Fig. 5. MMP2 and MMP9 levels (ng/mg protein). Total MMP2 amounted to 6.7 ± 2.6 in the control group and 3.6 ± 1.7 in KurozuM group. The corresponding values for activated MMP2 were 0.28 ± 0.12 and 0.12 ± 0.06, respectively (a). Total MMP9 amounted to 20.6 ± 4.6 in the control group and 12.0 ± 2.7 in KurozuM group. The corresponding values for activated MMP9 were 1.17 ± 0.38 and 0.51 ± 0.19, respectively (b). Total and activated MMP2 and MMP9 levels were all significantly ( p<0.001) reduced in the KurozuM group compared with the control group. Fig. 6. Survival rate by the KaplanMeier method. In the control group, all 8 rats died within 36 weeks after initial administration of DEN. On the other hand, the survival rate in the KurozuM group was 37.5% (3/8) at 48 weeks. The survival rate in the KurozuM group was significantly higher than that in the control group (p<0.001). development of HCC,(13) though exceptionally, HCC rarely develops in patients with autoimmune hepatitis. However, our study revealed that the extents of histological liver fibrosis or inflammatory change, as well as the serum levels of transaminases or hyaluronic acid as a marker of liver fibrosis, were not significantly different between the Kurozu-M group and the control. Therefore, inhibi- tion of HCC growth in the Kurozu-M group in this study was not associated with differences in background factors. It has been reported that iron overload, which induces oxidative stress, aggra- vates liver diseases(14) and increases the risk of malignancy.(15) However, the levels of iron in the diets of the control and Kurozu- M groups were essentially the same in this study. Increased levels of the gelatinases; MMP-2 and MMP-9 are considered prognostic for solid cancers, including HCC.(16) In fact, it has been suggested that inhibition of gelatinases can suppress development of HCC in an animal model.(17) Since activated MMP-2 promotes angiogenesis,(18) suppression of MMP-2 and MMP-9 by Kurozu-M may have led to inhibition of HCC growth in our model. We found that the number of GST-P-positive foci 34 (which were estimated foci corresponding to low-grade dysplastic nodules) in the Kurozu-M group showed no difference from the control, but the size of developed GST-P-positive foci was greatly decreased. Thus, a possible explanation is that Kurozu-M inhibited angiogenesis, which may not greatly influence the initial progres- sion from premalignant tumor, but may be an important promoting factor at the stage of high-grade dysplastic nodules or early HCC. This would be consistent with our findings. Activation of MMPs is important for carcinogenesis. MMP-2 is activated by membrane type (MT)-1-MMP.(19) It is also activated by peroxylnitrite(20) which exhibits cytotoxicity via direct oxida- tion of sulfhydryl groups, leading to severe tissue damage. We have already reported that Kurozu-M inhibits nitrotyrosine gener- ation in colon cancer cells.(3) Nitrotyrosine is produced in vivo via two pathways, i.e., from the reaction of tyrosine with peroxyl- nitrite, which is generated from O2 − and nitric oxide (NO),(21) and from the reaction of tyrosine with nitrite, catalyzed by myeloper- oxidase (MPO).(22) Therefore, Kurozu-M might reduce MMP-2 activation by inhibiting the generation of peroxylnitrite. In addi- tion, MMP-9 is activated by MMP-2,(23) as well as by several cytokines.(24) However, in our study, serum levels of TNF-α and IL-2 were not increased. The active component of Kurozu-M remains to be identified. Acetic acid, which is the main component of Kurozu, is not included in Kurozu-M. However, Kurozu-M contains amino acids, oligopeptide, organic materials including metabolites of lactic acid or Koji molds, minerals, saccharides, lipids, etc. Candidate active components include branched chain amino acid (BCAA), which are widely used for the treatment of protein malnutrition in LC patients. It was reported that BCAA suppressed insulin- resistance-based carcinogenesis in diabetic rats(7) and suppressed angiogenesis in a chemical carcinogenesis model.(7) Furthermore, various other amino acids have anti-oxidative activity, acting as radical scavengers.(25) Therefore, various amino acids may sup- press the generation of O2 − or peroxylnitrite, thereby suppressing activation of MMP-2. On the other hand, oligosaccharides are also candidate active components, because oligosaccharides from agar suppressed NO generation in vitro.(26) Oligosaccharides also reduced the activity of inducible NO synthase (iNOS),(27) which might lead to a decrease of peroxylnitrite generation. Organic materials, including metabolites of lactic acid or Koji formed in the earthenware jars are also potential anti-oxidants,(28) and anti- cancer agents such as bleomycin or mitomycin C or their deriva- tives are known to be produced as bacterial metabolites. doi: 10.3164/jcbn.10112 ©2011 JCBN