A Study On The Anti-tumor Effects Of Coeomic Fluid Extracts From Earthworm Eisenia Foetida

The most important immune barrier of earthworm is coelomic fluid, which contains of many kinds of leukocytes and biologically active molecules to participate in the immune defense. The coelomic fluid of earthworms exhibits different biological functions, including bacteriostatic, proteolytic, haemocytolysis, agglutination, cytolytic (hemolytic) and mitogentic activities. It hasn't been reported that the coelomic fluid of earthworm shows nuclease activity. Recently, there has been increased interest in the anti-tumor activity of earthworm, especially in the anti-tumor activity of serine protease. We get extracts from coelomic fluid which has high nuclease activity. Our aim is to analyze the anti-tumor effects of the extracts from coelomic fluid and to further reveal the important role of nucleases in the immune defense.We use cell-free coelomic fluid as the experiment materials, and the coelomic fluid extract (CFE) , which has high nuclease activity, was purified by ammonium sulphate sedimentation and acetone precipitation. To investigate the nuclease activity of CFE, the agarose gel eletrophoresis of DNA and the formaldehyde degenerating agarose gel eletrophoresis of RNA were applied. The fibrin plate assay was used to test the fibrinolytic activity, and the suspension of rabbit red blood cells were used to test the blood coagulation activity and haemolysis activity of the extract. The CFE was studied by MTT assay and SRB assay methods to test its in vitro anti-tumor activity on tumor cell strains (Siha, SW480, Colo205 and PC12) and normal cell strain Cos7. Inverted optical microscope and Hoechst 33258 fluorescent staining were used to observe tumor cell morphology changes; the DNA ladder gel and single cell gel electrophoresis were applied to analyze DNA damage of target tumor cell lines, and flow cytometry was used to test the cycle of the target cell lines. Then we use cooperation test, Transwell migration assay and Transwell invasion assay to further study the function of nucleases in the anti-tumor activity.It is found that CFE was fibrinolytic as well as plasminogen-activating, but it lost any coagulation and haemolysis activity. The extract has very high nuclease activity, which can degrade plasmid DNA, genomic DNA of tumor cells as well as baker's yeast RNA. In vitro, the CFE showed concentration dependent manner and time dependent manner, and different cell lines showed different sensitivity to the extract. The target cells performanced typical changes of apoptosis; DNA ladder gel and single cell gel electrophoresis displayed that the extent of damage to chromatin was time-dependent; flow cytometry was used to detect the presence of apoptotic cells, and the CFE can inhibit the proliferation of target cells by blocking them in S phase. PMSF, heating and proteinase K digestion dramatically decreased anti-tumor activity, while trypsin digestion caused no dramatic decrease in anti-tumor activity. The purified nucleases were used to test their anti-tumor activity. The ribonuclease from earthworm can dramatically inhibit the growth of tumor cells. The deoxyribonuclease has no changes on the growth of tumor cells, but it can enhance the activity of non-DNase chromatography components and cisplatin. We also found that the DNase can inhibit the migration and invasion of tumor cells.The coelomic fluid extract may play anti-tumor activity by following ways: (1) serine protease can inhibit the proliferation of tumor cells by hydrolyzing the structural protein compotents and destroy the integrity of tumor cells. (2) Ribonucleases can enter target tumot cells by receptor-mediated pathway, and inhibit protein synthesis by degrading rRNA, (3)Deoxyribonucleases can enter target tumor cells though defect made by other biologic moleculars ,then they degrade genetic materials in nuclear, so the replication of DNA was blocked and the cells were stayed in S phage, (4)Deoxyribonucleases can decrease the aggregation of tumor cells as well as waken the migration and invasion ability of tumor cells by degrading the biofilms made of DNA. It will effectively increase the contact area anti-tumor drug and tumor cells. In a word, the nucleases in earthworm coelomic fluid may take part in the anti-tumor effects of extract, and we assumed that the nucleases are the defense moleculars in innate immunity of earthworm.