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Circulating fibrocytes had been reported to migrate into the injured lungs, and contribute to fibrogenesis via chemokine-chemokine receptor systems including CXCL12-CXCR4 axis. Here we hypothesized that blockade of CXCR4 might inhibit the migration of fibrocytes to the injured lungs and the subsequent pulmonary fibrosis. To explore the antifibrotic effects of blockade of CXCR4, we used a specific antagonist for CXCR4, AMD3100, in bleomycin-induced pulmonary fibrosis model in mice. Administration of AMD3100 significantly improved the loss of body weight of mice treated with bleomycin, and inhibited the fibrotic lesion in subpleural areas of the lungs. The quantitative analysis demonstrated that treatment with AMD3100 reduced the collagen content and fibrotic score (Aschcroft score) in the lungs. Although AMD3100 did not affect cell classification in bronchoalveolar lavage fluid on day 7, the percentage of lymphocytes was reduced by AMD3100 on day 14. AMD3100 directly inhibited the migration of human fibrocytes in response to CXCL12 in vitro, and reduced the trafficking of fibrocytes into the lungs treated with bleocmycin in vivo. These results suggest that the blockade of CXCR4 might be useful strategy for therapy of patients with pulmonary fibrosis via inhibiting the migration of circulating fibrocytes.

The irradiation of fat-containing food forms 2-dodecylcyclobutanone (2-DCB) from palmitic acid (PA). In this study, we investigated whether 2-DCB and PA induce apoptosis in human lymphoma U937 cells. We found that cell viability decreased by 2-DCB and apoptosis was induced by 2-DCB and PA. 2-DCB and PA significantly enhanced the formation of intracellular reactive oxygen species (ROS). Apoptosis induced by 2-DCB and PA was strongly prevented by an antioxidant, N-acetyl-L: -cysteine. The treatment with 2-DCB and PA resulted in the loss of mitochondrial membrane potential, and Fas, caspase-8 and caspase-3 activation. Pretreatment with a pan-caspase inhibitor (z-VAD) significantly inhibited apoptosis induced by 2-DCB and PA. Moreover, 2-DCB and PA also induced Bax up-regulation, the reduction in Bcl-2 expression level, Bid cleavage and the release of cytochrome c from the mitochondria to the cytosol. In addition, an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) was observed after the treatment with 2-DCB and PA. Our results indicated that intracellular ROS generation, the modulation of the Fas-mitochondrion-caspase-dependent pathway and the increase in [Ca(2+)](i) involved in apoptosis are induced by 2-DCB and PA in U937 cells.

Background: Diagnosis of triple-negative breast cancer (ER-negative, PgR-negative, HER2-negative; TNBC) is performed by means of immunohistological staining. HER2-negative includes HER2(0) and HER2(1+), based on differences in the staining intensity, but there have been no reports on comparison of these two types in TNBC. Accordingly, this study was designed to investigate the possible differences in the biological characteristics of HER2(0) breast cancer and HER2(1+) breast cancer in TNBC., Methods: Tissue specimens from 89 TNBC patients were immunohistochemically stained for CK5/6, EGFR, p53, Ki67, E-cadherin, TOP2A and Bcl-2. The expressions of these markers and the clinicopathological findings were compared between the HER2(0) patient group and the HER2(1+) patient group. When either CK5/6 or EGFR was positive, the specimen was judged to be the basal-like phenotype of breast cancer., Results: The percentages of CK5/6- and/or EGFR-positive specimens in the HER2(0) and HER2(1+) groups were 44.9 and 16.8%, respectively, showing that there was a significantly greater number of basal-like phenotype patients in the HER2(0) group (p < 0.01). The percentage of E-cadherin-positive specimens in the HER2(0) group was 66.6%, which was significantly greater than the 40.0% recorded in the HER2(1+) group (p < 0.05). The respective percentages of TOP2A-positive specimens in the HER2(0) and HER2(1+) groups were 55.0 and 30.0%, and the difference was statistically significant (p < 0.05)., Conclusion: In TNBC, HER2(0) breast cancer showed a strong tendency to include more of the basal-like phenotype compared with HER2(1+) breast cancer. The staining results indicated the possibility that HER2(0) breast cancer and HER2(1+) breast cancer have different characteristics.

Background: Elucidation of the biological features of triple negative breast cancer (TNBC) is important for deciding treatment strategies. The expression of a number of biomarkers in TNBC was analyzed to elucidate those features., Patients and Methods: The subjects were 134 TNBC patients. Immunohistochemical staining was employed to analyze for eight biomarkers: cytokeratin 5/6 (CK5/6), epidermal growth factor receptor (EGFR), p53, Ki-67 antigen (Ki-67), E-cadherin, N-cadherin, topoisomerase 2 alpha (TOP2A) and B-cell lymphoma 2 (BCL-2), which were then correlated with the nuclear grade (NG), tumor diameter, and the presence/absence of lymph node metastasis, distant recurrence and lymphatic infiltration., Results: Significantly more high than low NG TNBC exhibited positive p53, Ki-67, E-cadherin and TOP2A. High N-cadherin and TOP2A expression was shown significantly in TNBC with lymphatic infiltration, and N-cadherin was also significantly positively expressed in node metastasis-positive cases. EGFR and CK5/6 were positively expressed in high NG TNBC, but not significantly., Conclusion: Analysis for expression of p53, Ki-67, E-cadherin, N-cadherin and TOP2A is meaningful for deciding treatment strategies for TNBC.

Sixty-three male 5-week-old Syrian hamsters received the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) s.c. in 5 weekly injections (the first, 70 mg/kg body, and the remaining, 20mg/kg each). The hamsters that received BOP were given intragastric administration of 0.2 ml of medium chain triglyceride (MCT) with or without 0.04 μg of 1α-hydroxyvitamin D3 [1α(OH)D3] through a feeding tube for 12 weeks. Thus, 3 groups were assigned:Group 1;BOP alone (n＝20), Group 2;BOP＋MCT (n＝18) and Group 3;BOP＋1α(OH)D3 (n＝25). The mean body weight of Group 3 was lower than those of Groups 1 and 2 at the end of the experiment (p＜0.001,Tukey-Kramer HSD test). At the end of week 12, all surviving hamsters were put to sleep. The incidences of liver tumors were 80%, 72% and 32% in Groups 1, 2 and 3, respectively. The incidence of tumors in Group 3 was significantly lower than in Group 1 and Group 2 (p＜0.05, χ2-test). All tumors were cholangiocarcinoma. These results indicated that BOP-induced cholangiocarcinogenesis was suppressed by the supplemental administration of 1α(OH)D3.

Chronic inflammation and oxidative stress are arguably associated with an increased risk of cancer. Certain diseases that are characterized by oxyradical overload, such as Wilson's disease (WD), have also been associated with a higher risk of liver cancer. The Long-Evans Cinnamon (LEC) rat, an animal model for WD, is genetically predisposed to the spontaneous development of liver cancer and has been shown to be very useful for studying the mechanisms of inflammation-mediated spontaneous carcinogenesis. Endonuclease III (Nth1) plays a significant role in the removal of oxidative DNA damage. Nth1 and a tumor suppressor gene Tuberous sclerosis 2 (Tsc2) are bi-directionally regulated in humans, mice, and rats by a common minimal promoter containing two Ets-binding sites (EBSs). In this study, we examined the expression of Nth1 and Tsc2 genes during disease progression in the LEC rat liver. During the period of acute hepatitis (16-17 weeks), we observed decreased Nth1 and Tsc2 mRNA levels and a continued decrease of the Tsc2 gene in 24 weeks in LEC rats, while the effect was minimal in Long-Evans Agouti (LEA) rats. This reduction in the mRNA levels was due to the reduced binding of EBSs in the Nth1/Tsc2 promoter. Increase in protein oxidation (carbonyl content) during the same time period (16-24 weeks) may have an effect on the promoter binding of regulatory proteins and consequent decrease in Nth1 and Tsc2 gene expressions during tumorigenesis.

Background/aims: Selenoprotein P (SeP), a plasma protein, is considered to have a protective effect against various organ damages. We investigated whether addition of SeP to storage solution could attenuate cold ischemia/reperfusion injury (IRI) in rat liver transplantation., Methodology: After 24 hrs cold preservation in either University of Wisconsin (UW) solution with or without SeP (1 micromol/L or 10 micromol/L), the liver was flushed with warm lactated Ringer's solution. Alanine aminotransferase (ALT) level in the venous effluent was measured. Orthotopic liver transplantation (OLTx) was then performed after the same preservation as above. Blood biochemical features and tissue lipid peroxide levels were measured after OLTx, and morphometric changes analyzed by hematoxylin and eosin, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining., Results: ALT levels in effluent in the SeP 10 group were significantly lower than those in other groups. Serum aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were significantly decreased in the SeP 10 group. Histological examinations showed amelioration of sinusoidal damage in the SeP 10 group at 1 hr after OLTx. Percentages of necrotic areas and apoptotic sinusoidal endothelial cells were decreased in the SeP 10 group., Conclusions: The addition of SeP to UW solution attenuates injury in OLTx.

Rationale: Imatinib is an inhibitor of platelet-derived growth factor receptors. We have reported that treatment with imatinib inhibited bleomycin-induced pulmonary fibrosis in mice. However, late treatment with imatinib had no effect., Objectives: To clarify why imatinib had no antifibrotic effect when its administration was delayed, we focused on alpha(1)-acid glycoprotein (AGP), because it was reported to bind imatinib and mediate drug resistance., Methods: The concentration of AGP in serum of mice and patients with idiopathic pulmonary fibrosis was measured by radial immunodiffusion testing. The effects of AGP in vitro were evaluated by assaying the growth of lung fibroblasts. We examined the combined effects of erythromycin (EM) or clarithromycin (CAM) on bleomycin-induced pulmonary fibrosis in mice., Measurements and Main Results: Addition of AGP abrogated imatinib-mediated inhibition of the growth of fibroblasts. However, treatment with EM or CAM restored the growth-inhibitory effects of imatinib. The elevated level of AGP was detected in serum and lung homogenates in bleomycin-exposed mice and reached a plateau on Day 14. Imatinib alone did not ameliorate pulmonary fibrosis when treatment was started on Day 15, whereas coadministration of imatinib and EM or CAM significantly reduced the fibrogenesis via inhibition of the growth of fibroblasts in vivo. Serum levels of AGP were higher in patients with idiopathic pulmonary fibrosis than in healthy subjects., Conclusions: AGP is an important regulatory factor modulating the ability of imatinib to prevent pulmonary fibrosis in mice, and combined therapy with imatinib and EM or CAM might be useful for treatment of pulmonary fibrosis.

With the aim of developing a medium-term assay for screening of environmental carcinogens, we exposed mammary carcinogen sensitive human c-Ha-ras proto-oncogene transgenic (Hras128) rats to various carcinogens, including compounds that do not normally induce mammary tumors. Seven-week-old Hras128 rats and wild-type littermates received administrations of 3-methylcholanthrene (3-MC), benzo[a]pyrene (B[a]P), anthracene, pyrene, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), dimethylarsinic acid (DMA), diethylnitrosamine (DEN) or azoxymethane (AOM) and were sacrificed at week 12 (females) (at week 10 for the 3-MC group) or week 20 (males). Female Hras128 rats receiving NNK, DEN, or DMA showed a significant increase in mammary tumor incidence and/or multiplicity compared to the respective values with olive oil or deionized distilled water (DDW) vehicles. In male Hras128 rats, a significant increase in mammary tumors was also observed in groups administered 3-MC, B[a]P, anthracene, IQ, and NNK. Mutations of transgenes were observed in codons 12 and/or 61 in the induced tumors by PCR-RFLP except in the DEN group in female and in the MeIQx group in male Hras128 rats. Thus various carcinogens, not necessarily limited to those normally targeting the breast, were found to induce mammary carcinomas in Hras128 rats, especially in females, pointing to potential use for medium-term screening.

IkappaB kinase (IKK) alpha exhibits diverse biological activities through protein kinase-dependent and -independent functions, the former mediated predominantly through a noncanonical NF-kappaB activation pathway. The in vivo function of IKKalpha, however, still remains elusive. Because a natural strain of mice with mutant NF-kappaB-inducing kinase (NIK) manifests autoimmunity as a result of disorganized thymic structure with abnormal expression of Rel proteins in the thymic stroma, we speculated that the NIK-IKKalpha axis might constitute an essential step in the thymic organogenesis that is required for the establishment of self-tolerance. An autoimmune disease phenotype was induced in athymic nude mice by grafting embryonic thymus from IKKalpha-deficient mice. The thymic microenvironment that caused autoimmunity in an IKKalpha-dependent manner was associated with defective processing of NF-kappaB2, resulting in the impaired development of thymic epithelial cells. Thus, our results demonstrate a novel function for IKKalpha in thymic organogenesis for the establishment of central tolerance that depends on its protein kinase activity in cooperation with NIK.

Adenocarcinoma of the lung is characterized by frequent aerogenous spread (AE) and advancement along the alveolar wall (BAC growth). To elucidate the mechanism of AE metastasis and BAC growth in human lung adenocarcinoma, we established an in vivo orthotopic animal model and an in vitro culture. Investigation of expression levels of integrins, laminins and Type IV collagens, which are the major regulating molecules for cell attachment and anoikis was carried out and a clear correlation between the expression level of laminin 5 (LN5) and the BAC growth was observed using an orthotopic animal model. Introduction of LN5 cDNA to A549 cells increased anoikis resistance in an expression dependent manner. Cells with LN5 overexpression resisted with anoikis after treatment with PI3K-Akt and ERK inhibitors. The amount of phosphorylated focal adhesion kinase (FAK) was also higher in LN5 overexpressing cells. Major tyrosine residues of the EGF receptor at 1068, 1086 and 1173, except at 1148, remained phosphorylated only in the LN5 overexpressing cells even without EGF stimulation, that indicates the ligand independent activation of EGF receptor. BAC growth ratio and AE was confirmed to be significantly correlated with LN5 expression in surgically resected human lung adenocarcinomas by immunohistochemistry. Our results indicate that the activation of the EGF receptor by overexpressing LN5-integrin-FAK signaling pathway may play a crucial role in BAC growth and AE metastasis in human lung adenocarcinoma.

Imatinib mesylate is a potent and specific tyrosine kinase inhibitor against c-ABL, BCR-ABL, and c-KIT, and has been demonstrated to be highly active in chronic myeloid leukemia and gastrointestinal stromal tumors. We examined the antifibrotic effects of imatinib using a bleomycin-induced lung fibrosis model in mice because imatinib also inhibits tyrosine kinase of platelet-derived growth factor receptors (PDGFRs). Imatinib inhibited the growth of primary murine lung fibroblasts and the autophosphorylation of PDGFR-beta induced by PDGF. Administration of imatinib significantly prevented bleomycin-induced pulmonary fibrosis in mice, partly by reducing the number of mesenchymal cells incorporating bromodeoxyuridine. Analysis of bronchoalveolar lavage cells demonstrated that imatinib did not suppress early inflammation on Days 7 and 14 caused by bleomycin. These results suggest that imatinib has the potential to prevent pulmonary fibrosis by inhibiting the proliferation of mesenchymal cells, and that imatinib might be useful for the treatment of pulmonary fibrosis in humans.

Kojic acid (KA) has been used as a food additive for preventing enzymatic browning of crustaceans and as a cosmetic agent for skin whitening. In the present experiments, effects of KA on the induction of hepatic pre-neoplastic lesions in N-bis(2-hydroxypropyl)nitrosamine-initiated (experiment 1) and non-initiated (experiment 2) models, and its promoting influence in a medium-term liver bioassay (experiment 3) were investigated at dietary doses of up to 2% in male F344 rats. In experiment 1, 2% KA feeding induced significant increases in numbers (22.3 +/- 13.0 vs 8.5 +/- 3.4 in the 0%) and areas (0.37 +/- 0.29 vs 0.05 +/- 0.03 in the 0%) of glutathione-S-transferase P form (GST-P)-positive foci and toxic changes such as vacuolation of hepatocytes and microgranulomas. The development of GST-P-positive foci was pronounced in the animals with hepatocellular toxic changes. In experiment 2, numbers (0.65 +/- 0.57 vs 0.17 +/- 0.28 in the 0%) and areas (0.005 +/- 0.005 vs 0.0007 +/- 0.0012 in the 0%) of GST-P-positive foci and hepatocellular proliferating cell nuclear antigen (PCNA) expression (3.8 +/- 2.3 vs 2.6 +/- 0.7 in the 0%) were significantly increased by the 2% treatment. The PCNA-positive hepatocytes were abundantly localized around the vacuolated and granulomatous legions in both experiments 1 and 2. In experiment 3, significant increases in numbers (16.9 +/- 3.2 vs 8.4 +/- 2.7 in the 0%) and areas (1.62 +/- 0.39 vs 0.77 +/- 0.34 in the 0%) of GST-P-positive foci were again observed with 2% KA. These results demonstrate tumor-promoting and possible hepatocarcinogenic activity of KA at 2%, but the carcinogenic potential is likely to be weak. This study also indicated that enhanced replication of hepatocytes related to toxic changes might be involved as an underlying mechanism.

Physical contact between thymocytes and the thymic stroma is essential for T cell maturation and shapes the T cell repertoire in the periphery. Stromal elements that control these processes still remain elusive. We used a mouse strain with mutant NF-kappaB-inducing kinase (NIK) to examine the mechanisms underlying the breakdown of self-tolerance. This NIK-mutant strain manifests autoimmunity and disorganized thymic structure with abnormal expression of Rel proteins in the stroma. Production of immunoregulatory T cells that control autoreactive T cells was impaired in NIK-mutant mice. The autoimmune disease seen in NIK-mutant mice was reproduced in athymic nude mice by grafting embryonic thymus from NIK-mutant mice, and this was rescued by supply of exogenous immunoregulatory T cells. Impaired production of immunoregulatory T cells by thymic stroma without normal NIK was associated with altered expression of peripheral tissue-restricted Ags, suggesting an essential role of NIK in the thymic microenvironment in the establishment of central tolerance.

Endothelin (ET)-1(1-31) is a novel vasoconstrictor peptide produced by human mast cell chymase, which selectively cleaves big ET-1 at the Try(31)-Gly(32) bond. We investigated the localization of ET-1(1-31) in various hamster tissues by immunohistochemistry and compared it to the distribution of ET-1(1-21). We found that the localization and amount of ET-1(1-31) were different from those of ET-1(1-21) in each tissue. ET-1(1-31)-like immunoreactivities (IR) in the heart, lung, and adrenal gland were observed in the same areas as ET-1(1-21) but were significantly weaker, suggesting that ET-1(1-31) might play a role only in mast cell/chymase-related pathological conditions in these tissues. In the liver, ET-1(1-31)-like IR was strongly detected in Kupffer cells where ET-1(1-21)-like IR was seen more weakly. In the kidney, ET-1(1-31)-like IR was slightly higher than ET-1(1-21). These results suggest that ET-1(1-31) might have physiological roles distinct from those of ET-1(1-21) in some hamster tissues.

Background: Glycogen-rich carcinoma is a rare special histologic subtype of breast cancer and its incidence is estimated to be 1.4% in breast malignancies. However, its precise characteristics in cytological specimens have not yet been fully clarified., Case: Fifty-nine-year-old and 53-year-old women underwent fine-needle aspiration biopsy cytology (FNABC) of a breast tumor, confirming malignancy. A mastectomy with axillary dissection was performed. Cytologically, a moderate amount of eosinophilic, finely granular cytoplasm was seen in the majority of the tumor cells, however, foamy and vacuolated cytoplasm was noted in some tumor cells. Histologically, the tumor cells of both cases had clear and granular cytoplasm, which showed a positive reaction with periodic acid-Schiff, eliminated by diastase., Conclusion: While clear cytoplasm in the tumor cells in the FNABC seemed to be a pivotal cytological characteristic of glycogen-rich carcinoma, it may not be a major component of cytological specimens. Routine periodic acid-Schiff staining may be required to diagnose glycogen-rich carcinoma in cytological methods.

Background: We examined the relationship between biological markers, apoptotic indices and pathologic subtypes of ductal carcinoma in situ (DCIS) of the breast. The tumor-biological factors can be divided into invasive and proliferative markers. We chose urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor type 1 (PAI-1) and Cathepsin D as invasive markers, and Ki-67 and C-erbB2 oncoproteins as proliferative factors for our study., Methods: We used immunohistochemical methods to investigate the expression of uPA, PAI-1, Cathepsin D, Ki-67, C-erbB2 and ssDNA (single-stranded DNA for apoptotic cells) in 20 cases of DCIS. Tumor histological grade and the immunohistochemical expression of invasive and proliferative markers were compared., Results: Histological grade is associated with C-erbB2, MIB-1, apoptotic index (AI) and expression of PAI-1 in cancer and stroma. The correlation coefficient of the MIB-1 index and AI was 0.867. Of these invasive markers, only expression of PAI-1 in tumor and in stroma was associated with C-erbB2., Conclusion: Our results show that the apoptosis index is closely related to the MIB-1 index, and also suggest that the immunohistochemical detection of PAI-1 in the cytoplasm of both carcinoma cells and stromal cells of DCIS is related to histological grade and expression of the proliferative markers MIB-1 and C-erbB2. Therefore, we infer that both invasiveness and proliferation are affected by the tumorigenesis of DCIS.

Mucocele-like lesions (MLL) of the breast have been reported as extremely rare as well as benign, but now it is believed they can be both malignant and benign. This paper describes two cases of malignant MLL, both subjected to immunohistochemical staining. Case 1: A 42-year-old woman with multiple malignant MLLs without evidence of a mass at presentation to our hospital after biopsy, but whose ultrasonogram showed small multiple hypoechoic lesions. Case 2: A 70-year-old woman, whose left breast cancer was found on routine mammography after modified radical mastectomy for right breast cancer. Pathologically, MLL with intraductal carcinoma was diagnosed. Case 1 underwent two lumpectomies in 3 years, but even now new lesions have developed. However, the patient refused to have another operation. C-erbB2 was negative in both cases. When malignant MLL is histologically of low grade, excisional biopsy is sufficient for a single MLL with intraductal carcinoma, while it may be necessary to perform a subcutaneous mastectomy for multiple lesions.