| Objective: The mechanism of apoptosis research is one of the highlights in the1990s. Apoptosis plays great roles on a variety of physiological and pathologicalprocess. Apoptosis is important aspect in chemotherapy-induced tumor cell death andrestore apoptosis deterred in tumor cell by many antitumor drugs and natural products.It is well known that caspase-dependent apoptosis is characterized by the activation ofeither the extrinsic pathway initiated by activation of death receptors leading to cleavageof caspase-8, or the intrinsic pathway triggered by mitochondrial depolarization, releaseof cytochrome c, and subsequent activation of caspase-9. Intracellular reactive oxygenspecies (ROS) is considered to be a death signal in apoptosis. It is well known that atphysiological low levels, ROS serve as signaling messenger to mediate variousbiological responses, including cell proliferation, angiogenesis, innate immunity, geneexpression, apoptosis and senescence.Fucoidan, isolated from brown algae and marine invertebrates, compared to othersulfated polysaccharides, the fucoidan extracted from Sporophyll of Undariapinnatifida has higher sulfate and L-fucose content. It has recently been reported thatfucoidan have wide variety of biological activities, including anticoagulant, antivirus,anti-angiogenesis, immuno-modulation and antitumor activities. ROS inducesdepolarization of the MMP and release of cytochrome c from mitochondria into thecytosol, where cytochrome c triggers caspase-9activation and initiates caspases cascadewhich terminates cell to apoptosis. Tumor cells are more sensitive to fluctuation of ROSlevel than normal cells; therefore, ROS is also considered as an important target forantitumor drug research.Method:1. The fucoidan was purified by alcohol precipitation and the contents oftotal carbohydrate was detected by the phenol-sulfuric acid reaction, the sulfate radical and uronic acid were measured by BaCl2-gelation and sulfuric acid-carbazolecolorimetric method, respectively. Meanwhile, the molecular weight of the sample wasevaluated by size exclusion chromatography using a TSK-gel G3000PWXL(TOSOH,Tokyo, Japan). The optical rotation was tested at589nm by the WZZ-1polarimeter at20oC.2. MTT assay was used to measure the effect of fucoidan on growth inhibition ofSMMC-7721cells. The distribution of cells in the different phases of the cell cycle,which treated with fucoidan for24h and48h, was assayed by measuring the DNAcontent of nuclei labeled with propidium iodide (PI). The AnnexinV/PI doublestaining was used to determine the apoptosis rate.3. Using Hoechst33258staining to observe the change of cell and mitochondrialultrastructure through inverted fluorescence microscope.4. The changes in mitochondrial function were evaluated to mitochondrialmembrane potential using fluorochrome dye JC-1and the generation of intracellularROS was monitored via the detection of the fluorescent probe DCFH-DA by flowcytometry.5. The release of cytochreme c was detected by immunofluorescence method.ELISA was used to show the release of cytochreme c from the mitochondria into thecytosol in SMMC-7721cells after treatment the fucoidan.6. Western blot was used to analyze the expression of Bcl-2and Bax proteins.RT-PCR was used to evaluate the level of livin and xiap expression in SMMC-7721cells to investigate the mechanism of fucoidan on apoptosis.7. The levels of GSH, MDA, T-AOC and the activity of SOD in the cells weredetected by spectro photometric assays.Results:1.The fucoidan sample consisted mainly of carbohydrates (79.14%)，sulfates (21%) and uronic acid (10.895%) with fucose and galactose constituting themonosaccharide composition. The molecular weight of fucoidan was about10.44×104Da. The optical rotation of the fucoidan (0.6mg/mL) showed a value of0.99°at20°C.2. SMMC-7721cells were exposed to fucoidan and observed by light invertedmicroscopy. In control group, cultured cells were in a good condition: grew to polygonwith excellent refraction ability; while cells in administration group showed shrink, badrefraction, and more dead cells in culture medium. MTT assays was applied to observethe inhibitory effects of fucoidan at6,12,24,48and72h. The results showed that theinhibitory rate of SMMC-7721cells increased significantly with the increasing dosage and time of exposure compared with control group (P<0.01).There exists an obvioustime-effect and dose-effect relationship. Apoptosis was measured by AnnexinV/FITCdouble staining using flow cytometry. The results showed that the percentage ofapoptotic cells was increased significantly with increasing dosage.3. SMMC-7721cells were treated with500and1000μg/mL fucoidan.Morphological features of apoptosis nucleus were stained by Hoechst33258andapoptotic changes were observed by microscopy. The results showed fucoidan couldinduce typical apoptosis: cell shrinkage, chromatin condensation.4. The number of mitochondria was decreased. The mitochondrion with JC-1probes staining was detected by FCM. The results showed the content of thecharacteristic light fluorescence was increased and MMP was decreased with increasingdosage. There was a significant difference between the fucoidan groups and controlgroup.5. Cyt C in the endochylema protein of SMMC-7721cells treated with fucoidan24h. Results showed that the content of Cyt C had an increasing trend with theincreasing dosage in endochylema. The relative activities of caspase-9, caspase-3,caspase-8were all increased.6. The Western blot assay was used to detect the Bax and Bcl-2expression of totalprotein in SMMC-7721cells treated with fucoidan for24h. Results showed that the Baxand Bcl-2expression and Ratio of Bax/Bcl-2all increased with the increasing offucoidan. Meanwhile, Livin and Xiap were all decreased in mRNA levels.7. Biochemical kits were used to study activities of ROS, SOD, GSH, T-AOC andcontent of MDA in SMMC-7721cells24h after of500and1000μg/mL fucoidantreatment. The results showed that the activity of SOD was decreased, and it had asignificant changes between fucoidan administration groups and control group (P<0.01);the content of MDA was increased significantly at different exposed groups comparedwith control (P<0.01). The content of GSH was decreased, ROS was increased in cells.Conclusions:1. Our results indicated that intracellular GSH depletion, ROSaccumulation, mitochondria oxidative damage may be closely correlated with the cellsapoptosis induced by fucoidan.2. These findings suggest that fucoidan isolated from U. pinnatifida might induceapoptosis of SMMC-7721cells via ROS-mediated mitochondrion pathway. |
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