Michiyo Sugiyama

Direct protection by a gonadotropin-releasing hormone analog from doxorubicin-induced granulosa cell damage.

Background/Aims: Recent clinical applications suggest a beneficial effect of gonadotropin-releasing hormone analog (GnRHa) as a gonadal protector from chemotherapy-induced premature ovarian failure. This study aimed to determine cellular mechanisms involved in the protective action of GnRHa against granulosa cell damage caused by doxorubicin. Methods: Granulosa cells were obtained by ultrasound-guided follicular aspiration from patients undergoing in vitro fertilization, and screened for GnRH receptor expression prior to analyses. The cellular function was assessed by measuring the conversion of exogenously supplied androstenedione to estradiol-β (E2) in response to follicle-stimulating hormone (FSH) (1 µM). Results: Exposing to doxorubicin for 12 h before FSH stimulation caused a concentration-dependent inhibition of the E2 secretion to a minimum level of 20% of control. When the cells were incubated with a GnRHa for 12 h before and during exposure to doxorubicin, granulosa cells produced an equal level of E2 to that of control cells. The protective action of GnRHa was dose-dependent; a half-maximal effect occurred at 10 nM. Preincubation with GnRHa alone had no effect on FSH-induced E2 production. Conclusion: These findings demonstrate that a GnRHa may retard doxorubicin-induced granulosa cell damage, suggesting an additional GnRH activity to protect the gonads during chemotherapy through GnRH receptor-mediated mechanism(s).

In vitro stimulation of granulosa cells by a combination of different active ingredients of unkei-to.

Unkei-to is widely used in traditional Japanese herbal medicine for its ovulation-inducing effect. In the present study, we investigated the in vivo effects of unkei-to and its compounds on the steroidogenesis and cytokine secretion in human granulosa cells. Unkei-to stimulated the secretions of 17beta-estradiol and progesterone from highly luteinized granulosa cells obtained from in vitro fertilization patients; the stimulated effect on estradiol secretion occurred with 0.3 microg/ml, while a significant effect on progesterone secretion was obtained at 10 microg/ml. The unkei-to stimulation of estradiol secretion could be accounted for by the effects of its ingredients, Shakuyaku (paeoniae radix, Paeonia lactiflora Pallas) and Keihi (cinnamomi cortex, Cinnamomum cassia Blume); while dose response curves for unkei-to and Keihi to induce progesterone production were superimposable. Exposure of the cells to unkei-to caused dose-dependent increases in the concentrations of interleukin (IL)-1beta, IL-6 and IL-8 in the culture medium. Similar results were obtained when cells were incubated with the ingredient Ninjin (ginseng radix, Panax ginseng C.A. Meyer), but not Shakuyaku and Keihi. These results indicate that unkei-to has direct stimulatory effects on human granulosa cells to stimulate the steroidogenesis and secretion of cytokines (IL-1, IL-6 and IL-8). The various beneficial actions of unkei-to on the ovary may result from a combination of different ingredient herbs with different stimulatory effects on both steroidogenesis and the ovulatory process within the ovary, as well as stimulatory effect on the hypothalamus-pituitary axis.

Gonadotrophin-releasing hormones agonist therapy increases peritoneal fibrinolytic activity and prevents adhesion formation after myomectomy

The aim of this study was to evaluate uterine adhesions after myomectomy and peritoneal fibrinolytic capacity in women treated with gonadotrophin-releasing hormone agonist (GnRHa) before surgery. A prospective observational study comprised 15 infertile women who underwent myomectomy. Before surgery, 10 were treated with buserelin acetate (900 μg/day) for 10 – 12 weeks followed by additional postoperative treatment with GnRHa for 4 weeks (GnRHa group) and five received no treatment (control group). Peritoneal fluid specimens were taken at the beginning of myomectomy and the adhesions were estimated by second-look surgery (caesarean section or laparoscopy). Levels of plasminogen activator (PA) and PA inhibitor (PAI) were determined by enzyme-immunosorbent assays. Pre- and postoperative GnRHa therapy significantly reduced adhesion formation compared with control groups (adhesion scores; 0.2 ± 0.4 vs. 2 ± 1, P < 0.0001). GnRHa group showed a significant decrease in PAI level (P < 0.0001) but no significant change in PA level, suggesting increased fibrinolytic capacity in peritoneal fluid from GnRHa-treated patients. These data suggest that GnRHa therapy is successful in preventing adhesion formation after myomectomy. GnRHa-induced shift to more fibrinolytic activity, mainly because of a decreased level of PAI, may play a critical role in the mechanism of the GnRHas action on postoperative adhesion development.

Treatment of perimenopausal women with uterine myoma: successful use of a depot GnRH agonist leading to a natural menopause

Gonadotrophin-releasing hormone agonists (GnRHa) reduce the size of uterine fibroids and relieve patients of myoma-related symptoms. However, rapid regrowth frequently occurs after the therapy is stopped. We attempted to determine whether GnRHa therapy could lead perimenopausal women carrying symptomatic myomas to the natural onset of the menopause. A retrospective analysis of 145 patients who received GnRHa for 24 weeks demonstrated that after cessation of therapy no menstruation occurred over 25 weeks in women aged over 45 years, with elevated levels of follicle-stimulating hormone (FSH) and luteinising hormone (LH) > 25 mIU/ml. To extend this study, we studied prospectively 21 women, aged 45 years and older who had regular menstruation with symptoms attributed to myomas and elevated days 3 - 5 FSH and days 3 - 5 LH levels ( > 25 mIU/ml). After discontinuation of GnRHa (leuprorelin acetate, 1.88 mg) therapy for 6 months, menstruation occurred in only two of 21 individuals but the remaining 19 cases had no menstrual bleeding. It is suggested from this study that the rise in early follicular phase serum gonadotrophins, in particular FSH > 25 mIU/ml, may precede the natural menopause following (or during) GnRHa therapy in older women. Measuring days 3 - 5 serum FSH concentrations may make it easier to decide on the optimal duration of therapy for symptomatic uterine fibroids in perimenopausal women aged > 45 years.

Gi protein-mediated translocation of serine/threonine phosphatase to the plasma membrane and apoptosis of ovarian cancer cell in response to gonadotropin-releasing hormone antagonist cetrorelix

Summary Serine/threonine protein phosphatase 2A (PP2A), a crucial enzyme in apoptosis control, has been demonstrated within the plasma membrane as well as in the soluble fraction. This study aimed to examine hormonal translocation of PP2A to the plasma membrane in gonadotropin-releasing hormone (GnRH)-responsive ovarian cancer cells. Apoptosis of ovarian cancer cell lines Caov-3 and SK-Ov-3 was quantified by nuclear morphology after staining with Hoechst 33342 dye. PP2A protein and activity in plasma membrane were assessed by immunohistochemical staining with PP2A-specific antibodies and by the measurement of the dephosphorylation of phosphopeptide highly selective for the PP2A, respectively. Incubation for 48 h with a GnRH antagonist cetrorelix caused parallel increases in the percentage of cells undergoing apoptosis and the membrane-associated PP2A activity; half-maximal effects occurred with 5 nmol/l cetrorelix. PP2A protein was also localised to the plasma membrane when the cell lines were exposed to cetrorelix. Pretreatment of the cells with pertussis toxin, but not cholera toxin, completely inhibited cetrorelix-stimulated apoptotic cell death and PP2A redistribution. These findings demonstrate that translocation of PP2A to plasma membrane is closely coupled to the onset of apoptosis in ovarian cancer cells exposed to GnRH antagonist. These GnRH-induced cellular events may be mediated through pertussis toxin-sensitive Gi protein-linked GnRH receptor.

Membrane-associated serine/threonine protein phosphatase in endometrial cancer

OBJECTIVE Reversible serine/threonine protein phosphorylation catalyzed by kinases and phosphatases plays a crucial role in cellular growth and differentiation. We attempted to determine the subcellular location of serine/threonine phosphatase (protein phosphatase type 2A [PP2A]) in endometrial cancer. STUDY DESIGN Endometrial cancers surgically removed were examined. PP2A activity was assessed by measuring the dephosphorylation of phosphopeptide highly selective for the PP2A in cytosol and membranes fractionated on a continuous sucrose density gradient. Its protein level was detected by immunoblotting with a specific antibody. RESULTS There were three peaks of PP2A enzyme activity and immunoreactivity corresponding by marker enzyme analysis to the cytoplasm, plasma membrane, and endoplasmic reticulum fractions. An enzyme kinetic analysis showed the different activity in cytosol and plasma membrane; K(m) values of 98+/-12 micromol/L for cytosol and 32+/-6.2 micromol/L for plasma membrane (P<.01), respectively. The membrane phosphatase was sensitive to inhibition by okadaic acid and sodium fluoride, characteristics suggestive of PP2A activity. CONCLUSION PP2A activity in the plasma membrane of endometrial cancers might be distinct from that present in the cytosol. The plasma membrane PP2A may be responsible for a rapid and initial decrease in intracellular level of phosphoserine and phosphothreonine, interfering with serine/threonine protein phosphorylation-mediated growth of endometrial cancer.

Efficacy of Raloxifene Hydrochloride for the Prevention of Health Care Problems in Patients Who Undergo Surgery for Endometrial Cancer A Multicenter Randomized Clinical Trial

Objective Removal of the ovaries is common during surgery for endometrial cancer. However, because loss of the ovaries can cause several health problems in patients, strategies for the prevention of such problems need to be established. Hence, we decided to conduct a multicenter randomized clinical trial to assess the effect of raloxifene on bone mineral density (BMD), bone metabolism, and the lipid profile of patients who had undergone surgery for patients with endometrial cancer. Materials and Methods Patients with endometrial cancer were enrolled after treatment. The participants were randomized into 2 groups: group 1 included 39 women who received alfacalcidol (1 μg/d) alone and group 2 included 37 women who received alfacalcidol and the test drug, raloxifene hydrochloride, at a dose of 60 mg/d. The BMD of lumbar spine and femoral neck, serum bone markers, as well as lipid profile parameters were evaluated at enrollment as well as 6, 12, and 24 months after the enrollment. The primary efficacy end point was the percentage change from baseline to 24 months in lumbar spine (L2-L4) and femoral neck BMD. Results Sixty-four women completed the 24-month study. At 24 months, the lumbar and femoral neck BMDs were significantly increased in group 2 compared with group 1 (3.5% vs −0.8% and 2.3% vs −2.8%, respectively). In group 2, low-density lipoprotein-cholesterol levels were significantly reduced by 13.6% and serum N-terminal telopeptide of type I collagen as well as bone-specific alkaline phosphatase values were significantly reduced by 16.7% and 25.7%, respectively. The patients who received adjuvant therapy for endometrial cancer showed a significantly higher response to raloxifene (5.8% vs 1.9%). Recurrence was detected in 2 (2.6%) patients in group 1. No severe adverse events were noted in any patient during the study period. Conclusions Raloxifene exerts positive effects on BMD, bone metabolism, and lipid profile parameters and could provide an improved therapeutic option for patients with endometrial cancer.

The occurrence of CTX-M-25-producing Enterobacteriaceae in day-old broiler chicks in Japan

Day-old chicks from 3 hatcheries were placed on bedding paper and brought to a commercial broiler farm between January and July 2016. Sixty-six samples of the paper, which were stained with meconium droppings of the chicks, were collected and examined for isolation of cephalosporin-resistant Enterobacteriaceae. Cefotaxime (CTX)-resistant Klebsiella pneumoniae (1 isolate) and Enterobacter cloacae (4 isolates) were isolated from 5 (7.58%) of the 66 samples. Conjugation experiments revealed that the blaCTX-M-25 gene conferring CTX resistance was transferred from the K. pneumoniae isolate and 2 of the 4 E. cloacae isolates to Escherichia coli DH5α via IncA/C plasmids carrying the gene. Our results suggested that the blaCTX-M-25 gene originating from chicks may be spread among commercial broiler farms.