Background The earthworm fibrinolytic enzyme (EFE) is a complex protein enzyme that is widely distributed in theearthworm’s digestive cavity.Possessing strong protein hydrolysis activity,EFE not only has a direct effect on fibrin,butalso can activate plasminogen.Its therapeutic and preventative effects on thrombosis-related disease have beenconfirmed clinically.Recently,there has been increased interest in the anti-tumor activity of EFE.In this study,theanti-tumor activity of EFE,isolated from Eisenia foetida,on human hepatoma cells was evaluated in vitro and in vivo.The potential mechanisms involved were also studied.Methods In vitro experiments were performed in four human hepatoma cell lines:HLE,HuhT,PLC/PRF/5 and HepG2.After treatment with EFE in various concentrations,the inhibition of the rate of cell proliferation was measured.For the invivo studies,tumor-bearing models xenografted with Huh7 cells were developed in nude mice,and then the mice werefed with EFE once a day for 4 weeks,and the control group received only saline.An inhibitory effect on tumor growth wasobserved.Also,apoptosis was observed with flow cytometric assay and fluorescent dye staining with acridine orange andethidium bromide (AO/EB).The expression of matrix metalloproteinase 2 (MMP-2) were detected by Western blottingassay.Results After treatment with various concentrations of EFE,the proliferation of all hepatoma cell lines was suppressedto varying degrees in vitro.The IC5o for HLE,Huh7,PLC/PCF/5 and HepG2 were 2.11,5.87,25.29 and 17.30 uku/ml,respectively.After administration of EFE orally for 4 weeks,the growth of tumor xenograft of Huh7 cells in nude mice wassignificantly inhibited in vivo.The tumor inhibitory rates in the EFE 500 uku/(kg.d) and 1000 uku/(kg.d) groups were46.08% (compared with control group,P=0.026) and 57.52% (compared with control group,P=0.002) respectively.Meanwhile,the average weight of body,spleen or thymus did not show any remarkable differences among the variousgroups.The population in sub-Gstage was more in the EFE treated groups than in the control group according to flowcytometric assay.After treatment with EFE 0,5,10 uku/ml for 72 hours,the apoptotic rates were 3.5%,10.9% and 12.3%in HLE cells,and 5.0%,24.7% and 34.5% in Huh7 cells respectively.Under fluorescent staining with AO/EB,theapoptotic morphological changes could be detected more significantly in the EFE treated groups than in the untreatedgroups.After treatment with EFE in doses of 0,5,10 uku/ml for 72 hours,the apoptotic rates were 3.02%,8.76%,10.54%in HLE cells,and 3.95%,18.27%,30.89% in Huh7 cells respectively.The apoptosis-inducing effects of EFE occurred in adose dependent manner.Western blotting assay showed that,after treatment with EFE,the secretions of MMP-2 weresignificantly inhibited in HLE and Huh7 cells.Conclusions EFE showed significant anti-tumor activity in hepatoma cells both in vitro and in vivo,which may bebecause EFE could induce apoptosis of hepatoma cells and inhibit the expression of MMP-2.This suggests that EFE hasa potential role in the treatment of hepatoma.
