Mitsuru Futakuchi

Inhibition of mammary gland carcinogenesis by green tea catechins and other naturally occurring antioxidants in female Sprague-Dawley rats pretreated with 7,12-dimethylbenz[a]anthracene

Effects of the naturally occurring antioxidants on mammary gland carcinogenesis were examined in female Sprague-Dawley rats pretreated with 7,12-dimethylbenz[alpha]anthracene (DMBA). Groups of 15-16 7-week-old rats received a 50 mg/kg body weight intra-gastric dose of DMBA, and starting one week thereafter placed on diet containing 0.4% catechol, 1.0% gamma-oryzanol, 2.0% phytic acid, 1.0% green tea catechins (GTC), 1.0% tannic acid or basal diet alone for 35 weeks. Although the final incidences and multiplicities of mammary tumors were not significantly different between DMBA-treated groups, the numbers of survivors in the antioxidant-treated groups at the end of the experiment at week 36 were significantly higher than in the basal diet group. In particular, the survival rate of the GTC group at 93.8% strongly contrasted with that of only 33.3% for rats on the basal diet. At the end of week 18, when all the animals were still alive, the average size of palpable mammary tumors was significantly smaller in the catechol, phytic acid and catechins groups. These results indicate that antioxidants, and GTC in particular, inhibit rat mammary gland carcinogenesis after DMBA initiation.

Strong promoting activity of phenylethyl isothiocyanate and benzyl isothiocyanate on urinary bladder carcinogenesis in F344 male rats

Post‐initiation effects of phenylethyl isothiocyanate (PEITC) and benzyl isothiocyanate (BITC) on hepatocarcinogenesis and urinary bladder carcinogenesis were examined in rats pretreated with diethylnitrosamine (DEN) and N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN). Groups of 21 rats received a single intraperitoneal injection of 200 mg/kg body weight of DEN. Starting 2 days thereafter, they were administered 0.05% BBN in the drinking water for 4 weeks. Three days after completion of the carcinogen treatment, they were placed on a diet containing PEITC or BITC at a dose of 0.1%, or a basal diet alone for 32 weeks and then killed for autopsy. Further groups of 6 rats each were similarly treated with PEITC, BITC or basal diet alone for 32 weeks without prior DEN and BBN exposure. In the liver, although the incidences of liver tumors were not significantly affected, the multiplicity of foci larger than 0.5 cm in diameter was slightly increased by PEITC. In the urinary bladder, the incidences of papillary or nodular (PN) hyperplasias and carcinomas were significantly elevated by PEITC or BITC after DEN and BBN initiation. In the groups without initiation, PN hyperplasia was found in all rats of both PEITC and BITC groups, along with papillomas and carcinomas in some animals. Tumors and PN hyperplasias in the groups treated with PEITC and BITC are characterized by downward growth. Our results thus showed PEITC and BITC to be strong promoters of urinary bladder carcinogenesis with some complete carcinogenic potential. Int. J. Cancer 77:773–777, 1998.

Effects of arctiin on PhIP-induced mammary, colon and pancreatic carcinogenesis in female Sprague-Dawley rats and MeIQx-induced hepatocarcinogenesis in male F344 rats.

Chemopreventive effects of arctiin, a lignan isolated from Arctium lappa (burdock) seeds, on the initiation or post initiation period of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced mammary carcinogenesis in female rats and on 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-associated hepatocarcinogenesis in male rats were examined. In experiment 1, female Sprague-Dawley (SD) rats were given intragastric doses of 100 mg/kg body wt of PhIP once a week for 8 weeks as initiation. Groups of 20 rats each were treated with 0.2 or 0.02% arctiin during or after PhIP initiation. Control rats were fed 0.2 or 0.02% arctiin, or basal diet alone during the experimental period. Animals were killed at the end of week 48. Although the incidence of mammary carcinomas did not significantly differ among the PhIP-treated groups, multiplicity was significantly decreased in rats given 0.2 (0.7+/-0.7, P<0.05) or 0.02% (1.0+/-1.1, P<0.05) arctiin after PhIP initiation as compared with the PhIP alone controls (2.1+/-2.5). The average number of colon aberrant crypt foci was also significantly decreased in these two groups. Pancreas acidophilic foci were induced in PhIP treated animals with slight decrease in the multiplicity with arctiin during the initiation phase. For liver carcinogenesis, groups of 15 male F344 rats were given a single intraperitoneal injection of diethylnitrosamine (DEN) and starting 2 weeks later, they were administered 0.03% MeIQx in the diet, MeIQx together with 0.5% arctiin, 0.1% arctiin or basal diet for 6 weeks. They were subjected to two-third partial hepatectomy 3 weeks after DEN initiation and killed at the end of week 8 for glutathione S-transferase placental form (GST-P) immunohistochemistry. The numbers and areas of preneoplastic GST-P positive foci were elevated by the treatment with MeIQx, and further increased by the simultaneous treatment with arctiin. These results indicate that arctiin has a protective effect on PhIP-induced carcinogenesis particularly in the mammary gland in the promotion period. On the other hand, it may have a weak co-carcinogenic influence on MeIQx-induced hepatocarcinogenesis. In addition, the results suggested that PhIP is a weak pancreatic carcinogen in female SD rats, targeting acinar cells.

Experimental prostate carcinogenesis - rodent models.

A number of rodent models of prostate carcinoma development have been established to study mechanisms and modifying potential. All except for transgenic mouse models need long experimental periods for generation of a high yield of cancers. Spontaneous prostate tumor models, while not practical in terms of time and tumor incidences, allow the natural course of multistep neoplasia to be followed without a need for chemical exposure. Carcinogens, especially in combination with testosterone, can induce prostate carcinomas in rats, but none are prostate-specific, so that tumor development in other organs is a complicating factor. Induction of invasive prostate carcinomas in the rat frequently requires long-term administration of a pharmacological dose of testosterone with or without application of a chemical carcinogen. While there are several transgenic mouse models, each also has strong and weak points, and it is therefore necessary to select the best model for the purpose of any experimental study.

Cathepsin G Enhances Mammary Tumor–Induced Osteolysis by Generating Soluble Receptor Activator of Nuclear Factor-κB Ligand

Breast cancer commonly causes osteolytic metastases in bone, a process that is dependent on tumor-stromal interaction. Proteases play an important role in modulating tumor-stromal interactions in a manner that favors tumor establishment and progression. Whereas several studies have examined the role of proteases in modulating the bone microenvironment, little is currently known about their role in tumor-bone interaction during osteolytic metastasis. In cancer-induced osteolytic lesions, cleavage of receptor activator of nuclear factor-kappaB ligand (RANKL) to a soluble version (sRANKL) is critical for widespread osteoclast activation. Using a mouse model that mimics osteolytic changes associated with breast cancer-induced bone metastases, we identified cathepsin G, cathepsin K, matrix metalloproteinase (MMP)-9, and MMP13 to be proteases that are up-regulated at the tumor-bone interface using comparative cDNA microarray analysis and quantitative reverse transcription-PCR. Moreover, we showed that cathepsin G is capable of shedding the extracellular domain of RANKL, generating active sRANKL that is capable of inducing differentiation and activation of osteoclast precursors. The major source of cathepsin G at the tumor-bone interface seems to be osteoclasts that up-regulate production of cathepsin G via interaction with tumor cells. Furthermore, we showed that in vitro osteoclastogenesis is reduced by inhibition of cathepsin G in a coculture model and that in vivo inhibition of cathepsin G reduces mammary tumor-induced osteolysis. Together, our data indicate that cathepsin G activity at the tumor-bone interface plays an important role in mammary tumor-induced osteolysis and suggest that cathepsin G is a potentially novel therapeutic target in the treatment of breast cancer bone metastasis.

Multi-walled carbon nanotubes translocate into the pleural cavity and induce visceral mesothelial proliferation in rats

Multi‐walled carbon nanotubes have a fibrous structure similar to asbestos and induce mesothelioma when injected into the peritoneal cavity. In the present study, we investigated whether carbon nanotubes administered into the lung through the trachea induce mesothelial lesions. Male F344 rats were treated with 0.5 mL of 500 μg/mL suspensions of multi‐walled carbon nanotubes or crocidolite five times over a 9‐day period by intrapulmonary spraying. Pleural cavity lavage fluid, lung and chest wall were then collected. Multi‐walled carbon nanotubes and crocidolite were found mainly in alveolar macrophages and mediastinal lymph nodes. Importantly, the fibers were also found in the cell pellets of the pleural cavity lavage, mostly in macrophages. Both multi‐walled carbon nanotube and crocidolite treatment induced hyperplastic proliferative lesions of the visceral mesothelium, with their proliferating cell nuclear antigen indices approximately 10‐fold that of the vehicle control. The hyperplastic lesions were associated with inflammatory cell infiltration and inflammation‐induced fibrotic lesions of the pleural tissues. The fibers were not found in the mesothelial proliferative lesions themselves. In the pleural cavity, abundant inflammatory cell infiltration, mainly composed of macrophages, was observed. Conditioned cell culture media of macrophages treated with multi‐walled carbon nanotubes and crocidolite and the supernatants of pleural cavity lavage fluid from the dosed rats increased mesothelial cell proliferation in vitro, suggesting that mesothelial proliferative lesions were induced by inflammatory events in the lung and pleural cavity and likely mediated by macrophages. In conclusion, intrapulmonary administration of multi‐walled carbon nanotubes, like asbestos, induced mesothelial proliferation potentially associated with mesothelioma development.

Matrix Metalloproteinase (MMP)-13 Regulates Mammary Tumor-Induced Osteolysis by Activating MMP9 and Transforming Growth Factor-{beta} Signaling at the Tumor-Bone Interface

The tropism of breast cancer cells for bone and their tendency to induce an osteolytic phenotype are a result of interactions between breast cancer cells and stromal cells and are of paramount importance for bone metastasis. However, the underlying molecular mechanisms remain poorly understood. We hypothesize that tumor-stromal interaction alters gene expression in malignant tumor cells and stromal cells creating a unique expression signature that promotes osteolytic breast cancer bone metastasis and that inhibition of such interactions can be developed as targeted therapeutics. Microarray analysis was performed to investigate gene expression profiling at the tumor-bone (TB) interface versus the tumor alone area from syngenic mice injected with three different syngenic mammary tumor cell lines that differ in their metastatic potential. We identified matrix metalloproteinase 13 (MMP13), receptor activator of NF-kappaB ligand (RANKL), and integrins binding sialoprotein to be genes upregulated at the TB interface and validated. To determine the functional role of MMP13 in tumor-induced osteolysis, mice with Cl66 mammary tumors were treated with MMP13 antisense oligonucleotides (MMP13-ASO) or control scrambled oligonucleotides (control-ASO). Knockdown of MMP13 expression at the TB interface leads to significant reduction in bone destruction and in the number of activated osteoclasts at the TB interface. Further analysis to evaluate the mechanism of MMP13-dependent osteolytic bone metastasis revealed that MMP13-ASO treatment decreased active MMP9, RANKL levels, and transforming growth factor-beta signaling at the TB interface. Together, our data indicate that upregulation of MMP13 at the TB interface is important in tumor-induced osteolysis and suggest that MMP13 is a potential therapeutic target for breast cancer bone metastasis.

Matrix Metalloproteinase (MMP)-13 Regulates Mammary Tumor–Induced Osteolysis by Activating MMP9 and Transforming Growth Factor-β Signaling at the Tumor-Bone Interface

The tropism of breast cancer cells for bone and their tendency to induce an osteolytic phenotype are a result of interactions between breast cancer cells and stromal cells and are of paramount importance for bone metastasis. However, the underlying molecular mechanisms remain poorly understood. We hypothesize that tumor-stromal interaction alters gene expression in malignant tumor cells and stromal cells creating a unique expression signature that promotes osteolytic breast cancer bone metastasis and that inhibition of such interactions can be developed as targeted therapeutics. Microarray analysis was performed to investigate gene expression profiling at the tumor-bone (TB) interface versus the tumor alone area from syngenic mice injected with three different syngenic mammary tumor cell lines that differ in their metastatic potential. We identified matrix metalloproteinase 13 (MMP13), receptor activator of NF-kappaB ligand (RANKL), and integrins binding sialoprotein to be genes upregulated at the TB interface and validated. To determine the functional role of MMP13 in tumor-induced osteolysis, mice with Cl66 mammary tumors were treated with MMP13 antisense oligonucleotides (MMP13-ASO) or control scrambled oligonucleotides (control-ASO). Knockdown of MMP13 expression at the TB interface leads to significant reduction in bone destruction and in the number of activated osteoclasts at the TB interface. Further analysis to evaluate the mechanism of MMP13-dependent osteolytic bone metastasis revealed that MMP13-ASO treatment decreased active MMP9, RANKL levels, and transforming growth factor-beta signaling at the TB interface. Together, our data indicate that upregulation of MMP13 at the TB interface is important in tumor-induced osteolysis and suggest that MMP13 is a potential therapeutic target for breast cancer bone metastasis.

Chemoprevention of heterocyclic amine-induced carcinogenesis by phenolic compounds in rats.

Chemopreventive effects of synthetic and naturally occurring antioxidants on heterocyclic amine (HCA)-induced rat carcinogenesis and mechanisms of inhibition were assessed. In a medium-term liver bioassay, combined treatment with 0.03% 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and synthetic antioxidants such as 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), BHA, BHT, tert-butylhydroquinone (TBHQ) and propyl gallate, each at a dose of 0.25%, and troglitazone at doses 0.5 and 0.1%, potently inhibited development of glutathione S-transferase placental form (GST-P) positive foci as compared with MeIQx alone values. Of these antioxidants, HTHQ showed the greatest activity. Green tea catechins tended to inhibit GST-P positive foci development, while quercetin, rutin, curcumin, daidzin, ferulic acid and genistin all exerted significant enhancing effects. HTHQ also inhibited 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced colon carcinogenesis in a two stage colon carcinogenesis model using 1,2-dimethylhydrazine (DMH) as an initiator. Immunohistochemically detected PhIP-DNA adduct positive nuclei in the colon induced by continuous oral treatment with 0.02% PhIP for 2 weeks decreased by the combined treatment with 0.5 or 0.125% HTHQ. Methoxyresorfin O-demethylase activity in rat liver microsomes in vitro was clearly inhibited by the addition of HTHQ, BHA, BHT, TBHQ or propyl gallate, with particularly strong inhibition being observed in HTHQ. However, the CYP1A2 level in rat liver increased after oral treatment with HTHQ for 2 weeks. These results indicate that synthetic antioxidants, HTHQ in particular, is a very strong chemopreventor of HCA-induced carcinogenesis. It is suggested that depression of metabolic activation rather than antioxidant activity is responsible for the observed effect. However, other mechanisms, including the effects on phase II enzymes cannot be ruled out.

Cancer prevention by bovine lactoferrin: from animal studies to human trial.

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers and, despite improved colonoscopic screening, CRC is a leading cause of death from cancer. Administration of bovine lactoferrin (bLF) suppresses carcinogenesis in the colon and other organs of test animals, and recently it was shown that ingestion of bLF inhibits the growth of adenomatous polyps in human patients. Here we review work which established bLF as an anti-carcinogenic agent in laboratory animals and the results of a clinical trial which demonstrated that bLF can reduce the risk of colon carcinogenesis in humans.