Biomedical subjects

Toshio Nikaido

Publications and source records attributed to Toshio Nikaido.

31 records · Page 2Linked to original sources

PubMed2003

Up-regulation of small GTPases, RhoA and RhoC, is associated with tumor progression in ovarian carcinoma.

To clarify the role of small GTPases Rho in the biologic behavior of ovarian carcinoma, we first examined the mRNA expression of RhoA, RhoB, and RhoC in benign, borderline, and malignant ovarian tumors using RT-PCR and real-time RT-PCR. The expression and localization of RhoA protein were also analyzed by Western blotting and immunohistochemistry. Finally, we examined whether up-regulation of Rho enhances the invasiveness of ovarian cancer cells in vitro. Analysis of mRNA levels of the Rho family genes revealed that levels of both RhoA and RhoC were significantly higher in carcinomas than in benign tumors (RhoA, p = 0.0035; RhoC, p = 0.0006). According to histologic subtype, both RhoA and RhoC mRNA levels in serous carcinomas were significantly higher than those in other histologic types. With regard to the International Federation of Gynecological and Obstetrics stage classification, both of RhoA and RhoC mRNA levels were significantly higher in tumors of Stages III+IV than in those of Stages I+II (RhoA, p = 0.0200; RhoC, p = 0.0057). In addition, analysis of matched pairs of primary and disseminated lesions demonstrated that expression of both RhoA and RhoC mRNA was significantly higher in metastatic than in primary tumors. Examination of the protein level showed that expression of RhoA was also increased in advanced ovarian carcinomas, especially those of serous histology. Accordingly, we hypothesized that up-regulation of Rho GTPases plays an important role in the progression of ovarian carcinoma. Matrigel invasion assay using the ovarian cancer cell line, SKOV3, showed that up-regulation and activation after treatment with lysophosphatidic acid was associated with enhanced invasion of the cancer cells. This increase in invasiveness was suppressed by the addition of C3, a specific inhibitor of Rho. These findings suggest that up-regulation of Rho GTPases is important in the tumor progression of ovarian carcinoma and that Rho family proteins could be a molecular target in cancer therapy.

Base Sequence↗

PubMed2003

Expression of replication-licensing factors MCM2 and MCM3 in normal, hyperplastic, and carcinomatous endometrium: correlation with expression of Ki-67 and estrogen and progesterone receptors.

Minichromosome maintenance (MCM) proteins are essential for cell cycling due to their function as replication-licensing factors. The aim of the present study was to investigate the clinicopathologic implications of the MCM2 and MCM3 in endometrial carcinogenesis. The authors investigated the immunohistochemical expression of MCM2 and MCM3, Ki-67, estrogen receptor, and progesterone receptor in 23 normal endometria, 9 endometrial hyperplasias, and 60 endometrial carcinomas. In the normal endometrial glands, the expression of MCM2 and MCM3 was significantly higher in the proliferative phase than in the secretory phase and was strongly correlated with Ki-67 expression. Similar correlation between the expression of MCMs and Ki-67 was also found in endometrial hyperplasia. In endometrial carcinomas, however, the expression of MCM2 and MCM3 was significantly lower than that in the normal proliferative endometrium. There was only a weak correlation between MCM2 and Ki-67, and no significant correlation between MCM3 and Ki-67 expression. These findings suggest that the expression of MCM2 and MCM3 directly reflects cell proliferation in normal and hyperplastic endometria. In endometrial carcinomas, however, there is a discrepancy between the expression of MCMs and cell proliferation, suggesting that the replication-licensing system may be aberrant in endometrial carcinomas.

Adult↗

PubMed2003

Efficient gene transfer to hepatoblastoma cells through asialoglycoprotein receptor and expression under the control of the cyclin A promoter.

Specific gene delivery into hepatoma cells by liposomes and specific gene expression under the control of the cyclin A promoter were examined in HepG2 cells, a hepatoblastoma cell line that overexpresses cyclin A. A plasmid carrying the luciferase gene under the cyclin A promoter sequence was condensed with poly-L-lysine and encapsulated into anionic asialofetuin-labeled liposomes (AF-liposomes), which were preferentially taken up by hepatocytes through the action of the asialoglycoprotein receptor (AgpR). AF-liposomes delivered plasmids to the hepatoma cells by receptor-mediated endocytosis through the AgpR, and transgene expression could be achieved under the control of the cyclin A promoter. Furthermore, a fusogenic lipid, DOPE, as a liposomal component was required for the enhancement of transfection efficiency of AF-liposomes.

Antimalarials↗

PubMed2003

Human amnion-isolated cells normalize blood glucose in streptozotocin-induced diabetic mice.

Whole pancreas or beta-cell transplantation has opened the way for the treatment of advanced stage of diabetes mellitus. However, it is always limited by the scarcity of transplantation materials. The amniotic membrane is part of the fetal membrane and is composed of amniotic epithelium (HAE) and mesenchymal (HAM) cells that are derived from the inner cell mass in the blastocyst. Thus, HAE and HAM cells may have the potential to differentiate into various organs. The aim of our study was to assess the possibility of HAE cells differentiating into insulin-producing cells. In vitro, HAE cells stimulated with nicotinamide induced insulin mRNA in the culture cells. In vivo, HAE cells were capable of normalizing the blood glucose level of diabetic mice after several weeks of implantation into streptozotocin-induced diabetic mice. The distribution of human cells and human insulin secretion in mouse tissue studied by immunohistochemistry for anti-human-specific beta-2-microglobulin and anti-human-specific insulin shows the same location in mouse tissue. These studies suggest that HAE cells have the potential to differentiate into beta-cells in vivo, and hence that HAE cells have therapeutic potential for the treatment of type I diabetes mellitus.

Amniotic Fluid↗

PubMed2002

Drug metabolic activity of cultured hepatocytes can synchronize with bile acid concentration in the medium.

The regulation of drug metabolic activity of cultured hepatocytes can be applied to the evaluation of pharmacokinetics, analysis of drug delivery and the bioartificial liver system. It is very difficult to maintain the drug metabolic activity mediated by cytochrome P-450 (CYP) 3A. Recently we found that the CYP3A aminopyrine N-demethylase (AMND) activity of hepatocytes cultured on collagen surface oscillated with culture time. This phenomenon was related to the concentration of bile acid in the culture medium. CYP3A, multidrug resistant gene 2 (MDR2) and heat shock protein 84 (HSP84) mRNA appeared in a manner corresponding to this oscillation. When a large quantity of bile acid was taken up into hepatocytes from the medium, low AMND activity was observed, and these proteins did not appear. When bile acid was secreted and the bile acid concentration inside the hepatocytes was low, high AMND activity was obtained, and these proteins appeared. In order to clarify the mechanism of oscillation between AMND activity and bile acid, 8 microM glycocholic acid was added to the culture medium 15 h before the measurement. No oscillation in AMND activity was observed in the presence of 8 microM glycocholic acid. Bile acid controls the AMND activity in the transcription of hepatocytes.

Aminopyrine N-Demethylase↗

PubMed2002

FK506 enhanced osteoblastic differentiation in mesenchymal cells.

Bone morphogenetic protein (BMP) is a bone-derived growth factor capable of promoting the differentiation of mesenchymal cells into osteogenic lineage pathways. Recently, immunosuppressants were reported to cause a moderate increase in osteoblastic differentiation in a rat osteoblast-like osteosarcoma cell line. If immunosuppressants can induce osteoblastic differentiation, it will be useful for bone tissue transplantation. We assessed the effect of immunosuppressants with or without BMP-4 on inducing osteoblastic differentiation in osteoblast-like and other mesenchymal cells. FK506, an immunosuppressant often used clinically, induced a dose- and time-dependent increase in alkaline phosphatase (ALP) activity, one of the markers of osteoblast differentiation, in cells derived from mesenchyma. In the presence of BMP-4, ALP activity, mRNA levels of ALP and osteocalcin increased. FK506 was found to not only stimulate osteoblastic differentiation, but also to enhance BMP-4 induced osteoblastic differentiation. These results suggest that FK506 promotes differentiation of osteoblastic cells.

3T3 Cells↗

PubMed2002

Growth inhibition and differentiation of pancreatic cancer cell lines by PPAR gamma ligand troglitazone.

INTRODUCTION: Ligand activation of peroxisome proliferator-activated receptor gamma (PPAR gamma) results in growth inhibition and differentiation of various cancer cells. AIMS: We determined whether the PPAR gamma ligand, troglitazone, inhibits the growth of pancreatic cancer cells and clarified the underlying mechanisms with a special focus on restriction point control of the late G1 phase of the cell cycle. METHODOLOGY: Nine pancreatic cancer cell lines were used to study a variety of troglitazone effects on cell growth by MTT assay, on cell cycle by flow cytometry, on cell cycle regulating factors of late G1 phase by Western and Northern blotting and CDK2 kinase assay, and on morphology by collagen gel culture and electron-microscopy. RESULTS: Troglitazone showed a potent dose-response effect on the growth inhibition of six pancreatic cancer cell lines, which were suppressed to less than 50% of control at the concentration of 10 microM. The growth inhibition was linked to the G1 phase cell cycle arrest through the upregulation of p21 mRNA and protein expression simultaneously with the inhibition of CDK2 kinase activity and the hypophosphorylation of Rb protein. The upregulation of expression of p21 mRNA was mainly due to stabilization of mRNA. Troglitazone induced significant morphologic changes of duct structure with apoptotic cells in the lumen. CONCLUSION: Troglitazone had growth inhibitory and differentiation induction effects on the pancreatic cancer cell lines through the upregulation of p21 expression, suggesting that ligand activation of PPAR gamma is a new molecular target for effective therapy against pancreatic cancer.

Adenocarcinoma↗

PubMed2002

Tranilast inhibits the proliferation of uterine leiomyoma cells in vitro through G1 arrest associated with the induction of p21(waf1) and p53.

Uterine leiomyoma is a mesenchymal tumor composed of smooth muscle cells with fibrous tissues and many mast cells. Tranilast is known to suppress fibrosis or to work as a mast cell stabilizer and is reported to inhibit proliferation of vascular smooth muscle cells. In this study, we examined the effects of tranilast on cultured human leiomyoma cells in vitro to evaluate whether this agent has the potential to inhibit the growth of uterine leiomyomas. Tranilast inhibited the proliferation of cultured leiomyoma cells in a dose-dependent manner without any cytotoxic effect or induction of apoptosis. In association with the inhibitory effect, tranilast induced the cyclin-dependent kinase (CDK) inhibitor p21(waf1) and tumor suppressor gene p53 and decreased CDK2 activity. These results suggest that tranilast arrests the proliferation of uterine leiomyoma cells at the G0/G1 phase, through the suppression of CDK2 activity via an induction of p21(waf1) and p53. Tranilast was concluded to be a potent agent to inhibit proliferative activity of uterine leiomyoma cells.

Blotting, Western↗

PubMed2002

Altered post-translational modification of redox factor 1 protein in human uterine smooth muscle tumors.

Uterine leiomyomas are the most common benign smooth muscle tumors in the myometrium. The expression of redox factor 1 (Ref-1), a DNA repair enzyme and redox-modifying factor, was studied in the myometrium and uterine smooth muscle tumors to investigate the relevance of Ref-1 in the growth regulation of the tumors. Two forms of Ref-1 protein were detected, using three antibodies against different epitopes of Ref-1. The abundance of the large form of Ref-1 was increased in leiomyoma extracts relative to myometrial tissue extracts, and the large form was dominant in cell lines derived from leiomyosarcomas. A single mRNA transcript was detected in the same samples, leading us to hypothesize that the differentially migrating forms are the result of posttranslational modification(s). In vitro incubation of leiomyoma tissue extract lead to a shift from the large form to the small form, and this conversion was inhibited by either protease or phosphatase inhibitors. Finally, the relative abundance of the large form of Ref-1 was found to correlate with proliferating cell nuclear antigen levels, suggesting a correlation with increased proliferation. These results indicate that altered posttranslational modification of Ref-1 is involved in uterine smooth muscle tumorigenesis.

Adult↗

PubMed2002

Soluble asialoglycoprotein receptors reflect the apoptosis of hepatocytes.

Cell death is thought to take place through at least two distinct processes: apoptosis and necrosis. There is increasing evidence that dysregulation of the apoptotic program is involved in liver diseases. However, there is no method to simply evaluate apoptosis in the liver tissue at present. It has been reported that the expression of asialoglycoprotein receptors (AGPRs) increases with apoptosis, but there is no report until now that investigates the influence of soluble AGPRs on apoptosis of hepatocytes. Soluble AGPRs have been reported to be present in human serum under physiological conditions. In the present study, in order to investigate the correlation between apoptosis of hepatocytes and soluble AGPR, mouse soluble AGPRs were detected using SDS-PAGE and Western blot analysis was conducted using anti-extracellular mouse hepatic lectin-1 (Ex-MHL-1) antiserum (polyclonal rabbit serum). The mouse soluble AGPRs were present in culture medium and mouse serum when hepatocytes were damaged. The soluble AGPRs increased proportionately, as the number of dead hepatocytes increased. In addition, soluble AGPRs existed more when apoptotic cell death was observed in in vitro and in vivo than when necrotic cell death was observed. The extracellular moiety of MHL-1 exists in the culture medium and mouse serum as a soluble AGPR, but the detailed mechanism of releasing soluble AGPR from hepatocytes has not been revealed yet. We described the first evidence for the relation between quantity of soluble AGPRs with two kinds of cell death: necrosis and apoptosis. Based on the results of our study, soluble AGPRs might become a new marker of apoptosis in the liver tissue and be useful for clinical diagnosis and treatment for liver diseases.

Animals↗

PubMedDate not supplied

Down-regulation of estrogen receptor by the methylation of the estrogen receptor gene in endometrial carcinoma.

BACKGROUND: Human uterine endometrial carcinomas are composed of estrogen receptor (ER)-positve and ER-negative subgroups, though the mechanisms that down-regulate the ER expression are not understood. The present study was undertaken to examine the involvement of methylation of the ER gene on the ER status. MATERIALS AND METHODS: DNA was extracted from 25 cases of endometrial carcinoma. Polymerase chain reaction (PCR), using the extracted DNA and methylation-sensitive restriction enzymes (Hpa II, Hha I), was performed to detect the methylation of a CpG island in the ER (alpha) gene. The results were compared with the ER expression by immunostaining. RESULTS: In the 25 endometrial carcinomas, methylation at the Hpa II site, or at both the Hpa II and Hha I sites, was found in 6 cases, while methylation was not found in the remaining 19 cases. Immunohistochemically, 5 of the 6 methylation-positive cases were negative for ER, whereas 14 of the 19 methylation-negative cases were ER-positive (p=0.02). CONCLUSION: Methylation of the CpG island of the ER gene is inversely correlated with ER expression in a subset of endometrial carcinomas.

Carcinoma↗

PubMedDate not supplied

Hypoxia-induced changes in the expression of VEGF, HIF-1 alpha and cell cycle-related molecules in ovarian cancer cells.

BACKGROUND: Hypoxia is important in cancer progression, and at the stage of detachment of the cancer cells from the primary lesion. This study was undertaken to analyze the effect of hypoxia on angiogenesis and cell proliferation in ovarian cancer. MATERIALS AND METHODS: We first used immunohistochemistry to examine the expression of VEGF in 42 cases of ovarian carcinoma, with relevance to the p53 expression. Then, the expression of VEGF, HIF-1 alpha, cell cycle-related molecules and cell numbers were examined in 4 ovarian cancer cell lines with various p53 gene status. RESULTS: Immunohistochemistry showed that there was no significant correlation between VEGF and p53 expression. Moreover, hypoxia increased the expression of VEGF via up-regulation of HIF-1 alpha irrespective of p53 gene status. However hypoxia did not change the cell numbers, but influenced the expression of cell cycle-related molecules (increased p27 and decreased cyclin D1 and Rb). CONCLUSION: Hypoxia increased VEGF expression in ovarian cancer cells irrespective of p53 gene status.

Cell Cycle Proteins↗

PubMedDate not supplied

Nuclear localization of beta-catenin is correlated with the expression of cyclin D1 in endometrial carcinomas.

BACKGROUND: beta-catenin has recently been reported to act as a cell growth promoter through cyclin D1 transcription. However, the correlation between beta-catenin and cyclin D1 expressions is not fully understood in endometrial tissues. MATERIALS AND METHODS: Immunohistochemical expression of beta-catenin was examined in normal endometria (32 cases) and endometrial carcinomas (82 cases), and its expression was compared with that of cyclins (D1, E, A, B1). RESULTS: Sporadic nuclear staining of beta-catenin and cyclins was observed from proliferative phase of early secretory phase endometria, however, spacial correlations between beta-catenin and cyclins were not evident. In endometrial carcinomas, positivity for nuclear beta-catenin and cyclins increased compared to the normal endometria. Topologically, the cyclin D1-positive cells were frequently found in nuclear beta-catenin-positive cells. In addition, Spearman's rank correlation analysis revealed that the nuclear expression of beta-catenin correlated positively with that of cyclin D1 (p < 0.0001). CONCLUSION: The beta-catenin-cyclin D1 pathway might be involved in the growth of endometrial carcinomas.

Adult↗