| 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. |
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