title: Earthworm fibrinolytic enzyme: anti-tumor activity on human hepatoma cells in vitro and in vivo.
abstract: BACKGROUND
  The earthworm fibrinolytic enzyme (EFE) is a complex protein enzyme that is widely distributed in the earthworm's digestive cavity. Possessing strong protein hydrolysis activity, EFE not only has a direct effect on fibrin, but also can activate plasminogen. Its therapeutic and preventative effects on thrombosis-related disease have been confirmed clinically. Recently, there has been increased interest in the anti-tumor activity of EFE. In this study, the anti-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, Huh7, PLC/PRF/5 and HepG2. After treatment with EFE in various concentrations, the inhibition of the rate of cell proliferation was measured. For the in vivo studies, tumor-bearing models xenografted with Huh7 cells were developed in nude mice, and then the mice were fed with EFE once a day for 4 weeks, and the control group received only saline. An inhibitory effect on tumor growth was observed. Also, apoptosis was observed with flow cytometric assay and fluorescent dye staining with acridine orange and ethidium bromide (AO/EB). The expression of matrix metalloproteinase 2 (MMP-2) were detected by Western blotting assay.
  
  
  RESULTS
  After treatment with various concentrations of EFE, the proliferation of all hepatoma cell lines was suppressed to varying degrees in vitro. The IC(50) 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 was significantly inhibited in vivo. The tumor inhibitory rates in the EFE 500 uku/(kgxd) and 1000 uku/(kgxd) groups were 46.08% (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 various groups. The population in sub-G(1) stage was more in the EFE treated groups than in the control group according to flow cytometric 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, the apoptotic morphological changes could be detected more significantly in the EFE treated groups than in the untreated groups. 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 a dose dependent manner. Western blotting assay showed that, after treatment with EFE, the secretions of MMP-2 were significantly inhibited in HLE and Huh7 cells.
  
  
  CONCLUSIONS
  EFE showed significant anti-tumor activity in hepatoma cells both in vitro and in vivo, which may be because EFE could induce apoptosis of hepatoma cells and inhibit the expression of MMP-2. This suggests that EFE has a potential role in the treatment of hepatoma.
authors.affiliation: Department of Gastroenterology, Affiliated Zhongda Hospital, Southeast University, Nanjing 210009, China. njchenhong66@yahoo.com.cn
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
authors.forename: Shoichi
authors.initials: S
authors.lastname: Takahashi
authors.forename: Michio
authors.initials: M
authors.lastname: Imamura
authors.forename: Eiko
authors.initials: E
authors.lastname: Okutani
authors.forename: Zhi-guo
authors.initials: ZG
authors.lastname: Zhang
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Chayama
authors.forename: Bao-an
authors.initials: BA
authors.lastname: Chen
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D005434:Flow Cytometry; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000188:drug therapy* / Q000473:pathology; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009835:Oligochaeta / Q000201:enzymology*; D014183:Transplantation, Heterologous
chemical_list: D000970:Antineoplastic Agents; D005343:Fibrinolytic Agents; D020778:Matrix Metalloproteinase 2