The Preliminary Studies On The Separation And Purification Of Earthworm Fibrinolytic Protein And Its Effects On MCF-7Cell Growth And Adhesion

Earthworm is one of traditional Chinese medicinal materials with the function of promoting blood circulation to remove blood stasis and clearing and activating the channels and collaterals. In1983, Japanese researcher purified a group of enzymes named Lumbrokinase with strong fibrinolytic activity from earthworm, and he thought they would be a new drug for dissolving thrombus. Since then, study on earthworm pharmacological components attracts more and more attention. With the development of study on the pharmacological effects of earthworm, its antitumor activity was noticed by researchers gradually. However, most of the reseach is about the antitumor effect of crude extracts from eathworm. What’s more, our preliminary study indicates that there is some kind of protein which can inhibit tumor’s growth in its body. Therefore, this paper separated and purified a kind of protein ingredient named EFP which has antitumor effcet on MCF-7cells(a kind of breast cancer cell) from fresh Eisenia fetida. And we also explored the mechanism of resistance of tumor cell adhesion caused by EFP. What’s more, in this paper, we studied the change of MCF-7cells’growth after dealing with paclitaxel companied with EFP, too.And here are the results of specific experiments followed:1. The separation, purification and identification of earthworm antitumor protein (EFP).We detected the MCF-7cell viability using CCK-8after being dealt with eathworm extracts and in this way we screened the protein ingredient which had the highest antitumor activity. The process of separation and purification is described like next. Prepare slurry of earthworm, salt out with saturated ammonium suliate, screen the antitumor activity of the protein precipitation with cell experiment. And then after a series of purification operation using DEAE anion exchange chromatography, S200gel chromatography and G10gel chromatography, we got a single protein with high antitumor activity finally.After detecting the properties of the protein, we got that its molecular weight was 25kDa approximately using electrophoretic analysis. And the fibrin plate experiment showed the protein had a very strong dissolution effect on fibrous protein. EFP was analyzed by Beijing huada protein research center, and the LC-MS result showed that EFP and Q8MX72(a kind of fibrinolytic enzyme from Eisenia fetida) have highly similarity.2. The study on antitumor activity of EFP and mechanism of its anti-adhension We used CCK-8to detect the MCF-7cell viability after being dealt with EFP, and the result showed that EFP could inhibit the growth and survival of MCF-7cells significantly and the effect was weaker than paclitaxel did. The wound healing assay showed that EFP could also inhibit the growth and migration of MCF-7cells significantly. What’s more, during the process of the experiments, we also found that EFP had a strong antiadhension effect on MCF-7cells. And after detecting the expression of cell adhension factors FAK、CD44v6using RT-PCR and Western Blot, the results showed that EFP could down-regrulate the expression of the two cell adhension factors significantly at the level of mRNA and protein. These results indicated that EFP playing a role in antiadhension activity may be related with down-regrulating the expression of FAK-. CD44v6.3. The antitumor activity after the joint use of EFP and paclitaxel We detected the change of antitumor activity after the joint use of EFP and paclitaxel using CCK-8, and the result showed that the two samples had synergistic effect after being used together and the activity of paclitaxel increased significantly. We detected the change of β-tublin after being dealt with EFP and paclitaxel solely as well as together using the method of immunofluorescent staining. The results showed that there were no significant difference between combination group and paclitaxel alone group, and when dealt with EFP alone, the β-tublin changed little. These results indicated that EFP could not increase the ability of paclitaxel in inhibiting microtubule depolymerization.In conclusion, EFP, the earthworm fibrinolytic enzyme A, could inhibit the growth of MCF-7cells, and also has the ability of inhibiting cell migration and adhension. What’ more, EFP could down-regrulate the expression of FAK and CD44v6. After using with paclitaxel together, the antitumor activity is increased, and this phenomenon has no significant relationship with EFP increasing the ability of paclitaxel in inhibiting microtubule depolymerization.In this paper, we separated and purified the antitumor activity protein (EFP) from earthworm, and studied the effect of EFP for the further step. Our work provides a experimental basis for the new application of earthworm in clinic.