Enhanced miR-210 expression promotes the pathogenesis of endometriosis through activation of signal transducer and activator of transcription 3.

STUDY QUESTION: What are the roles of the microRNA miR-210-an miRNA that is up-regulated in endometriotic cyst stromal cells (ECSCs)-in the pathogenesis of endometriosis? SUMMARY ANSWER: Up-regulated miR-210 expression in ECSCs is involved in their proliferation, resistance to apoptosis and angiogenesis through signal transducer and activator of transcription (STAT) 3. WHAT IS KNOWN ALREADY: In the pathogenesis of endometriosis, a number of roles for microRNAs (miRNAs) are becoming apparent. STUDY DESIGN, SIZE, DURATION: ECSCs and normal endometrial stromal cells (NESCs) were isolated from ovarian endometriotic tissues (patients aged 24-40 years undergoing salpingo-oophorectomy or evisceration for the treatment of ovarian endometriotic cysts, n = 10) and the eutopic endometrial tissues without endometriosis (premenopausal patients aged 35-45 years undergoing hysterectomies for subserousal leiomyoma, n = 13), respectively. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used a global gene expression microarray technique to identify downstream targets of miR-210, and we assessed the functions of miR-210 in the pathogenesis of endometriosis by using the miR-210-transfected NESCs. MAIN RESULTS AND THE ROLE OF CHANCE: Gene expression microarray analysis revealed that one of the key target molecules of miR-210 is STAT3. In the NESCs, in comparison to the control, miR-210 transfection resulted in the induction of cell proliferation (P < 0.0005), the production of vascular endothelial cell growth factor (VEGF) (P < 0.0005) and the inhibition of apoptosis (P < 0.05) through STAT3 activation [increased levels of mRNA (P < 0.0005), and protein (P < 0.005)]. In the ECSCs, inhibitors of STAT3 inhibited the cell proliferation and VEGF production (P < 0.05), and induced the apoptosis of these cells (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: The roles of aberrant miR-210 expression were investigated only in the stromal component of ectopic and eutopic endometrium. Control endometrial tissues were obtained from premenopausal patients who had subserosal leiomyoma and NESC gene expression patterns may be altered in these women. Furthermore, the effects of STAT3 inhibitors were evaluated only in ECSCs and not in NESCs. WIDER IMPLICATIONS OF THE FINDINGS: The present findings indicate that miR-210 induces NESCs to differentiate into the endometriotic phenotype and we speculate that up-regulated miR-210 expression in ECSCs is involved in the creation of the endometriosis-specific cellular dysfunctions through epigenetic mechanisms. The data indicate that STAT3 inhibitors may be promising candidates for the treatment of endometriosis. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 13237327 to K.N., no. 25861500 to Y.K. and no. 23592407 to H.N.). There are no conflicts of interest to declare.

The role of serum adiponectin levels in women with polycystic ovarian syndrome.

PURPOSE OF INVESTIGATION: The aim of this study was to measure serum adiponectin concentrations in women with polycystic ovarian syndrome (PCOS) and to assess possible correlations between adiponectin and the hormonal or metabolic parameters of this syndrome. MATERIALS AND METHODS: Serum adiponectin levels were evaluated in 20 women with PCOS and 22 women without PCOS whose age and body mass index (BMI) matched the patients. The levels of fasting blood glucose, fasting insulin, gonadotropin, and sex steroid hormones were evaluated in both groups. The homeostasis model assessment (HOMA) score was also calculated. The serum adiponectin levels were assayed by enzyme-linked immunoabsorbent assay (ELISA). RESULTS: Serum adiponectin levels were significantly lower in obese women than in normal-weight women, and they were also significantly lower in PCOS patients with HOMA scores greater than 1.7 compared with those with HOMA scores lower than 1.7. When the subjects were divided in two groups based on serum adiponectin levels (> 40 microg/ml, < 40 microg/ml), 65% of patients with PCOS were included in the lower adiponectin group (p < 0.05). In addition, gonadotropin levels were increased, dependent on the adiponectin levels in women with PCOS. CONCLUSION: Adiponectin is regarded as a possible link between adiposity and insulin resistance (IR). From this data, the secretions of gonadotropin are implicated in the levels of adiponectin in women with PCOS. It is suggested that adiponectin may play an important role in the pathogenesis of PCOS.

Regulation of interleukin-1alpha and tumor necrosis factor-alpha-induced interleukin-8 production by amnion-derived (WISH) cells.

PURPOSE OF INVESTIGATION: It has been reported that interleukin (IL)-8 is produced in the amnion and that its production is enhanced by the initiation of labor. The purpose of this study was to clarify the mechanism of IL-8 production by amnion-derived (WISH) cells. METHODS: Cells were cultured and treated with various concentrations of interleukin (IL)-1alpha, IL-1 receptor antagonist (ra), tumor necrosis factor (TNF)- alpha, C2-, C6-ceramide, mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor (U0126) and pyridinyl imidazole (p38 MAP kinase inhibitor, SB203580). IL-8 in the culture medium was measured by ELISA. RESULTS: The production of IL-8 was significantly increased by IL-1alpha or TNF-alpha, and the increase of IL-8 stimulated by IL-1alpha was suppressed by IL-1 ra in a dose-dependent manner. The increase in IL-8 production by IL-1alpha or TNF-alpha was further enhanced by simultaneous addition of C2-ceramide. The increase of IL-8 stimulated by IL-1alpha or TNF-alpha was also suppressed by treatment with U0126 or SB203580. The results of this study demonstrate that the production of IL-8 induced by IL-1alpha and TNF-alpha is enhanced by C2-ceramide, and suppressed by MEK inhibitor or P38 MAP kinase inhibitor. CONCLUSION: The results suggest that ceramide-mediated accumulation and MAP kinase-mediated suppression of inflammatory events in the amnion may play an important role in the maintenance of pregnancy and initiation of labor.

Malignant lymphoma of the vagina successfully treated with rituximab, adryamicin, cyclophosphamide, vincristine sulfate, and prednisolone.

PURPOSE: Primary malignant lymphoma of the vagina is extremely rare. The most common histologic subtype is diffuse large B-cell lymphoma (DLBCL). We report a case of vaginal DLBCL successfully treated with chemotherapy consisting of rituximab, adryamicin, cyclophosphamide, vincristine sulfate, and prednisolone (R-CHOP), followed by pelvic irradiation. CASE: A 44-year-old Japanese woman was admitted complaining of atypical genital bleeding and puruloid vaginal discharge. Gynecological examination showed an ulceration of the vaginal wall and a hard mass the size of a goose egg beneath the left vaginal wall, which had infiltrated to the left pelvic wall. The pathological diagnosis based on a punch biopsy taken from the vaginal tumor was non-Hodgkin's lymphoma. Based on immunohistochemical study, the tumor was subclassified as activated B-cell type DLBCL. The patient was diagnosed with Ann Arbor Stage IEA DLBCL and Stage III vaginal cancer, according to the International Federation of Gynecologists and Obstetricians (FIGO) classification system. She was successfully treated by six courses of R-CHOP, followed by radiation therapy. The patient is well without evidence of disease 13 months following the initial treatment. CONCLUSION: Little attention has been paid to the use of rituximab in addition to conventional chemotherapy and the importance of clinical and morphological subgrouping of DLBCL arising in the vagina. The present case indicates that the effects of rituximab on the prognosis of vaginal DLBCL must be evaluated, and that clinical use of immunophenotypic subgrouping should be considered for vaginal DLBCL.

Villoglandular papillary adenocarcinoma of the uterine cervix diagnosed during pregnancy.

Villoglandular papillary adenocarcinoma (VPA) is a very rare subtype of adenocarcinoma of the uterine cervix but a well recognized variant of cervical adenocarcinoma with a favorable prognosis generally occurring in women of child-bearing age. Only five cases of VPA and pregnancy have been reported. Herein, we report a case of VPA diagnosed during pregnancy and this patient delivered a healthy baby. A successful pregnancy can be completed in patients with VPA without lymph-vascular invasion, when treated conservatively. This management is particularly desirable in young women to preserve reproductive capability.

Primary mucinous adenocarcinoma of the vagina.

PURPOSE: Primary mucinous adenocarcinoma of the vagina is a rare disease which is characterized by aggressiveness and poor prognosis because of its rapid growth and recurrence, its frequent distant metastases, and its relative resistance to conventional treatment modalities including surgery, radiotherapy, and chemotherapy. We report a case of advanced stage primary mucinous adenocarcinoma of the vagina that showed a highly aggressive course and resistance to combination chemotherapy with paclitaxel and carboplatin. CASE: A 46-year-old multigravid Japanese woman was admitted to our hospital to be treated for Stage IVb primary mucinous adenocarcinoma of the vagina. She had no history of in utero exposure to diethylstilbestrol. She was treated by two courses of neoadjuvant chemotherapy with tri-weekly paclitaxel and carboplatin, which were not effective. Subsequently, total pelvic exenteration with pelvic lymphadenectomy was performed. However, the disease progressed rapidly and the patient died five months after the initial treatment. CONCLUSION: Because of its rarity, little is known about the behavior of primary mucinous adenocarcinoma of the vagina. Additional data about patients with this rare tumor should be collected and analyzed in an attempt to elucidate its prognostic factors, characteristics, optimal treatment, and outcome.

Involvement of resistance to apoptosis in the pathogenesis of endometriosis.

Endometriosis, a disease affecting 3-10% of women of reproductive age, is characterized by the ectopic growth of endometrial tissue. Increasingly, endometriosis is also becoming recognized as a condition in which ectopic endometrial cells exhibit abnormal proliferative and apoptotic regulation in response to appropriate stimuli. Apoptosis plays a critical role in maintaining tissue homeostasis and represents a normal function to eliminate excess or dysfunctional cells. Accumulated evidence suggests that, in healthy women, endometrial cells expelled during menstruation do not survive in ectopic locations because of programmed cell death, while decreased apoptosis may lead to the ectopic survival and implantation of these cells, resulting in the development of endometriosis. Both the inability of endometrial cells to transmit a 'death' signal and the ability of endometrial cells to avoid cell death have been associated with increased expression of anti-apoptotic factors and decreased expression of pre-apoptotic factors. This paper is a review of the recent literature focused on the differential expression of apoptosis-associated molecules in the normal endometria of women without endometriosis, and in the eutopic and ectopic endometria of women with endometriosis. The role of apoptosis in the pathogenesis of endometriosis and the basic and clinical research on the current medical treatment for endometriosis from the view of apoptosis will be discussed.

Primary malignant melanoma of the vagina.

Malignant melanoma originating in the vagina is considered extremely rare and has a very poor prognosis. We report a case of a 70-year-old woman with primary malignant melanoma of the vagina, and discuss the importance of prognostic factors and the efficacy of adjuvant chemotherapy.

Bowel obstruction due to endometriosis in the rectovaginal septum.

It is very rare that endometriotic lesions in the rectovaginal septum cause ileus. We report a case of bowel obstruction due to endometriotic lesions in the rectovaginal septum in a 22-year-old woman whose barium enema presented with apple-core-like findings. Diagnostic and treatment modalities were discussed. Preoperative and postoperative gonadotropin-releasing hormone analog and aromatase inhibitor therapy promote relief of clinical symptoms, a reduction of tumor volume and a better approach to radical surgery.

Human oviductal epithelial cells express Toll-like receptor 3 and respond to double-stranded RNA: Fallopian tube-specific mucosal immunity against viral infection.

BACKGROUND: The aim of this study was to evaluate the site-specific immunoregulatory mechanisms against viral infection in human Fallopian tubes. METHODS: We therefore investigated the effects of double-stranded RNA (dsRNA) on the production of interleukin (IL)-6, IL-8 and granulocyte chemotactic protein-2 (GCP-2) by cultured oviductal epithelial cells (OECs) using enzyme-linked immunosorbent assays. Phosphorylation of inhibitor kappaB-alpha (IkappaB-alpha) protein after dsRNA stimulation and the expression of Toll-like receptor (TLR) 3 in these cells were also evaluated by western blot analysis. RESULTS: Polyriboinosinic:polyribocytidylic acid (poly I:C), a synthetic dsRNA that antagonizes TLR3, stimulated the secretion of IL-6, IL-8 and GCP-2 by OECs. Poly I:C-induced production of these cytokines by OECs was inhibited by the pretreatment of these cells with anti-TLR3 antibody. The phosphorylation of IkappaB-alpha protein was detected in OECs after stimulation by poly I:C. The expression of TLR3 was also detected in OECs. CONCLUSION: These results suggest that the epithelial cells of the human Fallopian tube have evolved a unique, site-specific mechanism for recognizing viral infection. TLR3-mediated production of proinflammatory cytokines and chemokines in OECs in response to viral dsRNA may be important for antiviral immunity in the human female reproductive tract.

Beta-hydroxyisovalerylshikonin induces apoptosis and G0/G1 cell-cycle arrest of endometriotic stromal cells: a preliminary in vitro study.

BACKGROUND: Most of the current medical treatments for endometriosis aim to down-regulate the estrogen activity. However, a high recurrence rate after medical treatments has been the most significant problem. Beta-hydroxyisovalerylshikonin (beta-HIVS) is an ATP non-competitive inhibitor of protein-tyrosine kinases and is considered an apoptosis-inducing agent. The aim of this study is to evaluate the effects of beta-HIVS on the proliferation, cell cycle and apoptosis of endometriotic stromal cells. METHODS: We investigated the effects of beta-HIVS on cultured ovarian endometriotic cyst stromal cells (ECSC) by a modified methylthiazoletetrazolium (MTT) assay, a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay and internucleosomal DNA fragmentation assays. The effect of beta-HIVS on the cell cycle of ECSC was determined by flow cytometry. The expression of apoptosis-related molecules was examined in ECSC using western blot analysis. RESULTS: Beta-HIVS significantly inhibited the proliferation and DNA synthesis of ECSC and induced apoptosis and G0/G1 phase cell-cycle arrest of these cells. Down-regulation of the B-cell lymphoma/leukaemia-2 (Bcl-2) expression with the activation of caspase-3, caspase-8 and caspase-9 was observed in ECSC after beta-HIVS treatment. CONCLUSIONS: These results suggest that beta-HIVS induces apoptosis of ECSC by suppressing anti-apoptotic proteins. Although our present findings are preliminary, beta-HIVS could potentially be a therapeutic agent for the treatment of endometriosis.

Phase I/II study of docetaxel and S-1 in patients with advanced gastric cancer.

The aims of this phase I/II study of docetaxel and S-1 were to determine the dose-limiting toxicity (DLT), maximum-tolerated dose (MTD), and recommended dose (RD) in the phase I part and to explore the tumour response, survival and safety in the phase II part. Patients with histologically- or cytologically confirmed unresectable or recurrent gastric cancer were eligible. Treatment consisted of intravenous docetaxel on day 1 (starting dose 50 mg m(-2)) and oral S-1 at a fixed dose of 40 mg m(-2) twice daily on days 1-14, every 4 weeks up to six cycles. Nine patients took part in the phase I portion of the study. The MTD of docetaxel was determined to be 50 mg m(-2), with the DLTs of grade 3 infection associated with grade 3 neutropenia and grade 4 neutropenia during S-1 administration. The RD of docetaxel was 40 mg m(-2) in combination with S-1 40 mg m(-2) b.i.d. The efficacy and safety of this regimen was therefore assessed in 46 patients with at least one measurable lesion. The overall response rate and estimated median overall survival were 46% (95% CI, 31-61%) and 14.0 months (8.3-17.3 months), respectively. The most common grade 3/4 toxicity was neutropenia (67% of patients), which was predictable and manageable. This regimen showed promising activity with moderate toxicities in advanced gastric cancer.

Endoscopic resection with a two-channel videoendoscope for gastric carcinoid tumors.

BACKGROUND/AIMS: Endoscopic resection has been used to treat hypergastrinemia-associated early carcinoid tumors of the stomach. However, indications for endoscopic treatment of these tumors have not been established. Moreover, endoscopic resection of these tumors is often difficult with conventional polypectomy, because these tumors are often located in the submucosal layer. To completely remove these tumors, we used a two-channel videoendoscope with which both grasping forceps and a polypectomy snare could be used simultaneously. METHODOLOGY: At Osaka Medical Center for Cancer and Cardiovascular Diseases, eight carcinoid tumors in six patients were removed with a two-channel videoendoscope. Reports of early carcinoid tumor in Japanese literature were reviewed to analyze the relationship between lymph node metastasis and the size and depth of involvement of these tumors. RESULTS: Six carcinoid tumors were completely removed "en bloc", but two tumors were incompletely removed. In these two patients, submucosal tumor invasion was observed on the excision line. To completely remove these tumors, the oral side, but not the top, of the tumor should be strongly grasped and pulled toward the center of the lumen as far as possible by the grasping forceps, which had been passed through the snare loop. Endoscopic follow-up studies showed no local recurrence in any patients with and without complete tumor resection during the average observation period of 30 months. A review of histological reports in Japanese literature showed that lymph node metastasis did not occur when the tumors were less than 10 mm in diameter, and could be completely removed by an endoscope. CONCLUSIONS: Endoscopic resection with a two-channel videoendoscope is a useful and safe method for resection of small carcinoid tumors of the stomach.

The clinical role of interleukin-6 and interleukin-6 soluble receptor in human follicular fluids.

In order to investigate the role of interleukin-6 (IL-6) and interleukin-6 soluble receptor (sR) in human ovulation, we evaluated the concentrations in human follicular fluid and analyzed the correlation of IL-6 and IL-6 sR with oocyte maturation. The oocytes were obtained from the follicular fluid of 45 women undergoing in vitro fertilization and embryo transfer. The concentrations of IL-6 and IL-6 sR in follicular fluid were measured by ELISA. In addition, granulosa cells obtained from the follicular fluid were cultured and treated with forskolin and 12- o-tetradecanoylphorbol 13-acetate for 24-48 h. The concentration of IL-6 was significantly higher in the follicular fluid than in the serum (P<0.01). In contrast, the concentration of IL-6 sR was significantly lower in the follicular fluid than in the serum (P<0.001). The concentrations of IL-6 and IL-6 sR were significantly higher in the follicular fluid containing mature oocytes than in fluid containing immature oocytes (P<0.05). The production of IL-6 was markedly increased over the basal level after 24 h of treatment with forskolin (P<0.001) and 48 h of treatment (P<0.01) with cultured granulosa cells. Our data suggest that IL-6 and IL-6 sR may play an important role in follicular growth and development in human preovulatory processes. It is possible that IL-6 in particular may be regulated by cAMP. IL-6 and IL-6 sR might also be valuable biochemical markers in the evaluation of oocyte maturation.

Complete response to radiation therapy in a patient with chemotherapy-resistant ovarian clear cell adenocarcinoma.

Recurrent ovarian cancer after front-line chemotherapy is incurable. In most institutions, chemotherapy is continued as salvage therapy after primary chemotherapy failure and despite the fact that long-term survival and complete responses are infrequent. Radiation therapy for patients with recurrent ovarian cancer has often been done with palliative intent. A patient with ovarian clear cell adenocarcinoma received irradiation with palliative intent to the whole pelvis after chemotherapy (paclitaxel, carboplatin, and irinotecan) produced no effect. Although she developed a rectovaginal fistula due to cancer invasion during radiation therapy. One year and half after the therapy, she is still alive with no evidence of disease. In an effort to maximize salvage potential and quality of life while minimizing toxicity, selected patients with ovarian cancer should be treated with radiation therapy directed to residual or recurrent sites.

The effect of epidermal growth factor on vascular endothelial growth factor secretion by endometrial stromal cells.

Endometrial stromal cells undergo morphological and functional changes to facilitate oocyte implantation under regulation of various hormones and growth factors. We studied physiological induction by epidermal growth factor (EGF) of vascular endothelial growth factor (VEGF) in these cells. In human endometrial stromal cells, the effect of EGF, genistein, tryphostin AG1478 (a tyrosine kinase inhibitor), and wortmannin (a phosphatidylinositol 3-kinase inhibitor) on production of VEGF was examined. Total RNA was extracted and VEGF mRNA expression was quantified by Northern analysis. EGF induced production of VEGF by stromal cells in a time-dependent manner; the effect became significant after 12 h and increased further between 24 and 48 h (P<0.05). Dose dependency was also significant (P<0.01). Genistein, tryphostin AG1478, and wortmannin partially suppressed the increase in production induced by EGF (P<0.01, P<0.01, P<0.01), respectively. Production of EGF by fertilized oocytes and trophoblasts has been reported in early pregnancy. VEGF is believed to be induced by EGF through mechanisms involving tyrosine kinase and phosphatidylinositol 3-kinase. The increase in VEGF may contribute to neovascularization that promotes proliferation of endometrium and placentation.

Induction by bombesin of peritoneal metastasis of gastric cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.

BACKGROUND: We investigated the effect of the gastrointestinal regulatory peptide, bombesin, on the development of peritoneal metastasis from gastric cancers induced in rats by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and on Rho activity in the gastric cancers. METHODS: Rats were allocated to three groups. All groups received MNNG (100 micrograms/ml) solution for 25 weeks from the start of the experiment. Group 1 (controls) received olive oil injections from the start of MNNG treatment; group 2 animals received alternate-day s.c. injections of bombesin (40 micrograms/kg body weight) in olive oil from the start of the experiment until the end of the experiment at week 52; and group 3 received the s.c. bombesin injection on alternate days from week 26 until week 52. The effect of bombesin on Rho activity in gastric cancer was examined by Western blotting. RESULTS: Bombesin given from the start of the experiment (group 2) and after the MNNG treatment (group 3) both significantly increased the incidence of gastric cancer metastasis, compared with controls, at week 52: The incidence of metastasis was significantly higher in group 2 than in group 3. Bombesin from the start of the experiment (group 2) significantly increased the incidence of tumors with deeper invasion or more infiltrative growth pattern, or lymphatic vessel tumor invasion, while bombesin after MNNG treatment (group 3) significantly increased the incidence of lymphatic vessel invasion. Bombesin also increased the activity of Rho protein in the tumors. CONCLUSION: Bombesin significantly increased the incidence of peritoneal metastasis from gastric cancers through the activation of Rho protein.