[Effects of fucoidan inducing impairment of human osteosarcoma cell 143B and its mechanism].

Abstract

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.