Hemolytic Characteristic And Cytotoxic Effect Of Karenia Mikimotoi Extract On Some Tumor Cells

Karenia mikimotoi was one of the most notorious harmful algal blooms forming dinoflagellates in the world. It produced some second metabolites such as hemolytic toxin and cytolytic toxin etc., which might be responsible for the high mortality of fish and invertebrate during the K. mikimotoi bloom.In order to clarify the hemolytic and cytotoxic mechanisms of K. mikimotoi, the hemolytic properties of K mikimotoi was analyzed, and the effects of exogenous membrane lipid such as lecithin, sphingomyelin, L-a-phosphatidic acid, cholesterol, mixed gangliosides and four single ganglioside on the hemolysis induced by K. mikimotoi extract were studied. Secondly, the sensitivities of some erythrocytes from different animals such as rabbit, rat and fish to the K. mikimotoi extract were evaluated, and the total gangliosides in the erythrocytes membrane were detected by colorimetry. The potential relationship between the hemolytic activity of K. mikimotoi extract to different erythrocytes and level of LBSA in the erythrocytes was discussed. Finally, the effects of K. mikimotoi extract on three cancer cells such as Hepg2, Hela, and A549 were investigated by MTT assay and the relative amount of GM1 in the cancer cells membrane were detected by immunofluorescence assay.The results showed that K mikimotoi hemolysin had a strong hemolytic activity. Only gangliosides of the five membrane lipids assayed significantly inhibited the hemolysis induced by K. mikimotoi extract (P<0.05). Hemolytic percentage decreased to 16.05% after 10 min addition of gangliosides, while that of control was 35.65%. The GMl, GDla, GDlb and GTIb, and their mixure all significantly inhibited the hemolysis induced by K. mikimotoi extract (P<0.05). Hemolytic percentage decreased to 21.35% after 20 min addition of gangliosides, while that of control was 73.17%(P <0.05). The rabbit erythrocytes was the most sensitive to the hemolytic toxin. The hemolytic percentage of rabbit erythrocytes was significantly higher than those of rat (P<0.05) and fish (P<0.01). The amount of lipid-bind sialic acid (LBSA) on frozen dried membrane of rabbit was 672.08μg/g, significantly higher than that of rat (585.97μg/g) (P<0.05) and that of fish (431.52μg/g) (P<0.01). There was a significant correlation between the sensitivities of different erythrocytes to the hemolytic toxin and the amount of gangliosides on the erythrocytes membrane. The K. mikimotoi extract significantly inhibited the proliferation of Hela, Hepg2 and A549 cells in a concentration-dependent manner with some differences, suggesting the cytotoxicity of the extract. Hela and A549 cells were more sensitve to the the K. mikimotoi extract than Hepg2 cell. However, no evident correlation between the proliferation inhibition and amount of GM1 in the cancer cells membrane was found.In conclusion, exogenous gangliosides had a potent inhibition on the hemolysis induced by hemolytic toxin from K mikimotoi. There was a significant correlation between the sensitivities of different erythrocytes to the hemolytic toxin and the amount of gangliosides on the erythrocytes membrane. The K. mikimotoi extract significantly inhibited the proliferation of Hela, Hepg2 and A549 cells in a concentration-dependent manner. However, no evident correlation between the proliferation inhibition and amount of GM1 in the cancer cells membrane was found, suggesting the complexity in target of cytotoxicity of K. mikimotoi extract on cell membrane.