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ABSTRACT: Human carbonyl reductase 1 (CBR1) is an enzyme that catalyse the reduction of many compounds by using NADPH-dependent oxydoreductase activity. Although CBR1 is known to regulate the tumour progression, the molecular mechanisms of CBR1 in cancer progression and the clinical significance of CBR1 status remain unclear. Here, we investigated the molecular mechanisms by which CBR1 affects cancer cell behaviour in vitro and the clinical significance of CBR1 using immunohistochemical analyses in endometrial cancer. Here, the role of CBR1 in cancer cell invasion and metastasis, and its molecular mechanisms were investigated by transfection of sense and antisense CBR1 cDNAs into a human endometrial adenocarcinoma cell line. The relationship between CBR1 expression analysed by immunohistochemistry and prognosis such as progression free survival (PFS) and overall survival (OS) was examined in endometrial cancer tissues from FIGO stage I-IV (n=109). Suppression of CBR1 by antisense CBR1 cDNA increased cancer cell invasion, and suppressed E-cadherin expression and capacity for cellular aggregation. In contrast, over-expression of CBR1 by sense CBR1 cDNA increased E-cadherin expression and capacity for cellular aggregation, and suppressed cancer cell invasion. The expression of transcriptional suppressors of E-cadherin, Snail and ZEB1, were increased by CBR1 suppression, but suppressed by CBR1 over-expression. Immunohistochemical analyses showed that decreased CBR1 expression is significantly related with poor PFS and OS compared with strong CBR1 expression. In multivariate analyses, decreased CBR1 expression was an independent prognostic factor for PFS and OS. CBR1 regulates cancer cell invasion in endometrial adenocarcinomas by regulating the epithelial mesenchymal transition. A decreased CBR1 expression can be a useful marker of an unfavourable clinical outcome in patients with endometrial cancer.
Cancer letters 04/2012; 323(1):69-76. · 4.86 Impact Factor
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ABSTRACT: The present study investigated tumor proliferation in a tumor model using murine ovarian cancer cells with increased carbonyl reductase (CR) expression.
CR cDNA was transfected into murine T-Ag-MOSE ovarian cancer cells by lipofection. CR-transfected cells (CR induction group) or empty vector-treated cells (control group) were injected into the backs of 8-week-old nude mice at a concentration of 0.5 × 10(6) per 0.2 mL. Subsequent tumor proliferation in both groups was observed for 5 weeks.
The control group showed an increase in tumor volume during the 5 weeks of observation. However, tumor volume in the CR induction group increased up to the second week but then decreased continuously until the fifth week of observation. The tumor growth curves for the two groups showed a significant difference (Mann-Whitney U test, P < 0.001). Histological and biochemical experiments were performed using tumor tissues isolated in the third week. Necrosis and inflammatory cell infiltration were noted for tumors in the CR induction group. Also, the number of apoptotic cells was significantly increased in the CR induction group compared with the control group (P < 0.001). Milk fat globule EGF factor 8, an "eat-me" signal for phagocytes such as macrophages, was expressed extensively in the tumor cytoplasm and interstitial cells of the CR induction group, and engulfment of apoptotic cells by macrophages was observed. Vascular endothelial growth factor expression in tumors was notably decreased in the CR induction group compared with the control group.
Increased necrosis due to engulfing of apoptotic cells by phagocytes attracted by increased milk fat globule EGF factor 8 was considered to be the mechanism of spontaneous tumor regression in the CR induction group.
Clinical Medicine Insights: Oncology 01/2012; 6:107-15.
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Journal of Hepatology 08/2011; 56(3):744-5. · 9.26 Impact Factor
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ABSTRACT: Carbonyl reductase (CR) is an NADPH-dependent, mostly monomeric, cytosolic enzyme with broad substrate specificity for carbonyl compounds. CR appears to be involved in the regulation of tumour progression. However, molecular mechanisms of CR in tumour progression and clinical significance of CR status remain unclear in human uterine squamous cell carcinoma (SCC). Here, we investigated the clinical significance of CR using immunohistochemical analyses of human uterine cervical SCC tissues and how CR affects cancer cell behaviour in vitro. Paraffin sections from uterine cervical SCC tissues, FIGO stage Ib1-IIb (n = 67) were immunostained with anti-CR antibodies. Overall survival (OS) and progression-free survival (PFS) were analyzed by the Kaplan-Meier method. Sense and antisense CR cDNAs were transfected into a human uterine SCC cell line (SiHa) to investigate the role of CR in cancer cell invasion and metastasis. Immunohistochemical analyses showed that reduced CR expression patterns in primary cancer lesions were closely associated with a high incidence of pelvic lymph node metastasis, poor OS, and poor PFS. In an in vitro experiment, suppression of CR increased cancer cell invasion, secretion of MMP-2, -9 and cyclooxygenase-2 (COX-2) expression and decreased E-cadherin expression. On the other hand, over-expression of CR increased E-cadherin expression and decreased MMP-2, -9 secretion and COX-2 expression. The reduced CR expression pattern, as measured by immunohistochemistry, can be a useful predictor of lymph node metastasis and poor prognosis in patients with uterine SCC. This clinical result is supported by the in vitro data which show that suppression of CR expression promotes cancer cell invasion with decreased E-cadherin expression and increased MMP-2, -9 secretion.
Cancer letters 07/2011; 311(1):77-84. · 4.86 Impact Factor
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ABSTRACT: To examine the possible involvement of nonparenchymal cells in the development of preneoplastic hepatic lesions induced by clofibrate (CF), alterations of these cells were investigated immunohistochemically in glutathione S-transferase M1 gene polymorphic rats (KS and NC types) with different cancer susceptibilities. After CF administration for 8 weeks, α-smooth muscle actin (α-SMA)-positive hepatic stellate cells (HSC) were markedly decreased in sensitive KS-type rats, but not in the NC-type rats. Kupffer cells were decreased with similar extents between them. The sinusoidal endothelial cells were not changed in either type. The other markers for HSC, vimentin and CRBP1, also confirmed the decrease of HSC in the KS type. The decrease of HSC was not observed at 4 weeks of CF administration. Preneoplastic peroxisomal bifunctional enzyme-negative foci were detected in the KS-type rats at 8 weeks of CF administration, but not at 4 weeks. Human HSC were cultured in the presence of clofibric acid and expression of most HSC marker genes, such as vimentin and α-SMA (ACTA2), evaluated by a microarray, was not altered by the treatment, suggesting that HSC loss in the KS-type rats was not due to the direct toxic effect of CF. The expression levels of most HSC marker genes were low in both control and CF-treated rat livers. A possible link between HSC loss and the development of preneoplastic hepatic foci is discussed.
Cancer Science 01/2011; 102(4):735-41. · 3.33 Impact Factor
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ABSTRACT: To clarify the mechanism of persistent cholestasis after massive hepatectomy, the relationship between such cholestasis and the expression and localization of organic anion transporters for bile acids was examined in a rat model.
Male Sprague-Dawley rats were subjected to 90% hepatectomy, and tissues were harvested at 0, 1, 3, and 7 days for microarray analysis, quantitative real-time polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry to examine the expression of multidrug resistance protein 4 (Mrp4), bile salt export pump (Bsep), and sodium-dependent taurocholate cotransporting polypeptide (Ntcp).
Persistently elevated levels of serum bile acids were observed at days 3 and 7. RT-PCR and Western blotting indicated that the expression of Mrp4, a bile acid export pump located in the basolateral membrane, was increased at day 3. The expression of Ntcp, a transporter used to uptake bile acids from the sinusoids, was significantly decreased throughout the period. The levels of Bsep, an export pump localized to the canalicular membrane, were unchanged. Immunohistochemistry revealed the localization of Mrp4 and Bsep in the basolateral and canalicular membranes, respectively. On the other hand, at days 3 and 7, Ntcp was localized in the cytoplasm and was hardly detected in the basolateral membrane.
These results suggested that the sustained repression and translocation of Ntcp and the expression of Mrp4 at the basolateral membrane seem to be responsible for the high blood bile acids levels after massive hepatectomy.
Journal of Hepatology 12/2010; 55(2):407-14. · 9.26 Impact Factor
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Daiki Rokunohe,
Hajime Nakano,
Eijiro Akasaka,
Kazuyuki Kimura,
Noriko Takiyoshi,
Koji Nakajima,
Takayuki Aizu,
Takahide Kaneko,
Yasushi Matsuzaki, Shigeki Tsuchida,
Daisuke Sawamura
Journal of dermatological science 10/2010; 60(3):199-201. · 3.71 Impact Factor
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ABSTRACT: Peroxisome proliferators (PPs), non-genotoxic rodent carcinogens, cause the induction of the peroxisomal fatty acid beta-oxidation system, including bifunctional enzyme (BE) and 3-ketoacyl-CoA thiolase (TH), in the liver. GST M1 gene is polymorphic in Sprague-Dawley rats, NC- and KS-type. The KS-type rats showed enhanced susceptibility to ethyl-alpha-chlorophenoxyisobutyrate (clofibrate, CF), one of the PPs. The degree of BE induction was higher in the KS-type and preneoplastic foci developed after 6-8 weeks of treatment, whereas no foci developed in the NC-type. In the preset study, factors involved in different BE inducibility were investigated. There were no differences in hepatic peroxisome proliferator-activated receptor (PPAR) alpha levels between them. Among various coactivators for PPARalpha, only steroid receptor coactivator (SRC)-3 level was higher in the KS-type. To investigate the association between PPARalpha and SRC-3 or other proteins, nuclear extracts from CF-treated livers were applied to a PPARalpha column. In the KS-type, 110, 72, and 42 kDa proteins were bound and these were identified as SRC-3, BE, and TH, respectively. EMSA supported the binding of these proteins to PPARalpha associated to the BE enhancer in CF-treated KS-type, but not in the NC-type. Histone H3 acetylation was increased 11-fold in the KS-type by CF treatment but not in the NC-type. As BE and TH are responsible for acetyl-CoA production and SRC-3 possesses a histone acetyltransferase activity, these results suggest that enhanced BE induction in the KS-type livers is due to acetylation-mediated transcriptional activation and epigenetic mechanisms might be involved in CF-induced rat hepatocarcinogenesis.
Cancer Science 04/2010; 101(4):869-75. · 3.33 Impact Factor
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ABSTRACT: Peroxisome proliferators (PP), including clofibrate (CF), are non-genotoxic rodent carcinogens, and oxidative DNA damages are suggested as a causative event for carcinogenesis. Gene expression profiles differ between hepatic lesions induced by PP and genotoxic carcinogens. Our previous study revealed that expression of L-bifunctional enzyme (enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, BE) was repressed in preneoplastic lesions induced by PP, whereas it was enhanced in the surrounding tissues. In the present study, we immunohistochemically examined expression of the specific glutathione S-transferase (GST) form, GST-A4, which detoxifies 4-hydroxy-alkenal, the end-product of lipid peroxides, and nuclear factor-erythroid 2-related factor 2 (Nrf2), a transcription factor for many genes encoding drug-metabolizing enzymes and defending enzymes against oxidative stress, during rat hepatocarcinogenesis induced by CF and genotoxic carcinogens. GST-A4 and Nrf2 were not expressed in BE-negative foci at 8 weeks of CF administration, but were expressed in the foci at 60 weeks. GST-A4-positive foci appeared at later stages than BE-negative foci, but its localization was coincidental with that of the latter foci. The areas of GST-A4-positive foci were larger than those of BE-negative foci without GST-A4 expression. Most GST-A4-positive foci were also positive for Nrf2. In rat livers induced by genotoxic carcinogens, GST-P-negative foci as well as GST-P-positive foci were demonstrated. GST-A4 and Nrf2 were expressed in GST-P-negative foci, whereas they were not expressed in most GST-P-positive foci. Thus, GST-A4-positive foci developed in rat livers by CF and genotoxic carcinogen administration, indicating that the enzyme is a positive marker for hepatic foci induced by these different carcinogens.
Cancer Science 01/2010; 101(5):1093-8. · 3.33 Impact Factor
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Koichi Hashiguchi,
Hiroyuki Tsuchiya,
Akiko Tomita,
Chisa Ueda,
Yuji Akechi,
Tomohiko Sakabe,
Akihiro Kurimasa,
Masami Nozaki,
Toshiyuki Yamada, Shigeki Tsuchida,
Goshi Shiota
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ABSTRACT: CD437, a synthetic retinoid, has a potent antitumor activity, in which an RAR-independent mechanism may be involved. Our previous study showed that CD437 transcriptionally upregulates the expression of thioredoxin-binding protein 2 (TBP2), leading to c-Jun N-terminal kinase 1 (JNK1)-mediated apoptosis. In the present study, we addressed the mechanism, by which CD437 induces TBP2 mRNA expression. CD437 efficiently caused the cell death of human osteosarcoma cells via apoptosis. CD437 also induced JNK1 activation through the upregulation of TBP2 mRNA, in consistent with our previous observation. A luciferase reporter assay for TBP2 promoter activation suggested that CD437-regulated TBP2 mRNA transcription requires the region between -400 and -300, which contains multiple possible ETS-binding sites. Finally, we demonstrated CD437-dependent recruitment of ETS1 transcription factor to this region by chromatin immunoprecipitation assay. These data suggest that ETS1 is involved in CD437-induced TBP2 mRNA expression in human osteosarcoma MG-63 cells.
Biochemical and Biophysical Research Communications 11/2009; 391(1):621-6. · 2.48 Impact Factor
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ABSTRACT: Lead nitrate induces hepatocyte proliferation and subsequent apoptosis in rat livers. Iron is a constituent of heme and is also required for cell proliferation. In this study, the expression of ferritin light-chain (FTL), the major iron storage protein, was investigated in rat livers after a single intravenous injection of lead nitrate. Western blotting and immunohistochemistry revealed that FTL was increased in hepatocytes around the central veins and strongly expressed in nonparenchymal cells. Some FTL-positive nonparenchymal cells were identified as Kupffer cells that were positive for CD68. FTL-positive Kupffer cells occupied about 60% of CD68-positive cells in the periportal and perivenous areas. The relationships between FTL expression and apoptosis induction or the engulfment of apoptotic cells were examined. TUNEL-positive cells were increased in the treatment group, and enhanced expression of milk fat globule EGF-like 8 was demonstrated in some Kupffer cells and hepatocytes, indicating enhanced apoptosis induction and phagocytosis of apoptotic cells. FTL-positive Kupffer cells were not detected without lead nitrate treatment or in rat livers treated with clofibrate, which induces hepatocyte proliferation but not apoptosis. These results suggest that FTL expression in Kupffer cells after lead treatment is dependent on phagocytosis of apoptotic cells.
Toxicologic Pathology 02/2009; 37(2):209-17. · 1.91 Impact Factor
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ABSTRACT: To examine the possibility that ETS family transcription factors, PU.1, SPI-B, ELF-1, ERG-3, ETS-1 and TEL, and homeodomain proteins, HOXA10, HOXC13, MEIS1 and PBX1B, function cooperatively, we investigated their interactions. In luciferase assays, HOXA10 and HOXC13 augmented the activity of PU.1 and SPI-B while diminishing that of ELF-1 and ERG-3. MEIS1 diminished the activity of ETS-1. No clear effects were observed for other combinations. Immunoprecipitation assays showed protein-protein interactions among the combinations exhibiting functional interactions. A mutation of HOXC13, which abolished binding to ELF-1, also abolished the diminishing effect on ELF-1. The results suggest functional interaction through physical interactions.
Leukemia research 09/2008; 33(3):483-9. · 2.36 Impact Factor
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ABSTRACT: Most models of hereditary hypotrichosis are due to alterations in growth factors and transcription factors, and the examples of causative mutations in hair keratin genes are limited. The Hirosaki hairless rat (HHR) is a mutant strain spontaneously derived from Sprague-Dawley rats (SDRs). In this study, the locus of the responsible gene was examined by linkage analysis and mapped on chromosome 7q36. Because many basic keratin genes are clustered on 7q36, their expression was examined. Reverse transcription-PCR and genomic PCR indicated that the Kb21 (Krt81), -23 (Krt83), and -26 (Krt86) genes encoding basic hair keratins were not expressed and were deleted. Furthermore, 80-kb genomic DNA ranging from exon 9 of Kb25 (Krt85) to exon 9 of Krt2-25 was deleted. The breakpoints of these genes were within a 95-bp portion shared by the two genes, suggesting that deletion due to non-allelic homologous recombination occurred. Proteins identified as Kb21, Kb23, and Krt2-25 in SDR hairs by mass spectrometry were not detected in HHR. Instead, the product of a fusion gene became dominant in HHR. Because fusion occurred between the exons of the two genes with the same sequences, the product was identical to the wild-type Kb25 protein. By using immunohistochemistry, Kb21 was not detected in HHR hair follicles. Kb25 was expressed in the cortex in HHRs, whereas it was in the medulla in SDRs. This study clearly illustrates the importance of hair keratin genes in hair growth.
Journal of Biological Chemistry 07/2008; 283(24):16868-75. · 4.77 Impact Factor
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ABSTRACT: Glutathione S-transferase P (GST-P), a marker for rat hepatic preneoplastic lesions, is suggested to bind to Jun N-terminal kinase (JNK) to repress stress response, and GST-P gene expression is regulated by a transcription factor, nuclear factor-erythroid 2-related factor 2 (Nrf2). In this study, we examined by immunohistochemistry whether JNK2, p38 mitogen-activated protein kinase, and Nrf2 were expressed in GST-P-positive foci induced by the Solt-Farber protocol. At 2 weeks after partial hepatectomy, all GST-P-positive foci were negative for p38, and 86.4 +/- 5.6% and 64.7 +/- 6.3% of GST-P-positive foci were negative for JNK2 and Nrf2, respectively. Western blot analysis showed decreased p38 mitogen-activated protein kinase and JNK2 expression in livers treated with the protocol. In immunohistochemistry, besides GST-P-positive foci, GST-P-negative foci were detected as p38-negative foci in the surrounding tissues positive for p38. In contrast to GST-P-positive foci, most GST-P-negative foci showed enhanced Nrf2 expression. The number of GST-P-negative foci was 76 +/- 18/10 mm(2) of liver section at 2 weeks, but was undetectable at 1 week. The area of GST-P-negative foci was 0.09 +/- 0.05 mm(2), smaller than that of GST-P-positive ones (0.29 +/- 0.23). After treatment with carbon tetrachloride, small vacuoles due to liver injury were frequently observed inside GST-P-negative foci but less frequently in GST-P-positive foci. However, this treatment resulted in expression of JNK2, p38, and Nrf2 in both foci. These results showed development of GST-P-negative foci during the early stage of hepatocarcinogenesis and suggested that Nrf2 is not responsible for GST-P expression in rat hepatic preneoplastic foci.
Cancer Science 04/2008; 99(3):497-501. · 3.33 Impact Factor
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ABSTRACT: Some of homeodomain proteins and the ETS family of transcription factors are involved in hematopoiesis. RT-PCR analysis revealed that the HOXC13 and PU.1 genes were expressed in murine erythroleukemia (MEL) cells and their levels decreased during DMSO-induced differentiation into erythroid cells. HOXC13 bound to the ETS domain of PU.1 through a region encompassing the C-terminal part of the homeodomain and the most C-terminal region and enhanced the transcriptional activity of PU.1. Enforced expression of HOXC13 in MEL cells resulted in the suppression of beta-globin gene expression. In MEL cells overexpressing HOXC13 and PU.1, which also inhibits the differentiation of MEL cells, no synergistic effect on the suppression of beta-globin gene expression was observed. However, in the presence of DMSO, the expression levels of the beta-globin gene in the cells overexpressing HOXC13 and PU.1 were, unexpectedly, higher than those in the cells overexpressing PU.1 alone. The levels of PU.1 protein were markedly decreased despite that the levels of mRNA were preserved in the cells overexpressing PU.1 and HOXC13. It was, thus, suggested that although HOXC13 negatively regulates the differentiation of MEL cells into erythroid cells, it antagonizes PU.1 possibly by down-regulation of PU.1 protein in the presence of a differentiation stimulus.
Experimental Cell Research 03/2008; 314(4):847-58. · 3.58 Impact Factor
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ABSTRACT: Recent reports have shown that peroxisome proliferator-activated receptor (PPAR)alpha ligands reduce growth of some types of malignant tumors and prevent carcinogenesis. In this study, we investigated the inhibitory effect of clofibric acid (CA), a ligand for PPARalpha on growth of ovarian malignancy, in in vivo and in vitro experiments using OVCAR-3 and DISS cells derived from human ovarian cancer and aimed to elucidate the molecular mechanism of its antitumor effect. CA treatment significantly suppressed the growth of OVCAR-3 tumors xenotransplanted s.c. and significantly prolonged the survival of mice with malignant ascites derived from DISS cells as compared with control. CA also dose-dependently inhibited cell proliferation of cultured cell lines. CA treatment increased the expression of carbonyl reductase (CR), which promotes the conversion of prostaglandin E(2) (PGE(2)) to PGF(2alpha), in implanted OVCAR-3 tumors as well as cultured cells. CA treatment decreased PGE(2) level as well as vascular endothelial growth factor (VEGF) amount in both of OVCAR-3-tumor and DISS-derived ascites. Reduced microvessel density and induced apoptosis were found in solid OVCAR-3 tumors treated by CA. Transfection of CR expression vector into mouse ovarian cancer cells showed significant reduction of PGE(2) level as well as VEGF expression. These results indicate that CA produces potent antitumor effects against ovarian cancer in conjunction with a reduction of angiogenesis and induction of apoptosis. We conclude that CA could be an effective agent in ovarian cancer and should be tested alone and in combination with other anticancer drugs.
Molecular Cancer Therapeutics 05/2007; 6(4):1379-86. · 5.23 Impact Factor
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Toshihiro Kudo,
Jumpei Asano,
Takeshi Shimizu,
Naoki Nanashima,
Yang Fan,
Miki Akita,
Keizo Ookawa,
Makoto Hayakari,
Yoshihito Yokoyama,
Kohji Suto, Shigeki Tsuchida
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ABSTRACT: Although peroxisomal bifunctional enzyme (enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase; BE) is a positive marker for peroxisome proliferation, it is completely absent or expressed very weakly in rat hepatic preneoplastic and neoplastic lesions induced by peroxisome proliferators (PP). After administration of PP for 8-15 weeks, some rats exhibit BE-negative preneoplastic foci but other rats do not. In the present study, to investigate the involvement of glutathione S-transferase (GST) M1 gene polymorphism in interindividual differences in susceptibility to PP, we developed a method to determine the genotypes of rats. We then examined whether rats with one type encoding 198Asn-199Cys (NC-type) or another encoding 198Lys-199Ser (KS-type) exhibit differences in clofibrate (CF) susceptibility. After administration of 0.3% CF for 6 weeks or more, BE-negative foci were found immunohistochemically in KS/KS-type rats, but not in NC/NC-type rats. The number of BE-negative foci in KS/KS rats was 15.3 +/- 9.0 foci/cm2 of liver section after 6 weeks of CF administration, and the values did not alter thereafter. The mean areas of BE-negative foci in KS/KS rat livers increased during the period from 6 to 60 weeks. At weeks 30 and 60, almost all BE-negative foci exhibited a clear cell phenotype, a type of preneoplastic hepatic lesion. BE-negative foci were devoid of peroxisome proliferator-activated receptor alpha, whereas surrounding tissues were positive for the receptor. These results indicate that rats that are polymorphic for the GST M1 gene exhibit different susceptibilities to CF in vivo.
Cancer Science 09/2006; 97(8):703-9. · 3.33 Impact Factor
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ABSTRACT: Recently, we discovered that beta-thujaplicin (BT) induces metallothionein (MT) expression in mouse keratinocytes, both in vivo and in vitro. However, the molecular mechanisms by which BT exerts its biological effects have not been elucidated. The purpose of this study is to explore the signal transduction pathway involved in the MT mRNA induction by BT. Using a HaCaT keratinocyte cell line, Northern blotting was performed for analyzing the human MT-IIA mRNA expression levels in combination with BT and a number of protein kinase (PK) inhibitors including H7, HA1004 and a PKC-specific inhibitor chelerythrin. CAT assays with the MT-IIA gene promorter-CAT construct were conducted for examining the transcriptional regulation by BT of MT. A free radical scavenger N-acetylcysteine (NAC) was used for analyzing a role of oxidative stress for the MT gene induction by BT. BT increased MT-IIA gene transcript levels and CAT activity in a dose-dependent fashion in HaCaT cells. The increase in MT-IIA mRNA levels and CAT activity were completely suppressed by H7 but not by HA1004. In addition, chelerythrin prevented BT-inducible MT-IIA promoter activation. Furthermore, NAC suppressed BT-inducible MT-IIA promoter activation. These results demonstrate that BT is a potent activator of the MT-IIA gene promoter and that PKC activation and reactive oxygen species are implicated in BT-inducible MT-IIA gene expression. BT may be a useful tool for dissecting the signal transduction pathway mediating MT-IIA promoter activation.
Biological & Pharmaceutical Bulletin 02/2006; 29(1):55-9. · 1.66 Impact Factor
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ABSTRACT: Hirosaki hairless rat (HHR) is a mutant strain spontaneously derived from Sprague-Dawley rats (SDR), and its inheritance is autosomal recessive. In addition to hair loss, female HHRs show involution of the mammary gland at an early stage of lactation. In the present study we investigated the mammary gland development in HHR. Morphological examinations revealed that HHR mammary glands are underdeveloped in virgins and exhibit distended alveoli on day 1 of lactation (L1), followed by involution. Milk secretion was observed on L1 in HHR. Whey acidic protein and other proteins were increased in milk of HHR and heterozygous rats on SDS-polyacrylamide gel electrophoresis. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay revealed apoptosis induction in HHRs at an early stage of lactation. By Western blotting, signal transducer and activator of transcription (STAT) 5A levels in cytoplasmic and nuclear fractions of the mammary glands were not different between HHR and SDR on L1 and L7. Nuclear localization of STAT5A in HHR and SDR was confirmed by immunohistochemistry. Tyr-phosphorylated STAT5A was not detected in HHR but was detected in SDR nuclear fractions. Several proteins modified with O-linked N-acetylglucosamine (O-GlcNAc) were detected in HHR nuclear extract on L1, although not in SDR or heterozygous rats by Western blotting. When HHR nuclear extract was applied to wheat germ agglutinin-agarose, a part of STAT5A was recovered in bound fractions. STAT5A of SDR or heterozygous rat nuclei were not bound to the lectin. Electrophoretic mobility shift assay revealed that STAT5A modified with O-GlcNAc is bound to the STAT5-responsive element. These results indicate that the mammary glands of HHR showed terminal differentiation for a short period, followed immediately by involution. In HHR, STAT5A is modified with O-GlcNAc but is not Tyr-phosphorylated. This type of glycosylation is suggested to be involved in the transient activation of STAT5A in HHR.
Journal of Biological Chemistry 01/2006; 280(52):43010-6. · 4.77 Impact Factor
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ABSTRACT: Benastatin A, isolated from Streptomyces bacteria, is reported to inhibit mammalian glutathione transferases (GSTs). Since GST inhibitors such as ethacrynic acid are suggested to induce apoptosis in some cell lines, the effect of benastatin A on the survival of mouse colon 26 adenocarcinoma cells was compared with that of ethacrynic acid. When cells in stationary phase were treated with benastatin A, viable cells were found to be dose-dependently decreased after 3 days. In the case of ethacrynic acid, this became apparent within 24 h. Electrophoretic analysis revealed DNA fragmentation, indicating that cell loss was due to apoptosis in both cases. The dominant GST in colon 26 cells was identified as the class Pi-form (GST-II), and the activities in crude extracts as well as purified GST-II were almost completely inhibited by 50 μM ethacrynic acid. Immunoblot and northern blot analyses revealed increased GST-II protein and mRNA levels in cells treated with ethacrynic acid. Benastatin A did not significantly affect the activity in the crude extract even at 20 μM, a 10-fold higher concentration than that which almost completely inhibited the activity of purified GST-II. However, GST activity and GST-II protein were decreased in colon 26 cells treated with benastatin A for 5 days, no significant activity being detected in the range of 16–20 μM. In addition, β-actin and bax mRNAs were also decreased in a dose-dependent manner. Furthermore, flow cytometric analysis of colon 26 cells revealed that benastatin A blocked the cell cycle at the G1/G0 phase. Thus, benastatin A also induces apoptosis of colon 26 cells, but this is unlikely to be due to inhibition of GST activity.
Cancer Science 08/2005; 91(11):1161 - 1168. · 3.33 Impact Factor
