Kanako Nanbu

Expression of vascular endothelial growth factor (VEGF) in epithelial ovarian neoplasms: correlation with clinicopathology and patient survival, and analysis of serum VEGF levels.

Vascular endothelial growth factor (VEGF) is known to be produced by various solid tumours and is thought to be involved in microvascular permeability and/or angiogenesis. To examine the relationship between VEGF expression in ovarian neoplasms and clinicopathological factors or patient survival, expression of VEGF was analysed immunohistochemically in 110 epithelial ovarian tumours. In addition, VEGF levels in the tumour fluid (17 patients), ascites (12 patients) and sera (38 patients) were determined using enzyme immunoassay. Positive immunostaining for VEGF was observed in 97% (68 out of 70) of ovarian carcinomas, which was significantly higher than that of tumours of low malignant potential (LMP) (13 out of 25; 52%) and benign cystadenomas (5 out of 15; 33%) (P < 0.01). In ovarian carcinomas, strong VEGF immunostaining was also observed more frequently in tumours of clear cell type (P < 0.05) in the advanced stage of disease (P < 0.05) and with positive peritoneal cytology (P < 0.01). Patients with strong VEGF staining had poorer survival rates than those with weak or no immunostaining for VEGF (P < 0.01). These findings suggest that strong VEGF expression plays an important role in the tumour progression of ovarian carcinoma. The enzyme immunoassay revealed higher serum VEGF levels in carcinoma patients than those in patients with LMP or benign tumours (P < 0.01). Serum VEGF levels decreased after the successful removal of tumours in ovarian cancer patients and, in one patient, the serum VEGF level was re-elevated during relapse. Therefore, serum VEGF could be used as a marker for monitoring the clinical course of ovarian cancer patients.

Expression of vascular endothelial growth factor (VEGF) during folliculogenesis and corpus luteum formation in the human ovary

Vascular endothelial growth factor (VEGF) has been suggested to be involved in angiogenesis and microvascular hyperpermeability. We examined immunohistochemically the expression of VEGF in the granulosa and theca cells, along with that of proliferating cell nuclear antigen (PCNA), in the vascular endothelium during the course of follicular development and corpora lutea formation in human ovaries. The immunolocalization of VEGF in these cells was compared with that of another putative angiogenic factor, basic fibroblast growth factor (bFGF). The granulosa cells in the primordial and primary follicles were VEGF negative, but at the preantral stage, the granulosa cells showed weakly positive immunostaining for VEGF. However, the VEGF immunostaining in the granulosa cells was weak throughout the folliculogenesis. In contrast, the theca interna cells of developing follicles showed strong staining for VEGF, which was well correlated with the PCNA positivity in the vascular endothelial cells in the thecal layer. In the atretic follicles, the granulosa and theca cells were VEGF negative. In the corpora lutea, VEGF was strongly expressed in both granulosa and theca lutein cells in the early luteal phase when the PCNA positivity in the endothelium increased, but the VEGF staining in these cells became weak in the mid- and late luteal phases. Accordingly, the PCNA positivity in the vascular endothelium was well correlated with the expression of VEGF in the theca cells during follicular development and atresia, and that in the granulosa and theca lutein cells in corpora lutea formation and regression. In addition, the immunolocalization of VEGF was different from that of bFGF.

In vivo anti-tumor effect through the controlled release of cisplatin from biodegradable gelatin hydrogel.

This paper is an investigation to achieve the in vivo controlled release of cisplatin (CDDP) from a biodegradable hydrogel. Hydrogels with different water contents were prepared through the chemical crosslinking of gelatin by various concentrations of glutaraldehyde. The gelatin hydrogel incorporating CDDP (CDDP-hydrogel) was prepared by allowing CDDP aqueous solution to sorb into the freeze-dried hydrogel. Irrespective of the hydrogel water content, approximately 10-30% of incorporated CDDP was released from the hydrogel in phosphate-buffered saline solution (PBS) at 37 degrees C within the initial 6 h, while little release was observed thereafter. The amount of CDDP released initially decreased with an increase in the time period of CDDP sorption. When intratumorally applied into Meth-AR-1 tumor-bearing mice, CDDP-hydrogel suppressed in vivo tumor growth to a significantly higher extent than free CDDP at the same dose. The survival rate was significantly higher by the application of CDDP-hydrogel of 40 microg CDDP. The CDDP concentration in the tumor tissue was maintained at a higher level for a longer time period than that of free CDDP. However, no problematic change in the mouse body and blood biochemical parameters was observed on the application of the CDDP-hydrogel. The time course of in vivo CDDP retention was in a good accordance with that of hydrogel remaining. Larger CDDP release was observed from the front surface of hydrogel onto which free CDDP was sorbed, than the back surface of hydrogel. These findings demonstrate that the controlled release of CDDP was based on biodegradation of the hydrogel carrier, but not simple diffusion of CDDP. It is possible that the CDDP molecules immobilized in the gelatin hydrogel were released from the hydrogel only when the hydrogel was degraded to generate some water-soluble gelatin fragments.

Messenger ribonucleic acid expression of LH/hCG receptor gene in human ovarian carcinomas

The mRNA expression of luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptors was analysed by the RT-nested PCR method in five normal ovarian tissues, 62 ovarian tumours (5 benign, 7 borderline and 43 malignant epithelial tumours, 3 sex cord-stromal tumours and 4 germ cell tumours) and in 2 ovarian cancer cell lines. In normal ovaries, two cDNA fragments of different sizes were detected using primers designed to amplify a region including exon 9. Sequencing revealed that the larger fragment was derived from a full-length receptor, while the smaller fragment was a splice variant lacking exon 9. In ovarian tumours, the larger fragment of LH/hCG receptors was detected in 40% of the epithelial ovarian carcinomas, none of the germ cell tumours, all of the sex cord-stromal tumours and one of the 2 ovarian cancer cell lines. Immunohistochemistry confirmed the localisation of LH/hCG receptor protein in the tumour cells which correlated with mRNA expression. Patients with full-length LH/hCG receptors in carcinomas showed a better prognosis compared with those without the receptors.

Expression of heat shock proteins HSP70 and HSP90 in endometrial carcinomas: Correlation with clinicopathology, sex steroid receptor status, and p53 protein expression

It has been suggested that heat shock proteins HSP70 and HSP90 are involved in the functional modulation of sex steroid receptors and are expressed in normal endometrium. However, little is known about the expression of HSP70 and HSP90 in endometrial carcinomas.

Immunohistochemical localization of basic fibroblast growth factor (bFGF) during folliculogenesis in the human ovary

Basic fibroblast growth factor (bFGF) has been suggested to be one of the intraovarian regulators of ovarian folliculogenesis, but its localization in the human ovary remains to be determined. We examined the immunohistochemical reactivity for bFGF in the course of follicular development and corpora lutea formation in the human ovary. Pregranulosa cells in the primordial follicle were negative, but at the preantral stage both granulosa and theca cells showed weakly positive immunostaining for bFGF. In the antral follicles, both granulosa and theca interna cells showed stronger staining for bFGF with the increase in follicular diameter. In atretic follicles at various stages, granulosa cells were negative or weakly positive for bFGF, whereas luteinized theca cells showed strong immunoreactivity. In the corpora lutea during the early luteal phase, granulosa lutein cells were strongly positive for bFGF but in the late luteal phase became immunonegative. On the other hand, bFGF staining in theca lutein cells was strong throughout the course of corpora lutea formation and regression. These findings suggest that bFGF is localized not only in granulosa cells but also in theca cells, and in the latter, bFGF immunoreactivity is associated with luteinization.

Human chorionic gonadotropin (hCG) inhibits cisplatin‐induced apoptosis in ovarian cancer cells: Possible role of up‐regulation of insulin‐like growth factor‐1 by hCG

Gonadotropins have been suggested to play a role in the development or progression of ovarian cancer, and we have previously reported the expression of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor in 40% of epithelial ovarian carcinomas. To examine the biological effect of LH/hCG on ovarian cancer cells, apoptosis induced by cisplatin with or without hCG treatment was investigated in 2 ovarian cancer cell lines, OVCAR‐3 and SK‐OV‐3. Stimulation of cell proliferation by hCG was also studied. In addition, to analyze further the mechanism of hCG signaling involved in apoptosis‐inhibition, we examined the expression of LH/hCG receptors and the regulation by hCG for apoptosis‐inhibitory pathways, such as the bcl‐2/bax system and the insulin‐like growth factor‐1 (IGF‐1)/IGF‐1 receptor (IGFR) system. hCG did not increase cell proliferation in either cell line. However, hCG treatment suppressed cisplatin‐induced apoptosis by 58% in the OVCAR‐3 cells, as shown by immunofluorescent staining and quantitation of DNA fragmentation. LH/hCG receptor mRNA was expressed only in OVCAR‐3, and no apoptosis‐inhibitory effect of hCG was observed in the SK‐OV‐3 cells that did not express the receptor. In the OVCAR‐3 cells, hCG significantly increased mRNA expression of IGF‐1, but did not change mRNA levels of bcl‐2/bax. Our findings suggest that LH/hCG influences the chemosensitivity of ovarian cancer cells through an apoptosis‐inhibitory signal possibly via up‐regulation of IGF‐1 expression. Int. J. Cancer 76:571–578, 1998.© 1998 Wiley‐Liss, Inc.

Immunohistochemical analysis of p53 protein over-expression in endometrial carcinomas: inverse correlation with sex steroid receptor status.

Mutations of the tumour suppressor p53 gene have been reported in a variety of human malignant tumours, and are frequently associated with over-expression of p53 protein. To examine the significance of p53 gene alteration in endometrial carcinomas, we studied the immunohistochemical reactivity with a monoclonal antibody against p53 (PAb 1801) in 30 endometrial carcinomas as well as in 64 normal endometria. The presence or absence of correlation of p53 over-expression with the clinicopathological features and with the immunohistochemical expression of sex steroid receptors (oestrogen receptors; ER, progesterone receptors; PR) was also analysed. Expression of p53 was found in none of 64 normal endometria, but was identified in 5 of the 30 (16.7%) endometrial carcinomas. All 5 of the p53-positive tumours developed in women more than 3 years post-menopause, whereas the carcinomas in 5 pre-menopausal women and 3 women less than 3 years post-menopause were p53-negative. None of the 5 p53-positive carcinomas was associated with adjacent endometrial hyperplasia. Two of the 5 p53-positive tumours showed non-endometrioid histology: serous papillary and clear cell carcinomas. In contrast, 6 carcinomas accompanied by adjacent hyperplasia were p53-negative. In addition, ER and/or PR expression was found in none of the 5 p53-positive tumours, but was present in 21 of the 25 p53-negative tumours (p<0.01). These clinicopathological features of p53-positive carcinomas and the inverse correlation of p53 immunoreactivity with sex steroid receptor status suggest that p53 over-expression is frequent in a specific category of endometrial carcinoma, presumably oestrogen-unrelated tumours.

Extraovarian sex cord-stromal tumor : Case report and review of the literature

A 45-year-old woman was found at laparotomy to have a multinodular solid tumor within the broad ligament, which was removed by total hysterectomy and bilateral salpingo-oophorectomy. Both ovaries were unremarkable. Three years after the operation, retroperitoneal tumor was detected, which was associated with clinical evidence of estrogen production. Histological examination of the primary and retroperitoneal tumors showed histological features that resembled those of granulosa cell tumors. Previously reported sex cord-stromal tumors arising in extraovarian sites were reviewed with respect to the histogenesis of these tumors.

C-erbB-2 or mutant Ha-ras induced malignant transformation of immortalized human ovarian surface epithelial cells in vitro

Ovarian cancer is believed to develop from the ovarian surface epithelium through the accumulation of aberrations of oncogenes and/or tumor suppressor genes. However, it is unclear how the gene abnormalities are involved in ovarian carcinogenesis. To elucidate the process, we transfected genes reported to show their abnormalities in human ovarian cancers into human ovarian surface epithelial cells. Immortalization of the cells was achieved by the transfection of SV40 large T antigen (LT) and human telomerase reverse transcriptase (hTERT); however, the resultant cells showed no tumorigenesis. Additional transfection of either c-erbB-2 or mutant Ha-ras into the immortalized cells showed the anchorage-independent growth and tumorigenesis in mice with the incidence of 50% and 40%, respectively. Histologically, all the tumours were undifferentiated. In association with the tumorigenesis, the cells expressing c-erbB-2 or mutant Ha-ras demonstrated increased vascular endothelial growth factor secretion under hypoxia and enhanced resistance to apoptosis compared with the immortalized cells. Collectively, the introduction of either c-erbB-2 or mutant Ha-ras in the cells, which were efficiently immortalized by the transfection of LT and hTERT, showed tumorigenicity, suggesting that c-erbB-2 or mutant Ha-ras genes might be involved in ovarian carcinogenesis.