Masatoshi Kariya

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.

Suppression of natural killer cell activity by sera from patients with endometriosis

OBJECTIVE We determined the effect of sera from patients who have endometriosis on natural killer cell activity. STUDY DESIGN The natural killer cell activity of lymphocytes from healthy volunteers was examined after incubation with sera from patients who had endometriosis or from controls, with K562 cells used as targets. RESULTS Lymphocytes treated with sera from patients who had endometriosis expressed significantly lower levels of cytotoxicity compared with lymphocytes treated with control sera. This suppression of cytotoxicity was dose dependent, and the degree of suppression was proportional to the incubation time of the effector cells with the sera. Decreased cytotoxicity after serum treatment was also observed with sera from patients who had been treated with danazol. CONCLUSIONS These findings show that humoral factors that can inhibit natural killer cell activity in vitro are present in the peripheral blood of patients who have endometriosis; moreover, they suggest that the suppressed natural killer cell activity may allow the development of endometrial cells at ectopic sites.

Human ovarian surface epithelial (OSE) cells express LH/hCG receptors, and hCG inhibits apoptosis of OSE cells via up-regulation of insulin-like growth factor-1

Gonadotropins including luteinizing hormone (LH) or human chorionic gonadotropin (hCG) have been implicated as playing an important role in the development of epithelial ovarian carcinomas, most of which are believed to originate from the ovarian surface epithelium (OSE). To address this issue, we examined the expression of LH/hCG receptors and the influence of hCG on cell proliferation and on the apoptosis of cultured human OSE cells. RT‐PCR and binding assay revealed that OSE cells express the LH/hCG receptor mRNA and have specific binding activity for hCG. Treatment with hCG stimulated the proliferation of OSE cells in a dose‐dependent manner. In addition, hCG treatment inhibited the apoptosis of OSE cells induced by serum deprivation. Among the apoptosis‐related genes, hCG treatment did not change the mRNA levels of bcl‐2, bax and IGF‐1 receptor but significantly increased that of IGF‐1. Treatment with IGF‐1 alone also suppressed the apoptosis of OSE cells, and treatment by hCG along with neutralization antibody against IGF‐1 receptor reversed the anti‐apoptotic effect of hCG. Accordingly, LH/hCG signaling followed by up‐regulation of IGF‐1 is involved in the inhibition of apoptosis of OSE cells, the possible histogenetic origin of epithelial ovarian carcinomas.

Oncogenic Property of Acrogranin in Human Uterine Leiomyosarcoma: Direct Evidence of Genetic Contribution in In vivo Tumorigenesis

To identify potential oncogenes that contribute to the development of uterine leiomyosarcoma, we conducted a cDNA microarray analysis between normal uterine smooth muscle and uterine leiomyosarcoma. We found that acrogranin (also named PCDGF or progranulin) is overexpressed in uterine leiomyosarcoma. With immunohistochemical staining of 12 leiomyosarcoma cases, we verified acrogranin expression in tumor cells. Furthermore, the intensity of acrogranin expression correlated with high histologic grade and poor prognosis. To directly analyze the oncogenic properties of acrogranin, we established an immortalized uterine smooth muscle cell line by transfection of human telomerase reverse transcriptase into primary culture. This cell line retained the original characteristics of uterine smooth muscle cells, including spindle-shaped extension as well as expression of vimentin, estrogen receptor α, progesterone receptor, and α smooth muscle actin. Transfection of acrogranin into the immortalized uterine smooth muscle cells resulted in colony formation in soft agar, but the diameter of the colonies did not exceed 100 μm. Transfection of both acrogranin and SV40 early region (SV40ER) into the immortalized uterine smooth muscle cells resulted in an increased number of colonies and increased colony size in soft agar versus transfection of SV40ER alone. We show that only immortalized uterine smooth muscle cells expressing both acrogranin and SV40ER are capable of tumor formation in nude mice. Thus, acrogranin is overexpressed in uterine leiomyosarcoma cells, particularly in high-grade cases, and forced expression of acrogranin in immortalized uterine smooth muscle cells contributes to malignant transformation, which suggest that acrogranin plays an important role in the pathogenesis of uterine leiomyosarcoma.

Lymphocytes stimulate progesterone production by cultured human granulosa luteal cells

After follicular rupture, massive invasion of blood vessels with neovascularization of the developing corpus luteum takes place, providing many chances for direct contact of luteal cells with resident and migrating immune cells. We studied the effects of peripheral blood lymphocytes on progesterone production by human granulosa luteal cells isolated from women undergoing in vitro fertilization. During 6 days of culture, progesterone production by granulosa luteal cells was significantly increased when they were cultured together with autologous or allogenic peripheral blood lymphocytes. This stimulatory effect was also observed on the addition of medium conditioned with peripheral blood lymphocytes and was synergistic with gonadotropin stimulation. The activity was present in the fraction retained by ultrafiltration with a 30,000 molecular weight cutoff filter and was preserved after heating at 56 degrees C for 30 minutes but disappeared after heating at 70 degrees C for 15 minutes. These findings suggest that lymphocytes infiltrating the corpus luteum during early luteinization can stimulate the function of human granulosa luteal cells through the action of some protein-like humoral factor(s) of higher molecular weight than that of previously identified lymphokines and indicate a possible paracrinologic regulatory role for lymphocytes in ovarian function.

Combination effect of adenovirus-mediated pro-apoptotic bax gene transfer with cisplatin or paclitaxel treatment in ovarian cancer cell lines.

To develop a novel therapeutic strategy for ovarian cancer, we constructed a recombinant adenovirus which highly expresses pro-apoptotic Bax protein and examined its therapeutic effect on a series of ovarian cancer cell lines: A2780, A2780/cDDP, OVCAR-3 and SK-OV-3. A recombinant adenovirus carrying the Bax-alpha gene (AxCALNKYbax) induced high expression of the Bax-alpha protein in all the cell lines. The cytotoxic effect of Bax was observed in three ovarian cancer cell lines: the per cent reduction in the number of cells was 40.0% for cisplatin-sensitive A2780, 50.0% for cisplatin-resistant A2780/cDDP, and 64.8% for marginally cisplatin-resistant OVCAR-3. In contrast, it was only 12.3% for cisplatin-resistant SK-OV-3. Cisplatin-resistant A2780/cDDP had a p53 mutation and exhibited attenuated Bax induction after cisplatin treatment, which may explain why supplementation of Bax was effective in this chemoresistant ovarian cancer. Combination with cisplatin or paclitaxel enhanced the cytotoxic effect of Bax induction in all but one cell line including cisplatin-resistant A2780/cDDP. It appears that adenovirus-mediated Bax induction, with or without combination with conventional chemotherapy, useful strategy for the treatment of ovarian cancer.

Adenocarcinoma of the uterine cervix with choriocarcinomatous and hepatoid differentiation: report of a case.

A case of adenocarcinoma of the uterine cervix that showed choriocarcinomatous and hepatoid differentiation was encountered in a 65-year-old woman. She presented with genital bleeding and had multiple metastatic nodules in the lungs. At operation, a large, hemorrhagic, and necrotic tumor was found in the uterine cervix. The major portion of the tumor consisted of typical choriocarcinoma admixed with minor areas of hepatoid carcinoma and endocervical adenocarcinoma. Human chorionic gonadotropin and alpha-fetoprotein were detected in tumor cells in the choriocarcinomatous and hepatoid areas, respectively. The patient died of pulmonary metastasis 4 months after the operation. The coexistence of choriocarcinomatous and hepatoid carcinoma in an endocervical adenocarcinoma has not been reported previously. Both heterotopic components were probably derived from aberrant differentiation (or neometaplasia) of the somatic epithelial cells of the endocervical adenocarcinoma.

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.

Hormonal Regulation in the Production of Macrophage Colony-Stimulating Factor and Transforming Growth Factor-Beta by Human Endometrial Stromal Cells in Culture

The effects of gonadal steroids on the secretion and gene expression of macrophage colony-stimulating factor (M-CSF) and on the secretion of transforming growth factor (TGF)-beta 1 and TGF-beta 2 by human endometrial stromal cells (ESCs) were examined by an in vitro system of ESC differentiation (decidualization). M-CSF production by ESCs was dose-dependently enhanced by the addition of progesterone or testosterone, while estradiol treatment had no effect. TGF-beta 2 secretion by ESCs was inhibited by progesterone, estradiol and testosterone treatment, and on the contrary, slight enhancement by estradiol was observed in TGF-beta 1 secretion. These findings indicate that human ESCs produce cytokines of M-CSF and TGF-beta s, which are important for the growth and differentiation of the peri-implantation embryo as well as local immune cells under direct control of gonadal steroidal actions, and suggest a novel network between endocrine and immune systems in the human endometrium.

Expression of cold-inducible RNA-binding protein in the normal endometrium, endometrial hyperplasia, and endometrial carcinoma.

&NA; Cold‐inducible RNA‐binding protein (CIRP), an 18‐kD protein in the mouse and human, is induced by lowering the temperature of cultured cells. CIRP is possibly a cell cycle regulator because its overexpression results in prolongation of G1 phase in vitro. We investigated the immunohistochemical expression of CIRP in 39 endometrial carcinomas, 12 endometrial hyperplasias, and 27 normal endometria using polyclonal antibody against CIRP and confirmed by Western blot analysis. CIRP was localized in the nuclei of glandular, stromal, and endothelial cells. The intensity of CIRP expression in glandular cells during the menstrual cycle was inversely proportional to its proliferative (Ki‐67) activity, whereas it remained unchanged in stromal and vascular endothelial cells. The intensity of CIRP expression in hyperplastic glands was variable, whereas CIRP expression was absent or markedly reduced in most of the endometrial carcinomas. These results suggest that CIRP may participate in the cell cycle regulation of normal endometrium and the loss of its expression may be involved in endometrial carcinogenesis.