Satoshi Ohira

Hypoxia Attenuates the Expression of E-Cadherin via Up-Regulation of SNAIL in Ovarian Carcinoma Cells

Since ovarian carcinoma cells detach from the primary lesion and metastasize via peritoneal dissemination, we hypothesized that these cells are exposed to hypoxia, which may affect cell attachment and invasiveness. To address this hypothesis, we first examined in vivo the immunohistochemical expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and its topological correlation with E-cadherin expression in ovarian carcinomas. We then examined in vitro the effect of hypoxia on the mRNA and protein expressions of E-cadherin using two ovarian cancer cell lines, SKOV3 and OVCAR3, and normal ovarian surface epithelial (OSE) cells. In addition, hypoxia-induced change in the expression of SNAIL, a transcriptional factor repressing E-cadherin expression, was also analyzed. Finally, we examined the facilitation of invasiveness of ovarian cancer cells under hypoxia using Matrigel invasion assay. Immunohistochemically, nuclear localization of HIF-1alpha was observed in 32 of the 76 (42%) carcinomas studied, and showed a topological correlation with loss of E-cadherin expression. Northern blotting, real-time PCR and Western blotting demonstrated that E-cadherin expression was remarkably decreased under hypoxia in both SKOV3 and OVCAR3 cells, but not in normal OSE cells. mRNA expression of SNAIL was increased under hypoxia in both ovarian cancer cell lines. Invasion assay revealed that hypoxia increases the invasiveness of ovarian cancer cells. Accordingly, the present study demonstrated that hypoxia induces down-regulation of E-cadherin in ovarian carcinoma cells, via up-regulation of the transcriptional repressor SNAIL. These findings suggest that hypoxia plays an important role in the change in intercellular attachment, which may be involved in the initiation of tumor progression of ovarian cancer cells.

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.

Levels of oxidative stress and redox-related molecules in the placenta in preeclampsia and fetal growth restriction.

Recent evidence suggests that oxidative stress is involved in the pathophysiology of preeclampsia. Using immunohistochemistry and Western blotting, we investigated the oxidative stress- and redox-related molecules, such as 8-hydroxy-2′-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE), thioredoxin (TRX) and redox factor-1 (ref-1) in the placenta in preeclampsia, intrauterine growth restriction (IUGR), preeclampsia + IUGR and in normal pregnancy. Using immunohistochemistry, the level of 8-OHdG was significantly higher in IUGR (P=0.012) or preeclampsia + IUGR (P=0.0021) than in normal pregnancy, while TRX expression was significantly higher in preeclampsia (P=0.045), and ref-1 expression was significantly higher in preeclampsia (P=0.017), IUGR (P=0.016) and preeclampsia + IUGR (P=0.0038) than in normal pregnancy. The levels of 4-HNE did not differ significantly between either preeclampsia or IUGR and normal pregnancy. A significant positive correlation was observed between TRX and ref-1 expressions in both normal (ρ=0.52) and complicated (ρ=0.43) pregnancies. Using Western blotting, ref-1 expression tended to be higher in complicated pregnancies than in normal pregnancy (P=0.09). These results suggest that oxidative DNA damage is increased in IUGR and that redox function is enhanced in both preeclampsia and IUGR compared with normal pregnancy.

Expression of BRCA1 protein in benign, borderline, and malignant epithelial ovarian neoplasms and its relationship to methylation and allelic loss of the BRCA1 gene

BRCA1 is a putative tumour suppressor gene responsible for a hereditary ovarian cancer syndrome. To clarify the possible involvement of BRCA1 in the development of sporadic ovarian neoplasms, this study analysed the immunohistochemical expression of BRCA1 protein in normal ovarian surface epithelium and 119 epithelial ovarian tumours (19 benign, 24 borderline, and 76 malignant tumours). Loss of heterozygosity (LOH) of BRCA1 was examined using three microsatellite markers to analyse the relationship between BRCA1 expression and alterations of the BRCA1 gene. Methylation of the BRCA1 promoter was also analysed by methylation‐specific PCR. In ovarian carcinomas showing heterogeneous expression of BRCA1 protein in the same tumour, LOH and methylation status were analysed using microdissection techniques. Finally, the relationship of BRCA1 expression or its genetic alteration to clinicopathological parameters and patient survival was analysed. Ovarian surface epithelial cells expressed BRCA1 protein. Decreased expression of BRCA1 was found in 16% of benign tumours, 38% of borderline tumours, and 72% of carcinomas. LOH of BRCA1 was demonstrated in no benign tumours, 15% of borderline tumours, and 66% of carcinomas. Methylation of BRCA1 was not detected in benign or borderline tumours, but was present in 31% of carcinomas. Reduced expression of BRCA1 correlated with the presence of gene methylation. The frequency of BRCA1 methylation and LOH was higher in serous carcinomas than in other types. In one of the three serous carcinomas that showed heterogeneous expression of BRCA1, BRCA1‐positive borderline‐like tumour cells were LOH‐positive and methylation‐negative, whereas adjacent BRCA1‐negative carcinoma cells were LOH‐positive and methylation‐positive. The prognosis of carcinoma patients did not correlate with BRCA1 expression or genetic status. These findings suggest that reduced expression of BRCA1 protein along with genetic and epigenetic changes of the BRCA1 gene play an important role in the development of sporadic ovarian carcinomas, particularly those of serous histology. Copyright

Changes in the expression of E-cadherin repressors, Snail, Slug, SIP1, and Twist, in the development and progression of ovarian carcinoma: the important role of Snail in ovarian tumorigenesis and progression

Changes in the expression of E-cadherin have been reported to be important in the tumorigenesis and progression of epithelial ovarian carcinoma. To further examine the mechanisms regulating E-cadherin expression in ovarian tumorigenesis, we investigated the immunohistochemical expression of transcriptional repressors for E-cadherin, such as Snail, Slug, SIP1, and Twist, in the ovarian surface epithelium (OSE) and 95 cases of epithelial ovarian tumors. OSE cells were negative for SIP1 and Slug, whereas weak expression of Snail and Twist was observed in 8 (73%) and 3 (27%) cases, respectively. Of 95 ovarian tumors, the expression of Snail, Slug, SIP1, and Twist increased stepwise in benign, borderline, and malignant tumors. Among them, the expression of Snail showed significantly inverse correlation with that of E-cadherin. Regarding the FIGO stage classification, the expressions of Snail and Twist were significantly increased in advanced cases. The prognosis of ovarian carcinoma patients positive for Snail expression was poorer than that of negative patients. Our results indicate that the expression of E-cadherin transcriptional repressors increased with malignancy in ovarian epithelial neoplasms and that the expression of E-cadherin and its negative regulators is altered during ovarian cancer development and peritoneal dissemination.

Epithelioid trophoblastic tumor metastatic to the vagina: an immunohistochemical and ultrastructural study.

We describe an epithelioid trophoblastic tumor (ETT) metastatic to the vagina in a 30-year-old Japanese woman. A polypoid tumor in the vaginal orifice was composed of nests of intermediate trophoblastic cells that showed a striking epithelioid appearance. In the hysterectomy specimen, a tumor infiltrated through the myometrium and showed histologic findings similar to those of the vaginal tumor. The tumor cells were positive for cytokeratin, inhibin-alpha, and melanoma cell adhesion molecule (Mel-CAM, CD146) but were only focally positive for human placental lactogen. Electron microscopic examination revealed bundles of well-developed, intermediate-type filaments surrounding the nuclei.

Urachal adenocarcinoma metastatic to the ovaries: case report and literature review.

We report a urachal adenocarcinoma metastatic to both ovaries in a 50-year-old Japanese woman. Pelvic examination and imaging studies revealed a large cystic tumor occupying the pelvis and another cystic tumor between the umbilicus and the urinary bladder. A laparotomy was performed. Histopathological examination revealed a urachal tumor that was a well-differentiated invasive mucinous adenocarcinoma; the overlying urothelium was intact. The right and left ovarian tumors were well-differentiated mucinous adenocarcinomas. The urachal and ovarian tumors were immunoreactive for cytokeratin 20 and carcinoembryonic antigen, but negative for cytokeratin 7. The patient is alive with lymph node and bone metastases 6 months postoperatively. This is the eighth reported case of an adenocarcinoma of the bladder with ovarian metastasis.

Successful Pregnancy and Lactation Outcome in a Patient With Gaucher Disease Receiving Enzyme Replacement Therapy, and the Subsequent Distribution and Excretion of Imiglucerase in Human Breast Milk

BACKGROUND Enzyme replacement therapy (ERT) with imiglucerase is a well-established, effective treatment for Gaucher disease. However, there have been no published reports regarding the excretion of imiglucerase into human breast milk and its effects on the nursing infant. OBJECTIVE This letter reports on the successful pregnancy and lactation of a patient with Gaucher disease receiving treatment with imiglucerase, and the subsequent distribution and excretion of imiglucerase in human breast milk. METHODS A 39-year-old Japanese female (height, 164 cm; weight, 55 kg) with Gaucher disease had 2 successful pregnancies and continued ERT through both. The study was conducted 6 months after the first delivery. She was administered a 1-hour infusion of imiglucerase 60 U/kg that coincided with her regular every-2-week regimen. Serum and breast-milk samples were obtained before and up to 24 hours after administration. Breast-milk samples were also obtained from 10 nursing mothers with galactorrhea as controls. RESULTS The preinfusion level of breast-milk β-glucocerebrosidase was 0.008 nmol/h/mL. The peak of serum β-glucocerebrosidase activity (0.119 nmol/h/mL) was obtained at the end of the 1-hour infusion period. Slightly increased enzymatic activity (0.016 nmol/h/mL) was observed in the first breast milk sampled after imiglucerase infusion. CONCLUSIONS We report a case of successful pregnancy and breastfeeding in a Japanese patient with Gaucher disease. A small amount of imiglucerase was found to be excreted into human breast milk, but only in the first milk produced after infusion.

Placental Mesenchymal Dysplasia: Chronological Observation of Placental Images during Gestation and Review of the Literature

Placental mesenchymal dysplasia (PMD) is characterized by multiple hypoechoic vesicles which are similar to molar changes in the placenta; however, the process of such morphological changes of PMD during pregnancy has not been fully understood. We performed a review of all PMD cases published in English and identified 49 articles including 110 cases. With regard to the gestational age at which the multicystic pattern was seen, approximately 70% of cases were diagnosed at 13-20 weeks of gestation. Another characteristic feature of PMD is varicose dilation of fetal chorionic vessels. As many as 90% of cases were diagnosed as placenta with dilated fetal chorionic vessels in the third trimester. We also report a case of PMD which was found at 10 weeks of gestation according to ultrasonic molar patterns. Serial observations of the placenta using ultrasound and magnetic resonance imaging revealed that multicystic lesions became smaller after 23 weeks. In contrast, dilated placental vessels on the fetal side became apparent at 38 weeks. The present review highlights that placental vesicular lesions of PMD may precede dilation of fetal chorionic vessels during pregnancy. It also indicates the potential of a gradual reduction in size of PMDs placental vesicular lesions by serial study of placental images.

Lipocalin2 enhances the matrix metalloproteinase-9 activity and invasion of extravillous trophoblasts under hypoxia

OBJECTIVES The invasion of extravillous trophoblasts (EVTs) to the decidua and spiral arteries in early pregnancy is a crucial step for a successful pregnancy; however, its mechanisms are not fully understood. Lipocalin2 (LCN2), a multifunctional secretory protein known as neutrophil gelatinase-associated lipocalin (NGAL), reportedly enhanced invasiveness via the activation of matrix metalloproteinase-9 (MMP-9) in several cancer cells. In this study, the expression and function of LCN2 in early placenta were analyzed. METHODS Early placental tissues between 7 and 10 weeks of gestation were obtained from normal pregnant women who underwent elective termination. The expression of LCN2 was examined using immunostaining and RT-PCR. EVTs isolated from these placental tissues and a choriocarcinoma cell line (JAR) were used to investigate the effects of LCN2 on proliferation, invasion potential, and MMP-9 activity under hypoxia using a WST-1 assay, Matrigel invasion assay, and gelatin gel zymography, respectively. RESULTS The immunohistochemical expression of LCN2 was observed in the cytoplasm of EVTs, cytotrophoblasts and the decidua, but not in syncytiotrophoblasts. The addition of recombinant LCN2 did not affect proliferation, but enhanced the invasiveness (500 ng/mL, p < 0.01) and MMP-9 activity of primary cultured EVTs and JAR in a dose-dependent manner. Silencing LCN2 using shRNA reduced the invasiveness (p < 0.01) and MMP-9 activity of JAR. In addition, the hypoxic condition (2% O₂) increased LCN2 expression (p < 0.01), MMP-9 activity, and invasive ability (p < 0.01). CONCLUSIONS LCN2 was involved in the invasiveness of EVTs, especially under hypoxia, via increased MMP-9 activity.