羊栖菜褐藻糖胶CSFP-1抗肿瘤活性及机制研究
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摘要
目的分离纯化制备羊栖菜褐藻糖胶(CSFP-1),并对其抗肿瘤活性及可能作用机制进行初步研究。方法通过冷水浸提法制备羊栖菜褐藻糖胶(CSFP),经超滤系统分离获得CSFP-1,并采用苯酚-硫酸比色法、PMP衍生化、高效液相色谱法、红外光谱分析等多种方法对其理化性质进行分析;采用MTT法评价CSFP-1(0、10、100、300μg/m L)对宫颈癌细胞(He La)、肺癌细胞(A549)和肝癌细胞(Hep3B)生存能力的影响;应用流式细胞术评价CSFP-1对He La细胞细胞周期的影响;应用Western blotting技术评价CSFP-1对He La细胞凋亡和自噬相关蛋白表达的影响。结果 CSFP-1的糖含量为84.18%,糖醛酸含量为14.28%,硫酸根含量为10.02%,平均相对分子质量为5.6×104,主要由半乳糖、岩藻糖、甘露糖、葡萄糖醛酸和木糖组成。CSFP-1具有选择性细胞毒性,对He La细胞生长发挥显著抑制作用(P<0.05、0.001),对A549和Hep3B细胞存活率没有明显影响。与对照组比较,He La细胞经CSFP-1处理24 h后,G1期细胞比例明显增加(P<0.001);经CSFP-1处理48 h后,S期细胞比例显著增加(P<0.05、0.01、0.001),G2期细胞比例显著减少(P<0.01、0.001),均呈现剂量相关性。与对照组比较,CSFP-1 100、300μg/m L剂量组处理24 h,300μg/m L剂量组处理48 h Bcl-2蛋白表达显著减少(P<0.05);100、300μg/m L剂量组处理24、48 h Beclin-1蛋白表达显著增加(P<0.05、0.01),且存在时间和剂量相关性。结论 CSFP-1具有选择性抑制He La细胞增殖作用,作用机制可能与阻滞细胞周期、诱导细胞凋亡相关。
Objective To extract, purify, and prepare the fucoidin of Sargassum fusiforme(CSFP-1), and preliminary investigate its anti-tumor activity and underlying mechanism. Methods CSFP was extracted by cold water and CSFP-1 was obtained by ultrafiltration system. Phenol-sulfuric acid colorimetry, PMP derivatization, high performance liquid chromatography, infrared spectroscopy, and other methods were used to analyze the physical and chemical properties. The effects of CSFP-1(0, 10, 100, and 300 μg/m L) on the viability of cervical cancer cells(He La), lung cancer cells(A549), and hepatoma cells(Hep3 B) were evaluated by MTT assay, the effects of CSFP-1 on the cell cycle of He La were evaluated by flow cytometry, and the effects of CSFP-1 on apoptosis and autophagy related protein expression in He La cells were evaluated by Western blotting. Results CSFP-1 was mainly composed of galactose, fucose, mannose, glucuronic acid, and xylose with the sugar content of 84.18%, glucuronic acid content of 14.28%, sulfate content of 10.02% and average molecular weight of 56 k Da. CSFP-1 had selective cytotoxicity and significantly inhibited the growth of He La cells(P < 0.05, 0.001), but had no significant effect on the survival rate of A549 and Hep3 B cells. Compared with the control group, He La cells treated with CSFP-1 for 24 h increased the proportion of G1 phase cells significantly(P < 0.001); After treated with CSFP-1 for 48 h, the proportion of S phase cells increased significantly(P < 0.05, 0.01, 0.001), and the proportion of G2 phase cells decreased significantly(P < 0.01, 0.001). Compared with control group, the expression of Bcl-2 protein in 100 and 300 μg/m L CSFP-1 groups decreased significantly at 24 h, and that in 300 μg/m L group decreased significantly at 48 h(P < 0.05); The expression of Beclin-1 protein in 100 and 300 μg/m L group increased significantly at 24 and 48 h(P < 0.05 and 0.01), and there was a time-dose correlation. Conclusion CSFP-1 has selective inhibited effect on the proliferation of He La cells and its anti-tumor activity mechanism may be related with blocking cell cycle and inducing apoptosis.
Objective To extract, purify, and prepare the fucoidin of Sargassum fusiforme(CSFP-1), and preliminary investigate its anti-tumor activity and underlying mechanism. Methods CSFP was extracted by cold water and CSFP-1 was obtained by ultrafiltration system. Phenol-sulfuric acid colorimetry, PMP derivatization, high performance liquid chromatography, infrared spectroscopy, and other methods were used to analyze the physical and chemical properties. The effects of CSFP-1(0, 10, 100, and 300 μg/m L) on the viability of cervical cancer cells(He La), lung cancer cells(A549), and hepatoma cells(Hep3 B) were evaluated by MTT assay, the effects of CSFP-1 on the cell cycle of He La were evaluated by flow cytometry, and the effects of CSFP-1 on apoptosis and autophagy related protein expression in He La cells were evaluated by Western blotting. Results CSFP-1 was mainly composed of galactose, fucose, mannose, glucuronic acid, and xylose with the sugar content of 84.18%, glucuronic acid content of 14.28%, sulfate content of 10.02% and average molecular weight of 56 k Da. CSFP-1 had selective cytotoxicity and significantly inhibited the growth of He La cells(P < 0.05, 0.001), but had no significant effect on the survival rate of A549 and Hep3 B cells. Compared with the control group, He La cells treated with CSFP-1 for 24 h increased the proportion of G1 phase cells significantly(P < 0.001); After treated with CSFP-1 for 48 h, the proportion of S phase cells increased significantly(P < 0.05, 0.01, 0.001), and the proportion of G2 phase cells decreased significantly(P < 0.01, 0.001). Compared with control group, the expression of Bcl-2 protein in 100 and 300 μg/m L CSFP-1 groups decreased significantly at 24 h, and that in 300 μg/m L group decreased significantly at 48 h(P < 0.05); The expression of Beclin-1 protein in 100 and 300 μg/m L group increased significantly at 24 and 48 h(P < 0.05 and 0.01), and there was a time-dose correlation. Conclusion CSFP-1 has selective inhibited effect on the proliferation of He La cells and its anti-tumor activity mechanism may be related with blocking cell cycle and inducing apoptosis.
引文
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[3]Chen X,Nie W,Yu G,et al.Antitumor and immunomodulatory activity of polysaccharides from Sargassum fusiforme[J].Food Chem Toxicol,2012,50(3-4):695.
[4]Wang W,Lu J B,Wang C,et al.Effects of Sargassum fusiforme polysaccharides on antioxidant activities and intestinal functions in mice[J].Int J Biol Macromol,2013,58(7):127-132.
[5]Wu M,Wu Y,Qu M,et al.Evaluation of antioxidant activities of water-soluble polysaccharides from brown alga Hizikia fusiformis[J].Int J Biol Macromol,2013,56(5):28.
[6]Chen X,Nie W,Fan S,et al.A polysaccharide from Sargassum fusiforme protects against immunosuppression in cyclophosphamide-treated mice[J].Carbohyd Polym,2012,90(2):1114-1119.
[7]Ale M T,Mikkelsen J D,Meyer A S.Important determinants for fucoidan bioactivity:a critical review of structure-function relations and extraction methods for fucose-containing sulfated polysaccharides from brown seaweeds[J].Mar Drugs,2011,9(10):2106-2130.
[8]张海亮,唐圣松.细胞自噬与肿瘤耐药[J].中国生物化学与分子生物学报,2014,30(3):224-232.
[9]Du P,Cao H,Wu H R,et al.Blocking Bcl-2 leads to autophagy activation and cell death of the HEPG2 liver cancer cell line[J].Asian Pac J Cancer Prev,2013,14(10):5849-5854.
[10]Yousefi S,Perozzo R,Schmid I,et al.Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis[J].Nat Cell Biol,2006,8(10):1124-1132.
[11]Li X,Lu Y,Pan T,et al.Roles of autophagy in cetuximab-mediated cancer therapy against EGFR[J].Autophagy,2010,6(8):1066-1077.
[12]Miki H,Uehara N,Kimura A,et al.Resveratrol induces apoptosis via ROS-triggered autophagy in human colon cancer cells[J].Int J Oncol,2012,40(4):1020-1028.
[13]杨柳.岩藻多糖硫酸酯的抗肿瘤活性筛选和初步机理研究[D].济南:山东大学,2012.
[14]郭雷,吕明生,王淑军,等.苯酚-硫酸法测定樱桃酒中总糖[J].食品研究与开发,2010,31(6):130-132.
[15]马定远,陈君,李萍,等.柱前衍生化高效液相色谱法分析多糖中的单糖组成[J].分析化学,2002,30(6):702-705.
[16]王金霞,赵峡,于广利,等.柱前衍生高效液相色谱法分析海洋褐藻多糖药物的糖醛酸组成[J].分析化学,2009,37(5):648-652.
[17]张丽萍,王月秋,姜世成,等.离子色谱法测定高山红景天多糖硫酸酯中硫酸根含量的研究[J].分子科学学报,2000,16(3):161-164.
[18]徐晓霞,李炎,刘华,等.高效凝胶色谱法测定多花黄精多糖分子量与分子量分布[J].四川生理科学杂志,2008,30(3):102-103.
[19]季宇彬,高世勇,孔琪,等.羊栖菜多糖对肿瘤细胞P53基因蛋白表达的影响[J].哈尔滨商业大学学报(自然科学版),2001,17(2):1-3.
[20]季宇彬,高世勇.羊栖菜多糖体外抗肿瘤作用及其作用机制的研究[J].中草药,2003,34(12):1111-1114.
[21]岑颖洲,马夏军,王凌云,等.羊栖菜多糖的制备及其对Hep G-2细胞的抑制作用[J].中国海洋药物,2005,24(1):21-24.
[22]倪小芬,郑超,田吉来,等.羊栖菜多糖对H2O2诱导胰岛β细胞凋亡的保护作用及PI3K抑制剂的影响[J].中华中医药学刊,2009,(7):1506-1508.
[23]Chen P,He D,Zhang Y,et al.Sargassum fusiformepolysaccharides activate antioxidant defense by promoting Nrf2-dependent cytoprotection and ameliorate stress insult during aging[J].Food Funct,2016,7(11):4576.
[24]Chen P,Yang S,Hu C,et al.Sargassum fusiforme polysaccharide rejuvenates the small intestine in mice through altering its physiology and gut microbiota composition[J].Curr Mol Med,2017,18.
[25]Zhao T,Sun Q,del Rincon S V,et al.Gallotannin imposes S phase arrest in breast cancer cells and suppresses the growth of triple-negative tumors in vivo[J].Plos One,2014,9(3):e92853.