Yoshiro Toma

Regulation of estrogen activity in human endometrium: Effect of IL-1β on steroid sulfatase activity in human endometrial stromal cells

We investigated the effect of interleukin 1beta (IL-1beta) on steroid sulfatase (STS) activity and the expression of STS mRNA in human endometrial stromal cells. Endometrial tissue samples were obtained from patients undergoing hysterectomy to remove uterine fibroids. Stromal cells were isolated from the tissue preparation and cultured. IL-lbeta (1 approximately 100 ng/ml) was added into the culture medium and incubated for 24 h. The expression of STS mRNA was measured by competitive RT-PCR. The addition of IL-lbeta at 10 and 100 ng/ml suppressed STS mRNA expression to 55.2 +/- 12.8% and 25.1 +/- 10.9%, respectively, of the control sample to which no IL-lbeta had been added. STS activity was measured by radiolabelled steroid metabolite using thin layer chromatography, and this activity was also significantly suppressed in response to the administration of IL-lbeta in a dose-dependent manner. When IL-1 receptor antagonist (IL-1ra) was added together with IL-1beta to the culture medium, mRNA expression and STS activity were recovered. The present study is the first to demonstrate IL-1beta regulation of STS activity locally in human endometrium. IL-1beta suppressed mRNA and activity of STS in stromal cell culture. This initial demonstration of IL-1beta regulation of STS implies that IL-1beta may control the steroid microenvironment in human uterine endometrium by reducing biologic action of estrogen.

Localization and expression of steroid sulfatase in human fallopian tubes

Localization of steroid sulfatase, a membrane-bound microsomal enzyme, in human fallopian tubes was immunohistochemically investigated, and expression of RNA was confirmed by competitive RT-PCR. Human fallopian tubes were obtained from 10 patients in follicular and early luteal phases during gynecological laparotomy. An anti-human rabbit polyclonal antibody was prepared against sulfatase protein purified from human placenta. Total RNA was isolated from epithelium of fallopian tubes. A heterologous RNA competitor was designed, and competitive RT-PCR was carried out. Steroid sulfatase was localized to the cytoplasm of epithelial cells. With respect to the positive staining of cells, the number of positive secretory cells was higher than that of ciliated cells. A significantly higher number of positive cells was found in tissue obtained from the early luteal phase than that found in tissue from the follicular phase. An abundant expression of sulfatase mRNA in early luteal phase was also observed. This study demonstrates, for the first time, that steroid sulfatase is localized to human epithelial cells and that steroid sulfatase staining and mRNA expression changes with the menstrual cycle. These results suggest that sulfatase in the fallopian tube may be involved in controlling the local steroid environment, which appears to regulate aspects of the physiological reproductive function of the fallopian tube.

Lactoferrin and Interleukin-6 Interaction in Amniotic Infection

Lactoferrin (Lf) has been found in most biological fluids including amniotic fluid and cervical mucoids in pregnant women, and released from neutrophils in response to the inflammation. As Lf possesses antimicrobial properties, it is widely considered to be an important component of the host defence against microbial infections. It is known that premature labor is caused by amniotic infection with the increase of prostaglandin production. High concentration of the inflammatory cytokines: interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) in the amniotic fluid has been known. However, changes of Lf in amniotic fluid with infection has not been reported. In the present study, Lf concentrations in amniotic fluid were measured under the intra-uterine infections state and the biological significance of Lf was investigated. The effects of Lf on the IL-6 and IL-6mRNA production in cultured amnion cells were also investigated. The concentrations of Lf and IL-6 in amniotic fluid with CAM were 8.76 +/- 0.65 micrograms/ml and 6.92 +/- 4.88 ng/ml (n = 28) respectively and both were significantly higher (p < 0.01) than those without CAM [0.86 +/- 0.81 microgram/ml and 0.34 +/- 0.25 ng/ml (n = 31)]. Significant positive correlation (r = 0.91, p < 0.01) between Lf and IL-6 levels in amniotic fluid was found. IL-6 production induced by lipopolysaccharide (LPS) (100 ng/ml) in cultured amnion cells was significantly inhibited (p < 0.05) under the physiological concentration of Lf in amnion. Total RNA was extracted from the amniotic cells by guianizine solution. RT-PCR procedure and product analysis were performed from one microgram aliquote of total RNA. beta-actin was used as an international standard and c-DNA samples were followed by 30 cycles of PCR. RT-PCR product of IL-6 mRNA was detected by Southern hybridization. Expression of IL-6 mRNA was inhibited by the addition of Lf. From the results, the possibility that Lf might suppress amniotic IL-6 production under the condition of amniotic infection is suggested. It is also suggested that Lf might act as self defence mechanism from intra-uterine infection.

Inhibitory Effects of Catecholamines and Maternal Stress on Aromatase Activity in the Fetal Rat Brain

Objective: Aromatization in the the fetal brain is thought to be involved both in sex differentiation during early development and in adult sexual behavior. Although recently the relationship between aromatase and catecholamine has been disscussed, the effect of stress on aromatase in the fetal brain has not been clarified. Therefore, in the present study, localization of aromatase and the inhibitory effects of catecholamines and maternal stress on aromatase activity in the fetal rat brain were examined.

Effects of fadrozole and leuprorelin acetate on aromatase activity and cell proliferation in a human breast cancer cell line (SK-BR-3)

AbstractBackground. In recent years, aromatase inhibitors have been used to treat hormone-dependent breast cancer in postmenopausal women. Although gonadotropin-releasing hormone (GnRH) agonists inhibit the growth of breast cancers by estrogen deprivation, it is not known whether GnRH agonists have a direct effect on breast cancer cells. In the present study, we examined the direct effect of a GnRH agonist (leuprorelin acetate) on aromatase activity in a human breast cancer cell line, SK-BR-3. We also studied the synergistic effect of fadrozole (an aromatase inhibitor) and leuprorelin acetate on aromatase activity and cell proliferation in SK-BR-3 cells. Methods. Aromatase activity was determined by measuring [3H] water released upon the conversion of [1β-3H] androstenedione to estrone. Cell proliferation was estimated by determining the incorporation of 5-bromo-2′-deoxyuridine in cellular DNA (cell proliferation assay system). Results. Aromatase activity in SK-BR-3 was inhibited by fadrozole. In addition, SK-BR-3 aromatase activity was inhibited by leuprorelin acetate. Stimulation of cell proliferation by estradiol (10 nM) and testosterone (20 nM) was almost completely inhibited by the addition of an estrogen receptor antagonist, ICI 182780 (10 nM), and fadrozole (1 nM). When both these compounds were added, the most potent inhibition of aromatase activity (fadrozole, 0.1 nM; leuprorelin acetate, 1 nM) and cell proliferation (fadrozole, 10 nM; leuprorelin acetate, 100 nM) was observed. Conclusions. These results lead us to the conclusion that combination therapy with an aromatase inhibitor and a GnRH agonist may provide a new treatment for both pre- and postmenopausal patients with hormone-dependent breast cancer.