Objective: To investigate the effects of fucoidan inducing impairment of human osteosarcoma cell 143B, as well its mechanisms. Methods: After 143B cells were treated with different concentrations of FUC (0, 0.5, 1, 10, 100, 400, 800 μg/ml) for 48 h, the cell viability and dehydrogenase (LDH) level were detected by MTT assay and chemical colorimetry with six multiple wells for each concentration. Based on MTT results, we determined the value of IC50 was 244.5 μg/ml. The follow-up experiments were divided into control group (without FUC), FUC (10 μg/ml)-treated group, FUC (100 μg/ml)-treated group, FUC (400 μg/ml)-treated group and positive group (resveratrol, 40 μmol/L). There were four multiple wells for each concentration, and each experiment was repeated at least three times. Flow cytometry was performed to detect cell apoptosis and intracellular reactive oxygen species (ROS) level; acridine orange (AO) staining and lyso-tracker red staining were used to observe the autophagolysosome formation; chemical colorimetric analysis was performed to determine malondialdehyde (MDA) content and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px); Western blot was used to detect protein expressions of nuclear factor E2-associated factor 2 (Nrf2), heme oxygenase 1 (HO-1) and autophagy-associated proteins including microtubule-associated light chain protein 3 (LC-3), Atg7, Beclin-1 and p62. Results: Compared with control group, the cell viability was decreased significantly in FUC (100～400 μg/ml)-treated groups (P＜0.01); LDH levels in the supernatant (P＜0.05 or P＜0.01), the percentage of cell apoptosis (P＜0.01), intracellular ROS level and MDA content (P＜0.01) were increased remarkably; protein expressions of Atg7 and Beclin-1 were upregulated (P＜0.05 or P＜0.01); the conversion from LC-3I to LC-3II was significant (P＜0.01) together with elevation of autophagolysosome formation (P＜0.05 or P＜0.01); while the activities of SOD and GSH-Px and protein expressions of Nrf2, HO-1 and p62 were decreased remarkably (P＜0.05 or P＜0.01). Conclusion: FUC (100～400 μg/ml) treatment induces oxidative damage and autophagic death in osteosarcoma 143B cells.
目的: 探讨岩藻多糖(FUC)诱导人骨肉瘤细胞143B损伤作用及分子机制。方法: 采用不同浓度FUC(0、0.5、1、10、100、400、800 μg/ml)处理143B细胞48 h后,通过MTT法和化学比色法检测FUC对143B细胞活性和乳酸脱氢酶(LDH)释放的影响,每个浓度设6个复孔。根据MTT结果计算出IC50为244.5 μg/ml。后续实验分组为对照组(不加FUC)、FUC(10 μg/ml)组、FUC(100 μg/ml)组、FUC(400 μg/ml)和阳性对照组(白藜芦醇,40 μmol/L),每个浓度设4个复孔,每组实验至少重复3次。流式细胞术检测细胞凋亡情况和细胞内活性氧(ROS)含量的变化;吖啶橙(AO)染色和Lyso-Tracker Red染色观察细胞自噬溶酶体形成情况;化学比色法检测丙二醛(MDA)含量、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的活性; Western blot法检测细胞中核因子E2相关因子2(Nrf2)、血红素氧合酶1(HO-1)和自噬相关蛋白微管相关轻链蛋白3(LC-3)、Atg7、Beclin-1和p62等蛋白表达情况。结果: 与对照组比较,FUC(100和400 μg/ml)处理组143B细胞活性明显降低(P＜0.01),上清液中LDH水平(P＜0.05或P＜0.01)、细胞凋亡率(P＜0.01)、细胞内ROS水平和MDA含量显著增加(P＜0.01)、Atg7和Beclin-1等蛋白表达明显上调(P＜0.05或P＜0.01),LC-3I向LC-3II转换显著及自噬溶酶体形成增加(P＜ 0.01),而细胞中SOD活性、GSH-Px活性和Nrf2、HO-1及p62蛋白表达水平明显降低(P＜0.05或P＜0.01)。结论: 100和400 μg/ml的FUC处理骨肉瘤细胞143B,可诱导其氧化损伤和自噬性死亡。.
Keywords: autophagy; cell culture; fucoidan; osteosarcoma 143B cells; oxidative damage.