research 2000 Apr; 20(4):585-98
10.司徒镇强,吴军正。细胞培养西安：世界图书出版公司,1996：1 27
11.张延坤,张东祥.生物活性多糖的抗肿瘤作用及其机制研究进展,解放军预防医学杂志2006 May；24(5)：382·5
12. Hyun JH, Kim SC, Kang JI, Kim MK, Boo HJ, Kwon JM, Koh YS, Hyun JW, Park DB, Yoo ES, Kang HK. Apoptosis inducing activity of fucoidan in HCT-15 colon carcinoma cells, Biological & pharmaceutical bulletin 2009 Oct; 32(10):1760-64
13. Philchenkov A, Zavelevich M, Imbs T, Zvyagintseva T, Zaporozhets T. Sensitization of human malignant lymphoid cells to etoposide by fucoidan, a brown seaweed polysaccharide, Experimental oncology 2007 Sep; 29(3):181-5
14. Haneji K, Matsuda T, Tomita M, Kawakami H, Ohshiro K, Uchihara JN, Masuda M, Takasu N, Tanaka Y, Ohta T, Mori N. Fucoidan extracted from Cladosiphon okamuranus Tokida induces apoptosis of human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells, Nutrition cancer. 2005 Feb; 52(2):189-201
15. Nagamine T, Hayakawa K, Kusakabe T, Takada H, Nakazato K, Hisanaga E, Iha M. Inhibitory effect of fucoidan on Huh7 hepatoma cells through downregulation of CXCL12, Nutrition and cancer 2009 Mar; 61(3):340-7
16. Eriksson D, Stigbrand T. Radiation-induced cell death mechanisms, Tumour biology 2010 May 20 [Epub ahead of print]
17. Fulda S. Modulation of Apoptosis by Natural Products for Cancer Therapy, Planta medica 2010 May 19. [Epub ahead of print]
18. Han S, Roman J. Targeting Apoptotic Signaling Pathways in Human Lung Cancer, Current cancer drug targets.2010 May 18. [Epub ahead of print]
19. Xiao Y, Fan Y, Chen B, Zhang Q, Zeng H. Polysaccharides from Scurrula parasitica L. inhibit sarcoma S180 growth in mice, Zhongguo Zhong Yao Za Zhi. 2010 Feb; 35(3):381-4
20. Hu Y, Cheng SC, Chan KT, Ke Y, Xue B, Sin FW, Zeng C, Xie Y. Fucoidin enhances dendritic cell-mediated T-cell cytotoxicity against NY-ESO-1 expressing human cancer cells, Biochemical biophysical research communi-cations 2010 Feb; 392(3):329-34
21. Aisa Y, Miyakawa Y, Nakazato T, Shibata H, Saito K, Ikeda Y, Kizaki M. Fucoidan induces apoptosis of human HS-sultan cells accompanied by activation of caspase-3 and down-regulation of ERK. pathways, American journal of hematology.2005 Jan; 78(1):7-14
22. Zhang Y, Song S, Song D, Liang H, Wang W, Ji A. Proliferative effects on neural stem/progenitor cells of a sulfated polysaccharide purified from the sea cucumber Stichopus japonicus, Journal of bioscience and bioengineering 2010 Jan; 109(1):67-72
23. Lopez-Guerra M, Colomer D. NF-kappaB as a therapeutic target in chronic lymphocytic leukemia, Expert opinion on therapeutic targets 2010 Mar; 14(3): 275-88
24. Nagamine T, Hayakawa K, Kusakabe T, Takada H, Nakazato K, Hisanaga E, Iha M. Inhibitory effect of fucoidan on Huh7 hepatoma cells through downregulation of CXCL12, Nutrition and cancer 2009 Mar; 61(3):340-7
25. D'Urso V, Collodoro A, Mattioli E, Giordano A, Bagella L. Cytometry and DNA ploidy:clinical uses and molecular perspective in gastric and lung cancer, Journal of cellular physiology 2010 Mar; 222(3):532-9
26. Young EW, Simmons CA. Macro-and microscale fluid flow systems for endothelial cell biology, Lab Chip 2010 Jan; 10(2):143-60
27.吴善芳.人体肺腺癌细胞系：SPC-A-I的建立及其生物学特性,中国科学B辑1982(10)： 913-21
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13. Hyun JH, Kim SC, Kang JI, Kim MK, Boo HJ, Kwon JM, Koh YS, Hyun JW, Park DB, Yoo ES, Kang HK. Apoptosis inducing activity of fucoidan in HCT-15 colon carcinoma cells, Biological & pharmaceutical bulletin 2009 Oct; 32(10):1760-64
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16. Cumashi A, Ushakova NA, Preobrazhenskaya ME, D'Incecco A, Piccoli A, Totani L, Tinari N, Morozevich GE, Berman AE, Bilan MI, Usov AI, Ustyuzhanina NE, Grachev AA, Sanderson CJ, Kelly M, Rabinovich GA, Iacobelli S, Nifantiev NE; Consorzio Interuniversitario Nazionale per la Bio-Oncologia, Italy. A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds, Glycobiology 2007 May; 17(5):541-52
17. Yamasaki-Miyamoto Y, Yamasaki M, Tachibana H, Yamada K. Fucoidan induces apoptosis through activation of caspase-8 on human breast cancer MCF-7 cells, Journal of agricultural and food chemistry 2009 Sep; 57(18):8677-82
18. Nagamine T, Hayakawa K, Kusakabe T, Takada H, Nakazato K, Hisanaga E, Iha M. Inhibitory effect of fucoidan on Huh7 hepatoma cells through downregulation of CXCL12, Nutrition and cancer 2009 Mar; 61(3):340-7
19. Teruya T, Konishi T, Uechi S, Tamaki H, Tako M. Anti-proliferative activity of oversulfated fucoidan from commercially cultured Cladosiphon okamuranus TOKIDA in U937 cells, International journsl of biological macromolecules 2007 Aug; 41(3):221-6
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25. Lang K, Entschladen F, Weidt C, Zaenker KS. Tumor immune escape mechanisms: impact of the neuroendocrine system, Cancer Immunology Immunotherapy 2006 Jul; 55(7):749-60
26. Maruyama H, Tamauchi H, Iizuka M, Nakano T. The role of NK cells in antitumor activity of dietary fucoidan from Undaria pinnatifida sporophylls (Mekabu), Planta medica 2006 Dec; 72(15):1415-7
27. Haneji K, Matsuda T, Tomita M, Kawakami H, Ohshiro K, Uchihara JN, Masuda M, Takasu N, Tanaka Y, Ohta T, Mori N. Fucoidan extracted from Cladosiphon okamuranus Tokida induces apoptosis of human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells, Nutrition cancer. 2005 Feb; 52(2):189-201
28. Aisa Y, Miyakawa Y, Nakazato T, Shibata H, Saito K, Ikeda Y, Kizaki M. Fucoidan induces apoptosis of human HS-sultan cells accompanied by activation of caspase-3 and down-regulation of ERK pathways, American journal of hematology.2005 Jan; 78(1):7-14
29. Yang C, Chung D, Shin IS, Lee H, Kim J, Lee Y, You S. Effects of molecular weight and hydrolysis conditions fucoidan on anticancer activity of fucoidans from sporophyll of Undaria pinnatifida, International journsl of biological macromolecules 2008 Dec; 43(5):433-7
30. Byon YY, Kim MH, Yoo ES, Hwang KK, Jee Y, Shin T, Joo HG. Radioprotective effects of fucoidan on bone marrow cells:improvement of the cell survival and immunoreactivity, Journal of veterinary science 2008 Dec; 9(4):359-65
31. Philchenkov A, Zavelevich M, Imbs T, Zvyagintseva T, Zaporozhets T. Sensitization of human malignant lymphoid cells to etoposide by fucoidan, a brown seaweed polysaccharide, Experimental oncology 2007 Sep; 29(3):181-5
1. Li B, Lu F, Wei X, Zhao R. Fucoidan:structure and bioactivity, Molecules 2008 Aug;13(8):1671-95
2. Yang C, Chung D, Shin IS, Lee H, Kim J, Lee Y, You S. Effects of molecular weight and hydrolysis conditions fucoidan on anticancer activity of fucoidans from sporophyll of Undaria pinnatifida, International journsl of biological macromolecules 2008 Dec; 43(5):433-7
3. Hu T, Liu D, Chen Y, Wu J, Wang S. Antioxidant activity of sulfated polysaccharide fractions extracted from Undaria pinnitafida in vitro, International journal of biological macromolecules 2010 Mar; 46(2):193-8
4. Chen JH, Lim JD, Sohn EH, Choi YS, Han ET. Growth-inhibitory effect of a fucoidan from brown seaweed Undaria pinnatifida on Plasmodium parasites, Parasitology research 2009 Jan; 104(2):245-50
5. Kolb N, Vallorani L, Milanovic N, Stocchi V. Evaluation of marine algae wakame (Undaria pinnatifida) and kombu (Laminaria digitata japonica) as food supplements, Food Technology and Biotechnology 2004 Jan; 42(1):57-61
6. Okai Y, Higashi-Okai K, Ishizaka S, Yamashita U. Enhancing effect of polysac-charides from an edible brown alga, Hijikia fusiforme (Hijiki), on release of tumor necrosis factor-a from macrophages of endotoxin-nonresponder C3H/HeJ mice. Nutrition and cancer 1997;27:74-79
7. Byon YY, Kim MH, Yoo ES, Hwang KK, Jee Y, Shin T, Joo HG. Radioprotective effects of fucoidan on bone marrow cells:improvement of the cell survival and immunoreactivity, Journal of veterinary science 2008 Dec; 9(4):359-65
8. 高梦祥,叶森。海带多糖的提取工艺研究,长江大学学报(自然科学版)2005 Feb；25(2)：73-7
9. Jimenez-Escrig A, Sanchez-Muniz FJ. Dietary fibre from edible seaweeds: Chemical structure, physicochemical properties and effects on cholesterol metabolism, Nutrition Research 2000 Apr; 20(4):585-98
10.司徒镇强,吴军正。细胞培养西安：世界图书出版公司,1996：1 27
11.张延坤,张东祥.生物活性多糖的抗肿瘤作用及其机制研究进展,解放军预防医学杂志2006 May；24(5)：382·5
12. Hyun JH, Kim SC, Kang JI, Kim MK, Boo HJ, Kwon JM, Koh YS, Hyun JW, Park DB, Yoo ES, Kang HK. Apoptosis inducing activity of fucoidan in HCT-15 colon carcinoma cells, Biological & pharmaceutical bulletin 2009 Oct; 32(10):1760-64
13. Philchenkov A, Zavelevich M, Imbs T, Zvyagintseva T, Zaporozhets T. Sensitization of human malignant lymphoid cells to etoposide by fucoidan, a brown seaweed polysaccharide, Experimental oncology 2007 Sep; 29(3):181-5
14. Haneji K, Matsuda T, Tomita M, Kawakami H, Ohshiro K, Uchihara JN, Masuda M, Takasu N, Tanaka Y, Ohta T, Mori N. Fucoidan extracted from Cladosiphon okamuranus Tokida induces apoptosis of human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells, Nutrition cancer. 2005 Feb; 52(2):189-201
15. Nagamine T, Hayakawa K, Kusakabe T, Takada H, Nakazato K, Hisanaga E, Iha M. Inhibitory effect of fucoidan on Huh7 hepatoma cells through downregulation of CXCL12, Nutrition and cancer 2009 Mar; 61(3):340-7
16. Eriksson D, Stigbrand T. Radiation-induced cell death mechanisms, Tumour biology 2010 May 20 [Epub ahead of print]
17. Fulda S. Modulation of Apoptosis by Natural Products for Cancer Therapy, Planta medica 2010 May 19. [Epub ahead of print]
18. Han S, Roman J. Targeting Apoptotic Signaling Pathways in Human Lung Cancer, Current cancer drug targets.2010 May 18. [Epub ahead of print]
19. Xiao Y, Fan Y, Chen B, Zhang Q, Zeng H. Polysaccharides from Scurrula parasitica L. inhibit sarcoma S180 growth in mice, Zhongguo Zhong Yao Za Zhi. 2010 Feb; 35(3):381-4
20. Hu Y, Cheng SC, Chan KT, Ke Y, Xue B, Sin FW, Zeng C, Xie Y. Fucoidin enhances dendritic cell-mediated T-cell cytotoxicity against NY-ESO-1 expressing human cancer cells, Biochemical biophysical research communi-cations 2010 Feb; 392(3):329-34
21. Aisa Y, Miyakawa Y, Nakazato T, Shibata H, Saito K, Ikeda Y, Kizaki M. Fucoidan induces apoptosis of human HS-sultan cells accompanied by activation of caspase-3 and down-regulation of ERK. pathways, American journal of hematology.2005 Jan; 78(1):7-14
22. Zhang Y, Song S, Song D, Liang H, Wang W, Ji A. Proliferative effects on neural stem/progenitor cells of a sulfated polysaccharide purified from the sea cucumber Stichopus japonicus, Journal of bioscience and bioengineering 2010 Jan; 109(1):67-72
23. Lopez-Guerra M, Colomer D. NF-kappaB as a therapeutic target in chronic lymphocytic leukemia, Expert opinion on therapeutic targets 2010 Mar; 14(3): 275-88
24. Nagamine T, Hayakawa K, Kusakabe T, Takada H, Nakazato K, Hisanaga E, Iha M. Inhibitory effect of fucoidan on Huh7 hepatoma cells through downregulation of CXCL12, Nutrition and cancer 2009 Mar; 61(3):340-7
25. D'Urso V, Collodoro A, Mattioli E, Giordano A, Bagella L. Cytometry and DNA ploidy:clinical uses and molecular perspective in gastric and lung cancer, Journal of cellular physiology 2010 Mar; 222(3):532-9
26. Young EW, Simmons CA. Macro-and microscale fluid flow systems for endothelial cell biology, Lab Chip 2010 Jan; 10(2):143-60
27.吴善芳.人体肺腺癌细胞系：SPC-A-I的建立及其生物学特性,中国科学B辑1982(10)： 913-21