3.EXPERIMENTAL PATHOLOGY AND CHEMOTHERAPY DIVISION
Analysis of the Carcinogenic Process Using Transgenic (Tg) Rats
Further to the previous report of the high susceptibility of Hra128 rats to chemically induced mammary and bladder carcinogenesis, a recent study indicated that a similar situation exists with regard to development of esophageal tumors (62). Regarding the mammary gland susceptibility, increase in numbers of terminal endbuds (TEBs), higher levels of c-myc, cyclin D1, cyclin D2 and active MAP kinase in mammary glands have been noted. Using this rapid mammary carcinogenesis model, suppressive effects of d-limonene, a Ras farnesylation inhibitor (63), and octylphenol, a environmental endocrine disrupting agent (64), could clearly be shown in terms of both tumor incidence and multiplicity. Thus, this model may have advantages for the screening of chemopreventive and modifying agents of mammary carcinogenesis. Recent studies have indicated that the Hras128 rats could be used in screening for various environmental carcinogens not targeting the mammary glands.
To confirm the role of trans gene mutations, Tg rats harboring the mutated human c-Ha-ras gene, regulated by Cre recombinase for conditional expression, were established (Hras250). Mammary adenocarcinomas were found on day 15 after infection of the adenovirus-carrying Cre recombinase gene. Analysis of early lesions indicated that mammary tumors develop from the duct epithelium and acinar cells.
The neuronal leucine-rich repeat protein-3 (NLRR-3) belongs to the LRR superfamily. The rat NLRR-3 gene isolated from c-Ha-ras transgenic rat mammary tumors was cloned and shown to be regulated mainly through the Ras-MAPK signaling pathway. It was found to enhance phosphorylation of MAPK when COS-7 cells were transfected with NLRR-3 and stimulated with a low concentration of epidermal growth factor, but the amplification was no longer observed when the C-terminal 30 amino acid stretch containing clathrin-mediated endocytosis motifs was deleted. NLRR-3 may amplify MAPK activity downstream of receptor tyrosine-kinase resulting in growth stimulation of malignant tumors (65).
TGFa transgenic rats, conditionally inducible by Cre recombinase, were also established. The number of spontaneous and chemically-induced liver glutathion S-transferase (GST-P) positive foci, a preneoplastic lesion, significantly increased following injection of the Cre recombinase coded adenovirus (AdeX/Cre), indicating that the TGFa transgene plays an important role in enhancing susceptibility to liver carcinogenesis.
Prevention of Carcinogenesis and Anti-metastasis by Bovine Lactoferrin (bLF)
Oral administration of bovine lactoferrin (bLF) has been found to markedly inhibit carcinogenesis and lung metastatic colony formation. bLF down-regulates expression of one of the drug-metabolizing enzymes, CYP1A2,induced in the liver by MeIQx (66). Furthermore, protein levels of caspase-1 and IL-18 remarkably increased in epithelial cells in the small intestine on treatment with bLF (67). An in vitro study using peritoneal macrophages also indicated that bLF markedly increases caspase-1 activity and IL-18 levels. These results indicate that the elevation of caspase-1 activity induced by bLF may be important for production of IL-18 in vivo, which potentiates the killing activity of T and NK cells against preneoplastic cells and also tumor cells in the blood stream.
In concert with these in vivo studies, in vitro transfection technologies were employed to identify mechanisms by which lactoferrin prevents tumorigenesis. Lactoferrin binds to membrane receptors and may activate specific signal transduction cascades that affect cell behavior. Normally, after receptor binding, lactoferrin is internalized into the cytoplasm, but does not enter the nucleus. However, in contrast to standard lactoferrin, alternate forms of the protein can be produced that do not bind to membrane receptors, but do enter the nucleus. Interestingly, nuclear localization of these alternate forms of lactoferrin may cause apoptosis in tumor cells. Currently molecular events that
underlie these important observations are being elucidated.
The results suggest that lactoferrin may be used as an effective anti-cancer food supplement. In addition, on the basis of positive preliminary data, clinical studies on intestinal polyps and HCV induced chronic active hepatitis are now under way in the National Cancer Center Hospital in collaboration with other hospitals.
Prevention of Carcinogenesis
A polyphenolic antioxidant flavonoid, silymarin, showed suppressive effects on AOM-induced colon carcinogenesis in rats (68). While b-carotene was found to inhibit liver carcinogenesis, it adversely promoted lung tumor development (69), in line with epidemiological results pointing to enhancement of lung carcinoma development in humans.
Risk Assessment Study and Others
Large-scale studies on the dose-dependence of carcinogenesis were carried out in collaboration with other research groups, showing a lack of any linear dose-response relationship at very low dose range (70). A new classification of rat renal tumor was proposed in the project supported by the Japanese Society of Toxicological Pathology (71). Recently, in collaboration with a group in Thailand, the possibility of using wild rodents as environmental monitors has been explored, captured rats demonstrating GST-P positive lesions and other changes in the liver and lung which might point to contamination (72). Furthermore, over the last year a number of reviews and editorials were published regarding international cancer prevention efforts, screening and tobacco control (73-77).