Isoliquiritigenin (ILTG), a flavonoid group compound, exists in some foodstuffs and herbal medicines such as licorice (Glycyrrhiza uralensis Fisher). Previously, we showed that ILTG can suppress azoxymethane (AOM)-induced colon carcinogenesis in ddY mice. In the present report, we present evidence that ILTG markedly decreases both prostaglandin E 2 (PGE 2 ) and nitric oxide (NO) production in RAW264.7 mouse macrophage cells. The decrease of PGE 2 was dependent on cyclooxygenase-2 (COX-2) expression and the decrease of NO appeared due to a decrease in inducible nitric oxide synthase (iNOS) protein expression. In mouse and human colon carcinoma cells, ILTG treatment suppressed cell growth and caused apoptosis. soliquiritigenin (ILTG), a flavonoid with a chalcone structure ( Fig. 1), is reported to have vasorelaxant, 1) anti-platelet aggregation, 2) and anti-allergic activity.3) With regard to effects on carcinogenesis and related processes, ILTG has been shown to inhibit ornithine decarboxylase activity and to suppress 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin carcinogenesis, 4) to reduce 32 P-incorporation into phospholipids of HeLa cells promoted by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), 5) to inhibit growth of prostate cancer cell lines 6) and to decrease tube formation in vascular endothelial cells.7) Recently, it was reported that ILTG can stimulate apoptotic activity by inhibition of glucose transmembrane transport and promotion of Bax expression in mouse melanoma cells. 8) We previously found that ILTG markedly inhibited induction of aberrant crypt foci (ACF) and colon carcinoma development in azoxymethane (AOM)-treated ddY mice. 9) To clarify the inhibition mechanisms, we conducted the present examination of the effects of ILTG on cyclooxygenase (COX) and inducible nitric oxide synthase (iNOS)-related parameters considered relevant to colon carcinogenesis 10, 11) using the RAW264.7 mouse macrophage cell line, in which COX and iNOS are induced by LPS stimulation. Furthermore, the effects of cell proliferation and induction of apoptosis in three colon cancer cell lines were examined. In order to determine whether ILTG is actually effective as a chemopreventer in other species, we also conducted an in vivo study in rats with putatively preneoplastic ACF lesions as the end-point. Materials and MethodsChemicals. ILTG was synthesized at the Department of Natural Medicine and Phytochemistry, Meiji Pharmaceutical University, Tokyo (Fig. 1). AOM, 5-fluorouracil (5-FU) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co (St. Louis, MO). Lipopolysaccharide (LPS) was purchased from Difco Labs (Detroit, MI). Propidium iodide was purchased from Nacalai Tesque (Kyoto).Cell culture. Mouse macrophage RAW264.7 cells, and RCN-9, rat colon cancer cells, were provided by the RIKEN Cell Bank (RCB) (Ibaraki). COLO-320DM, human colon cancer cells, were kindly provided by Ms. Mari Onozuka (Kyoto Prefectural University of Medicine, Kyoto). The Colon 26 line, mo...

Epidemiologic studies have found that obesity is associated with malignant grade and mortality in prostate cancer. Several adipokines have been implicated as putative mediating factors between obesity and prostate cancer. Fatty acid binding protein 4 (FABP4), a member of the cytoplasmic fatty acid binding protein multigene family, was recently identified as a novel adipokine. Although FABP4 is released from adipocytes and mean circulating concentrations of FABP4 are linked with obesity, effects of exogenous FABP4 on prostate cancer progression are unclear. In this study, we examined the effects of exogenous FABP4 on human prostate cancer cell progression. FABP4 treatment promoted serum-induced prostate cancer cell invasion in vitro. Furthermore, oleic acid promoted prostate cancer cell invasion only if FABP4 was present in the medium. These promoting effects were reduced by FABP4 inhibitor, which inhibits FABP4 binding to fatty acids. Immunostaining for FABP4 showed that exogenous FABP4 was taken up into DU145 cells in three-dimensional culture. In mice, treatment with FABP4 inhibitor reduced the subcutaneous growth and lung metastasis of prostate cancer cells. Immunohistochemical analysis showed that the number of apoptotic cells, positive for cleaved caspase-3 and cleaved PARP, was increased in subcutaneous tumors of FABP4 inhibitor-treated mice, as compared with control mice. These results suggest that exogenous FABP4 might promote human prostate cancer cell progression by binding with fatty acids. Additionally, exogenous FABP4 activated the PI3K/Akt pathway, independently of binding to fatty acids. Thus, FABP4 might be a key molecule to understand the mechanisms underlying the obesity-prostate cancer progression link.

Licorice, the roots and long-stalks of various species of Glycyrrhiza genus, has been used as a traditional folk medicine and foodstuff all over the world.2) Isoliquiritigenin is one of the plant pigments of licorice, and is a simple chalcone derivative, 4,2Ј,4Ј-trihydroxy-chalcone (see Fig. 1). We have reported the isolation of this compound from Egyptian licorice (Glycyrrhiza grabla), along with new and known 3-arylcoumarins.3) Shibata reported the anti-tumor promoter effects of various chalcone derivatives in vitro, and isoliquiritigenin exhibited potential activity. 4) Other biological activities for this compound have also been reported. For example, antitumor promoting activity on two-stage mouse skin carcinogenesis, 5) inhibitory effect on aldose reductase activity, 6) antiplatelet aggregation effect, 7) antioxidative and superoxide scavenging activities, 8) etc. However, all these experiments were in vitro or topical application in vivo, and there is no reported data of the effect by oral administration in vivo.Repetitive treatment with the organotropic colon carcinogen, azoxymethane (AOM), produces colon tumors in rodents exhibiting pathological features that are similar to sporadic forms in human colon cancer.9) The AOM-induced colon cancer model in rat has been used to evaluate the potency of chemopreventive agents against colon cancer. 10)Aberrant crypt foci (ACF) were first identified in methylene blue stained, whole-mount preparations of colonic mucosa from carcinogen-treated rodents, 11) and ACF have also been found in the colons of patients both with or without colorectal cancer.12,13) McLellan et al. reported that ACF may be a significant biological lesion in the development of colon tumors, and aberrant crypt (AC) formation is specific for exposure to colon carcinogens. 14) From these findings, the colonspecific, carcinogen-induced ACF model in rodents has been used as a short term screening to identify chemopreventive agents for colon cancer. 15,16) In the present studies, the effect of isoliquiritigenin on the AOM-induced ACF formation model in mice was studied. Based on the data obtained, AOM-induced murine colon carcinogenesis experiment was also studied. MATERIALS AND METHODSChemicals AOM was purchased from Sigma Chemical Co., Ltd. (St. Louis, MO, U.S.A.). Isoliquiritigenin (ILG) was synthesized by the methods of Hulle et al. 17) and purified by column chromatography (silica gel with a mixture of nhexane and ethyl acetate). After recrystallization, the purity was quantificated by HPLC (octadecyl silica (ODS) with a gradient of acetonitrile and water) as over 99.8%. The yield of this material was 38.6%.Animals Five-weeks old ddY male mice were obtained from Shizuoka Laboratory Animal Center (Hamamatsu, Shizuoka, Japan). Effect of Isoliquiritigenin on Murine Colon Aberrant Crypt FociFive-weeks old ddY mice were housed in temperature-and humidity-controlled animal quarters with a 12 h light/dark cycle and fed CE-2 diet. (CLEA Japan Co., Ltd.) On the 1st and 8th days, AOM (10 mg/kg b.w.) was...

Prostaglandin E 2 (PGE 2 ) plays a key role in osteolytic bone metastasis as well as roles in inflammation, cell growth, and tumor development. PGE 2 exerts its effects by binding and activating E-prostanoid receptor (EP). In this study, we propose a new approach for blocking EP-mediated cell signaling using a soluble chimeric EP2 fragment. Mammalian expression vectors encoding several human EP2 cDNAs were introduced into 293 cells and the culture medium was tested for their function as a decoy receptor for PGE 2 . PGE 2 binding assays revealed that culture medium containing the second extracellular region of EP2 (FuEP2/Ex2) had binding activity. FuEP2/Ex2 neutralized PGE 2 -induced cyclic AMP production, cyclic AMP -responsive element binding protein phosphorylation, and subsequent induction of cyclooxygenase-2, interleukin (IL)-1B, and IL-6 mRNAs. In human osteoblasts, this culture medium neutralized the induction of receptor activator of nuclear factor-KB ligand mRNA. A stable transfectant expressing FuEP2/Ex2 was established from human prostate cancer PC-3 cells (PC3-FuEP2/Ex2). PC3-FuEP2/Ex2 cells grew at similar rates to vector control cells under normal culture conditions, although PGE 2 -induced growth stimulation was suppressed. Intraosseous injection of PC3-FuEP2/Ex2 cells into the tibia of athymic nude mice revealed that the degrees of tumor growth and osteolysis were decreased compared with control cellinjected mice, with decreased osteoclasts and increased apoptotic cells. Furthermore, the cyclooxygenase-2, IL-1B, and IL-6 mRNA levels were reduced in the tumor lesions. These data suggest that FuEP2/Ex2 is useful for treating osteolytic bone metastasis and cancers that depend on EP signaling for their growth and development.

New drugs that inhibit the osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL)/RANK pathway have demonstrated efficacy for the treatment of bone metastasis. Toxicities induced by these drugs, however, including osteonecrosis of the jaw and hypocalcemia, may adversely affect therapy. The aim of this study was to identify additional therapeutic targets that can be combined with OPG/RANKL/RANK pathway inhibition in the treatment of prostate cancer bone metastasis. We established a stable transfectant that produces high levels of OPG mRNA and protein from PC-3 human prostate cancer cells (PC3-OPG). The culture medium of PC3-OPG cells significantly inhibited the differentiation of mouse monocytes into mature osteoclasts. Furthermore, when PC3-OPG cells were injected into the bones of nude mice, bone destruction and tumor-induced osteoclast formation were reduced. Injection into bone of the mixtures containing equal amounts of green fluorescent protein (GFP)-expressing PC-3 cells (PC3-GFP) and PC3-OPG cells also reduced bone destruction, compared to the control mixture. PC3-GFP cells were subsequently isolated from bone tumors and used for microarray analysis to assess changes in gene expression following osteolytic tumor growth inhibition by OPG. We selected the top 10 upregulated genes based on results from microarrays and confirmed mRNA expression of each gene by RT-PCR. The expression patterns of retinol-binding protein 4 (RBP4) and placenta-specific 8 (PLAC8) were consistent with microarray results. Expression of these genes was also increased in the bone tumors of PC3-GFP/PC3-OPG-injected mice. Knockdown of both RBP4 and PLAC8 by siRNA inhibited the growth of PC-3 cells in vitro. Thus, RBP4 and PLAC8 may become new therapeutic targets for prostate cancer bone metastasis, in combination with OPG/RANKL/RANK pathway inhibition.