Nobutaka Nagai

Increased MCL–1 Expression Is Associated with Poor Prognosis in Ovarian Carcinomas

To investigate the potential role of the BCL–2 gene family (BAX, BCL–2, MCL–1, and BCL‐XL) in ovarian cancer development and progression, mRNA expression levels of these genes were measured using semi‐quantitative PCR in epithelial ovarian tumor tissues and normal ovaries. The immunohistochemical expression of MCL–1 in ovarian tumors was also examined. The expression levels of BAX and MCL–1 mRNA were significantly higher in ovarian cancers and in adenomas than in normal ovaries (P<0.05). In contrast, the BCL–2 mRNA expression level in ovarian cancers was significantly lower than in ovarian adenomas and in normal ovaries (P<0.05). Expression of BCL‐XL mRNA was no different between normal ovaries and ovarian tumors. Log‐rank testing showed that low BAX mRNA expression and high MCL–1 mRNA expression significantly correlate with poor survival for patients with stage III ovarian carcinomas (BAX, P=0.05; MCL–1, P=0.02). Immunohistochemical analysis showed that diffuse‐positive expression of MCL–1 protein in mucinous carcinomas was significantly higher than in mucinous low malignant potential (LMP) tumors (P=0.03). In ovarian cancer cases, diffuse‐positive expression of MCL–1 protein significantly correlates with advanced clinical stage, high histologic grade, and poor survival (stage, P<0.01; grade, P=0.01; survival, P=0.01). These results suggest that increased MCL–1 expression may play an important role in replacing the functions of increased BAX and decreased BCL–2 in ovarian carcinoma cells, thereby promoting cell survival, and resulting in a poor prognosis for patients with ovarian cancer.

Relationship between the angiotensin-converting enzyme genotype and the forearm vasodilator response to estrogen replacement therapy in postmenopausal women

OBJECTIVES We sought to evaluate the relationship between the angiotensin-converting enzyme (ACE) genotype and the change in forearm vasoreactivity in response to a three-month course of oral estrogen in postmenopausal women. BACKGROUND The ACE genotype is a known predictor of the response to an ACE inhibitor drug; however, it is not clear whether it can modify the effect of estrogen replacement therapy (ERT) on endothelial function in postmenopausal women. METHODS Fifty-five postmenopausal women received 0.625 mg of conjugated equine estrogen daily for three months. Forearm blood flow (FBF) was measured by strain-gauge plethysmography. RESULTS Twenty-one, 25 and 9 patients had the insertion/deletion (ID), II and DD genotypes, respectively. Plasma ACE activity was significantly higher at baseline in patients with either the DD or ID genotype than in those with the II genotype (p < 0.05). A significant decrease in plasma ACE activity with ERT was seen in the ID and II genotypes (p < 0.05), but not in the DD genotype. There were no significant differences in the FBF responses to reactive hyperemia at baseline between the three groups. Estrogen replacement therapy did not alter the FBF response to reactive hyperemia in the DD genotype (4.0 +/- 1.3%), although ERT significantly increased the FBF response in the ID and II genotypes (32.6 +/- 7.5% and 30.6 +/- 6.5%, respectively; p < 0.05). Forearm blood flow after administration of sublingual nitroglycerin did not change over three months in any of the three groups. CONCLUSIONS These findings suggest that the effect of ERT in postmenopausal women on forearm endothelial function may be determined in part by the genotype of the ACE gene.

Induction of matrix metalloprotease-7 is common in mucinous ovarian tumors including early stage disease

Matrix metalloproteases are known to play an important role in tumor invasion by mediating degradation of the extracellular matrix. In this study, we have investigated the immunohistochemical expression of matrix metalloprotease-7 (MMP-7) in 44 mucinous ovarian tumors (9 adenomas, 13 low malignant potential tumors, 22 adenocarcinomas) and 6 normal ovaries. Positive staining of MMP-7 is observed in all mucinous ovarian tumors, whereas little or no staining was observed in surface epithelium as well as the epithelial cells of germinal inclusion cyst of the normal ovary. Positive immunostaining of MMP-7 is also observed in the secreted mucin in the tumor glands, which suggests the secretion of the MMP-7 protein from tumor cells. mRNA expression of MMP-7 was confirmed using RT-PCR. The MMP-7 gene was amplified in parallel with an internal control gene β-tubulin using a thermal cycler. mRNA expression levels of MMP-7 were significantly elevated in mucinous tumor samples compared with that in normal ovaries. Our results suggest that MMP-7 is frequently overexpressed in mucinous ovarian tumors and secreted with the mucin which is produced from the tumor cells. MMP-7 may therefore contribute to mucinous ovarian tumor development or enhanced growth capacity of mucinous ovarian tumors. MMP-7 may also serve as a target for therapeutic intervention in the down regulation of tumor progression.

Prediction of individual response to platinum/paclitaxel combination using novel marker genes in ovarian cancers

We attempted to identify potent marker genes using a new statistical analysis and developed a prediction system for individual response to platinum/paclitaxel combination chemotherapy in ovarian cancer patients based on the hypothesis that expression analysis of a set of the key drug sensitivity genes for platinum and paclitaxel could allow us to predict therapeutic response to the combination. From 10 human ovarian cancer cell lines, genes correlative in the expression levels with cytotoxicities of cisplatin (CDDP) and paclitaxel were chosen. We first selected five reliable prediction markers for the two drugs from 22 genes already known as sensitivity determinants and then identified another 8 novel genes through a two-dimensional mixed normal model using oligomicroarray expression data. Using expression data of genes quantified by real-time reverse transcription-PCR, we fixed the best linear model, which converted the quantified expression data into an IC50 of each drug. Multiple regression analysis of the selected genes yielded three prediction formulae for in vitro activity of CDDP and paclitaxel. In the same way, using the same genes selected in vitro, we then attempted to develop prediction formulae for progression-free survival to the platinum/paclitaxel combination. We therefore constructed possible formulae using different sets of 13 selected marker genes (5 known and 8 novel genes): Utility confirmation analyses using another nine test samples seemed to show that the formulae using a set of 8 novel marker genes alone could accurately predict progression-free survival (r = 0.683; P = 0.042). [Mol Cancer Ther 2006;5(3):767–75]

Comparison of forearm endothelial function between premenopausal and postmenopausal women with or without hypercholesterolemia.

We sought to determine whether menopausal status or postmenopausal hypercholesterolemia affects forearm resistance artery endothelial function. We studied the forearm resistance artery endothelial function in 75 Japanese women: 25 premenopausal volunteers, 25 postmenopausal women with normal serum low-density lipoprotein (LDL) cholesterol concentrations, and 25 hypercholesterolemic postmenopausal women. Excluded from the study were patients with hypertriglyceridemia, hypertension, or diabetes, cigarette smokers. The forearm blood flow (FBF) during reactive hyperemia and after sublingual nitroglycerin (NTG) administration was measured by strain-gauge plethysmography. The serum concentrations of lipoprotein (a) [Lp(a)] were significantly higher in the hypercholesterolemic postmenopausal group than in the other two groups (P<0.01). These lipid parameters were similar between the premenopausal and postmenopausal women with normal cholesterol. The FBF responses to reactive hyperemia were significantly lower in the postmenopausal hypercholesterolemic women than in the other two groups (P<0.01). The reactive hyperemia also was impaired in the postmenopausal group with normal cholesterol as compared with the premenopausal group (P<0.01). Increases in FBF after NTG were similar between the three groups. By stepwise multivariate analysis, menopausal status and serum LDL cholesterol was the significant predictor of forearm endothelial function. These findings suggest that reactive hyperemia is impaired in forearm resistance arteries after menopause, especially in postmenopausal women with hypercholesterolemia.

Macrophage-colony stimulating factor inhibits the growth of human ovarian cancer cells in vitro.

The effect of macrophage-colony stimulating factor (M-CSF), which regulates the growth and differentiation of haematopoietic progenitor cells on the growth of ovarian cancer cells was investigated in three ovarian cancer cell lines in vitro. The spontaneous growth of these cells was significantly inhibited by the addition of M-CSF in a concentration-dependent manner over 96 h of culturing. The maximum response was obtained with 10 ng/ml (3857 U/ml) of M-CSF by counting the viable cell number using the trypan blue exclusion assay. [(3)H]-thymidine incorporation by these cells was also suppressed following a 96-h incubation with M-CSF. The inhibitory effect of M-CSF was reversed by the addition of anti-M-CSF monoclonal antibody. Flow cytometric analysis revealed that the treated ovarian cancer cells arrested at the G0/G1 phase of the cell cycle. These cells expressed M-CSF receptors on their surface as detected by Scatchard plot analysis using (125)I-labelled M-CSF. These results indicate that M-CSF has an antitumour activity for ovarian cancer cells and suggest that it can be applied for the treatment of this disease.

Expression of Tumor-Associated Differentially Expressed Gene-14 (TADG-14/KLK8) and Its Protein hK8 in Uterine Endometria and Endometrial Carcinomas

To clarify the biological behavior of TADG-14/KLK8, we investigated TADG-14/KLK8 mRNA by semiquantitative RT-PCR and hK8 expression by immunohistochemistry using 37 normal endometria and 44 endometrial carcinoma tissues. TADG-14/KLK8 mRNA expression levels were significantly higher in proliferative compared to secretory phase endometria (p = 0.0143). Levels of TADG-14/KLK8 mRNA expression correlated with hK8 protein levels. hK8 was detected in 73.3% (11/15) of endometria with a significantly higher detection rate in the proliferative compared to secretory and atrophic phase endometria (p = 0.0002). High expression of hK8 was found in 61.4% of endometrial carcinomas compared to 35.1% of endometrial tissue samples (p = 0.0187). hK8 expression was significantly higher in stage I (p = 0.0433, 0.0038) and grade 1/2 (G1/2) of the tumors (p = 0.0195, 0.0044). We suggest that expression of TADG-14/KLK8may be regulated by sex steroid hormones in endometria. Our results indicate that elevated TADG-14/KLK8 expression is an early event in endometrial carcinogenesis, and may potentially serve as a useful early biomarker for the detection of endometrial carcinomas in menopausal women.

Predicting the chemosensitivity of ovarian and uterine cancers with the collagen gel droplet culture drug-sensitivity test

We investigated the utility of the collagen gel droplet culture drug-sensitivity test (CD-DST) for predicting the response of gynecological cancers to chemotherapy. Eighty-three cancer patients were enrolled in this study: 26 ovarian, 29 cervical and 31 endometrial cancers. The CD-DST was performed at various concentrations of drugs. We calculated the T/C ratio, where T is the total volume of the treated culture and C is the total volume of the control culture, and a T/C ratio of 50% or less was defined as sensitive in vitro. The efficacy rate (%) was defined as the number of cultures with a T/C ratio of 50% or less, divided by the total number of evaluable cultures. True-positive cases were defined as clinical responders (complete+partial responses) and true-negative cases were defined as clinical non-responders. The overall tumor evaluation rate was found to be 79.1%. The appropriate drug concentrations were selected as 1.0 μg/ml for cisplatin, 20.0 μg/ml for carboplatin, 1.0 μg/ml for paclitaxel and 0.1 μg/ml for docetaxel by the linear regression equations. The in vitro sensitivity for each drug showed a significant correlation with clinical response rates (r=0.592, p=0021). We therefore conclude that the CD-DST can be used to predict the response to anti-cancer drugs, and may also provide important information by contributing to the development of new chemotherapy regimens.

Validation of a simple gas chromatographic–mass spectrometric method for the determination of gamma-butyrolactone in human plasma

A gas chromatographic-mass spectrometric (GC-MS) method is described for the determination of human plasma levels of gamma-butyrolactone (GBL) is described. The method is sensitive and simple. The plasma sample spiked with the internal standard was extracted by dichloromethane (CH(2)Cl(2)) in acidic conditions, and the concentrated organic layer was injected into GC-MS. Because of endogenous GBL in human plasma, the method used a standard calibration curve. The calibration curve was linear from 10 to 1000 ng/ml. The method has been validated for accuracy and precision with the relative error and C.V. for intra- and inter-day within 10%. GBL-spiked plasma samples stored at -80 degrees C were stable for a 3-month period. The stability of plasma samples after three cycles of freezing and thawing and of prepared samples on an autosampler for 48 h were demonstrated. Plasma concentrations of GBL before and after administration of UFT were 24.3+/-14.2 and 84.9+/-22.4 ng/ml, respectively.

Vaginal hemangiopericytoma: a light microscopic and ultrastructural study

A case of a vaginal hemangiopericytoma in a 20-year-old woman was studied by light and electron microscopy. The neoplastic cells had round-to-oval nuclei with one or two nucleoli; fine chromatin; a foamy, cyanophilic cytoplasm; and an increased nuclear cytoplasmic ratio. Light microscopically, the tumor was composed of spindle-shaped or round cells proliferating around vascular spaces. Mitotic figures, necrosis, and hemorrhage were difficult to find. Electron microscopically, the neoplastic cells occurred in clusters, each of which was enclosed by a basal lamina. The cytoplasm contained abundant free ribosomes, flattened elements of rough endoplasmic reticulum, small Golgi apparatus, round or elongated mitochondria, lysosomes, glycogen particles, and sometimes pinocytotic vesicles and bundles of microfilaments with large dense spots. Desmosomes were also seen. In addition, the cytoplasmic process of the benign vascular pericytes was directly in contact with the neoplastic cells which had varying degrees of cytodifferentiation. The neoplastic cells in this lesion are consistent with an origin from pericytes, confirming the findings of this neoplasm when it arose in other sites.