Nattokinase Crude Extract Inhibits Hepatocellular Carcinoma Growth in Mice

J Microbiol Biotechnol. 2019 Aug 28;29(8):1281-1287. doi: 10.4014/jmb.1812.12058.


Nattokinase (NK, E.C. is a serine protease produced by Bacillus subtilis natto that shows promise for the treatment of thrombotic disease. In this study, we assessed the effects of NK on the development of hepatocellular carcinoma (HCC), a principal malignancy of the liver that causes morbidity and mortality worldwide. Crude extracts of NK (NCE) were isolated from fermentation medium by centrifugation and separated into three fractions (<10 K, 100~30 K and >30K). Orthotopic HCC mouse models were established and NCE was administered by oral gavage. H&E staining was performed to examine the pathology of HCC livers. Immunohistochemistry and immunofluorescence were used to evaluate FOXM1, CD31, CD44 and vimentin expression in the liver. Compared to PBS groups, NCE increased the survival rates of HCC-bearing mice to 31% and decreased ascites. Low-intensity ultrasound imaging showed that the hypoechoic mass area was lower in NCE-treated mice and that tumor growth significantly decreased. IHC staining showed that the expression of FOXM1 was inhibited by NCE treatment. Immunofluorescence results revealed lower levels of CD31, CD44 and vimentin in the NCE groups. Taken together, these data demonstrate that NCE from Bacillus subtilis natto improves survival and inhibits tumor growth in HCC mice.

Keywords: Hepatocellular carcinoma; cancer; nattokinase; tumor growth.

MeSH terms

  • Animals
  • Bacillus subtilis / enzymology
  • Carcinoma, Hepatocellular / drug therapy*
  • Carcinoma, Hepatocellular / pathology
  • Complex Mixtures / isolation & purification
  • Complex Mixtures / pharmacology*
  • Disease Models, Animal
  • Fermentation
  • Fibrinolytic Agents / pharmacology
  • Forkhead Box Protein M1 / analysis
  • Hyaluronan Receptors / analysis
  • Liver / metabolism
  • Liver Neoplasms / drug therapy*
  • Liver Neoplasms / pathology
  • Mice
  • Mice, Inbred C57BL
  • Platelet Endothelial Cell Adhesion Molecule-1 / analysis
  • Subtilisins / isolation & purification
  • Subtilisins / pharmacology*
  • Vimentin / analysis


  • Cd44 protein, mouse
  • Complex Mixtures
  • Fibrinolytic Agents
  • Forkhead Box Protein M1
  • Foxm1 protein, mouse
  • Hyaluronan Receptors
  • Pecam1 protein, mouse
  • Platelet Endothelial Cell Adhesion Molecule-1
  • Vimentin
  • Subtilisins
  • nattokinase