Original Article PtjJ01KOhcroC980byreu.18iBoon3grS20nt1Ni-ona0605,catl-40iJl1oe/08a1jeAftc96py2bCratfnciloci.n1rleti0Fc-ra1iel1evB2Rioaecdhiceaml eiRstfersyefa nrecdhNcJuapttraitnsion of fermented rice vinegar sediment (Kurozu moromimatsu) in a diethylnitrosamineinduced hepatocellular carcinoma animal model Toru Shizuma,1,* Kazuo Ishiwata,1 Masanobu Nagano,2 Hidezo Mori1 and Naoto Fukuyama1 1Department of Physiology, School of Medicine, Tokai University, 143, Shimokasuya, Isehara, Kanagawa 2591193, Japan 2Sakamoto Kurozu Inc., 2115, Uenosonochou, Kagoshimashi, Kagoshima 8900052, Japan (Re?ce?i(vRedec1e4ivSeepdte1m4beSre,p20te1m0; bAecrc,ep2t0e1d02;5AOcctcoebpetre,d202150; OPucbtloisbheedr, o2n0li1n0e;17 June, 2011) svCKtiurdi2TCerceoa0hotdtze0ipusdvt?yheimreusigsooaCehnrro,oitgmodm©ipinmisem2atnorl0ainw0batss?ucouctJriAekoCissntsBit,straNhiapbrentruiodctsipeloerdneridplmyirLseotcinrdciittuebecnoudtstf.ieoKd,nuuwrinohdziaeucnr,hytahpmejaeterrefmderimriutmmseo,unfpntetrhodee- Japanese black vinegar produced from unpolished rice. Here, we examined the protective effects of Kurozu moromimatsu in a diethylnitrosamineinduced model of hepatocellular carcinoma. Thirtytwo F344 rats were divided into two groups; the control group received basal CE2 diet, and the Kurozu moromimatsu group received CE2 diet containing Kurozu moromimatsu. At 16 weeks after initial intraperitoneal administration of diethylnitro samine (150 mg/kg/week), serum was collected from half the rats. These rats were sacrificed and the liver was resected for histo logical examination of hematoxylineosinstained sections and assay of matrix metalloproteinase2 and matrix metalloproteinase 9 levels in tumor tissues. Glutathione Stransferase placental form positive foci were evaluated by immunostaining for glutathione Stransferase placental form. The remaining rats were maintained for evaluation of survival. There were no significant differences of serum transaminases, tumor necrosis factoralpha, and also no marked hepatic histological differences, between the two groups. However, the size of hepatocellular carcinomas was greatly decreased and the levels of activated matrix metalloproteinase2 and 9 were significantly reduced in the Kurozu moromimatsu group. Further, survival was significantly prolonged in the Kurozu moromimatsu group compared with the control. These results indicate that Kurozu moromimatsu inhibited the growth of hepatocellular carcinoma. Key Words: Kurozu moromimatsu, hepatocellular carcinoma K KIunrtorzoudisuacttiroadnitional black vinegar produced from unpolished rice and has been widely used in Japan as seasoning for food or as a health supplement. Kurozu is produced mainly in Kagoshima, Japan, by fermentation of vinegar with several bacilli for more than one year in earthenware jars. The supernatant is Kurozu, and the sediment at the bottom of the jars is called Kurozu moromimatsu (Kurozu-M), and contains high levels of amino acids, minerals, organic materials and so on. Anti-cancer effects of ethyl acetate extract of Kurozu have been reported in vitro(1) and in an animal model of colon cancer.(2) We previously reported that Kurozu-M inhibited development of human colon cancer cells, LoVo cells, in a transplanted rodent model.(3) On the other hand, the therapeutic effect of Kurozu-M on hepatocellular carcinoma (HCC) has not yet been reported in animals or humans. Recently, an anti-cancer effect of coffee drink on HCC has been reported in humans,(4,5) and the presence of dietary factors that inhibit HCC has been detected in an animal doi: 10.3164/jcbn.10112 ©2011 JCBN model.(6) However, effective dietary factors to inhibit the develop- ment of HCC remain to be identified. The most common cause of HCC is thought to be hepatitis C virus (HCV), followed by hepatitis B virus (HBV) infection in Japan, and the number of deaths from HCC is more than 30,000/ year; it is the third most important cause of cancer death in males in Japan. Moreover, HCC is the fifth most common cancer world- wide.(7) Recently, drugs directed against HCC-related molecular targets have entered clinical trial.(8) However, at present, the impact on the survival rate in HCC patients is limited owing to high frequencies of recurrence and metastasis and the background of often-severe liver dysfunction. Several matrix metalloproteinase (MMP)s, which are zinc- dependent endopeptidases degrading the extracellular matrix, are related to development and metastasis of various solid cancers, including HCC.(8) Moreover, MMPs can promote the activation of insulin-like growth factor, which promotes tumor development and growth.(9,10) In particular, MMP-2 and MMP-9 (gelatinases) play important roles in tumor angiogenesis and cancer growth. HCC is typically a hypervascular cancer, and angiogenesis is important in its early stages.(11) Therefore, inhibition of angio- genesis, for example, by inhibition of activation of MMPs, might improve the prognosis of HCC patients. In this work, we examined the protective effects of Kurozu-M in a dietheylnitrosamine (DEN)-induced HCC experimental model, focusing on suppression of activated MMPs. Materials and Methods Experimental animal model. The experimental procedures were approved by the Animal Experimentation Committee, School of Medicine, Tokai University, Japan. Thirty-two F344 male rats (8 weeks of age) were obtained from CLEA Japan Inc. (Tokyo, Japan) and bred under specific pathogen-free conditions at a room temperature of 24–25°C, under constant humidity, with a 12-h light/dark cycle. The rats were randomized into two groups; the control group received basal CE-2 diet (CLEA Japan Inc., Tokyo, Japan) (n = 16) and the Kurozu-M group received CE-2 diet including 2% Kurozu-M (Sakamoto Kurozu Inc., Kagoshima, Japan) (n = 16). The volume of Kurozu-M added to the diet of rats was chosen based on the typical volume of ingestion by humans, adjusted for body weight. Basal CE-2 diet and special diet *To whom correspondence should be addressed. Email: shizuma@is.icc.utokai.ac.jp J. Clin. Biochem. Nutr. | July 2011 | vol. 49 | no. 1 | 31–35