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Purpose and experimental design: Despite the therapeutic utility of progestin in invasive and preinvasive endometrial neoplasias, the molecular mechanisms through which it exerts inhibitory effects on endometrial epithelial growth are largely unknown. The aim of the study was to clarify the molecular mechanisms of progestin action to endometrial epithelial cells using originally established in vitro and in vivo treatment models for immortalized and transformed endometrial epithelial cell lines that express progesterone receptor.Results: In this model, progestin effectively inhibited the cell growth, inducing G0/G1 arrest rather than apoptosis without p21/WAF-1 induction. Using DNA microarray analysis, we identified 24 genes whose expression increased more than 10-fold on progestin treatment. Of these genes, we paid special attention to forkhead box transcription factor FOXO1, known as a key gene for endometrial decidualization. Progestin markedly induced FOXO1 gene expression mainly in the nuclei in vitro and in vivo. This induction was not due to the canonical activation of FOXO1 via protein dephosphorylation but due to FOXO1 promoter activation and mRNA induction. siRNA inhibition of FOXO1 significantly attenuated the effects of progestin to inhibit endometrial epithelial cell growth. Disrupting Akt activity by the introduction of the dominant negative form of Akt increased nuclear FOXO1 accumulation and enhanced the effect of progestin.Conclusion: These findings suggest that FOXO1 is a direct target of progestin, implicating novel molecular mechanisms of progestin to eradicate endometrial neoplasia. Clin Cancer Res; 17(3); 525–37. ©2010 AACR.

Supplementary Figures S1-S3; Supplementary Table S1.

We report a case of synchronous high-grade cervical intraepithelial neoplasia (CIN) and metastatic squamous cell carcinomas (SCCs) of unknown primary in the rectum. A 74-year-old woman was diagnosed with CIN3 by biopsy of the uterine cervix. Magnetic resonance imaging showed two masses in the outer rectal wall. They were diagnosed as SCCs by transrectal biopsy from one mass. On surgical treatment, CIN3 and SCCs in the rectum were identified, respectively. Pathological analysis revealed that SCCs were observed in serosa of the rectum, not mucosa, indicating that these tumors were metastatic SCCs. Gene analysis showed HPV31-positive and TP53 mutation in CIN3, and HPV16-positive in rectal SCCs. Pretreatment examination did not detect the primary site of metastatic SCCs in the rectum. We diagnosed the patient with synchronous CIN3 and metastatic SCCs of unknown primary in the rectum. In this case, gene analysis was useful to clarify the relationship between CIN3 and SCCs.

Although the human papillomavirus (HPV) vaccine is effective for preventing cervical cancers, this vaccine does not eliminate pre-existing infections, and alternative strategies have been warranted. Here, we report that FOXP4 is a new target molecule for differentiation therapy of cervical intraepithelial neoplasia (CIN). An immunohistochemical study showed that FOXP4 was expressed in columnar epithelial, reserve, and immature squamous cells, but not in mature squamous cells of the normal uterine cervix. In contrast with normal mature squamous cells, FOXP4 was expressed in atypical squamous cells in CIN and squamous cell carcinoma lesions. The FOXP4-positive areas significantly increased according to the CIN stages from CIN1 to CIN3. In monolayer cultures, downregulation of FOXP4 attenuated proliferation and induced squamous differentiation in CIN1-derived HPV 16-positive W12 cells via an ELF3-dependent pathway. In organotypic raft cultures, FOXP4-downregulated W12 cells showed mature squamous phenotypes of CIN lesions. In human keratinocyte-derived HaCaT cells, FOXP4 downregulation also induced squamous differentiation via an ELF3-dependent pathway. These findings suggest that downregulation of FOXP4 inhibits cell proliferation and promotes the differentiation of atypical cells in CIN lesions. Based on these results, we propose that FOXP4 is a novel target molecule for nonsurgical CIN treatment that inhibits CIN progression by inducing squamous differentiation.

Recently, it was demonstrated that the expression of

Objectives To investigate neuroendocrine carcinoma (NEC) of the uterine cervix cases for MRI features and staging, as well as pathological correlations and survival. Results FIGO was I in 42, II in 14, III in 1, and IV in 5 patients. T2-weighted MRI showed homogeneous slightly high signal intensity and obvious restricted diffusion (ADC map, low intensity; DWI, high intensity) throughout the tumor in most cases, and mild enhancement in two-thirds. In 50 patients who underwent a radical hysterectomy and lymphadenectomy without neoadjuvant chemotherapy (NAC), intrapelvic T staging by MRI overall accuracy was 88.0% with reference to pathology staging, while patient-based sensitivity, specificity, and accuracy for metastatic pelvic lymph node detection was 38.5%, 100%, and 83.3%, respectively. During a mean follow-up period of 45.6 months (range 4.3-151.0 months), 28 patients (45.2%) experienced recurrence and 24 (38.7%) died. Three-year progression-free and overall survival rates for FIGO I, II, III, and IV were 64.3% and 80.9%, 50% and 64.3%, 0% and 0%, and 0% and 0%, respectively. Materials and methods Sixty-two patients with histologically surgery-proven uterine cervical NEC were enrolled. Twelve received NAC. Clinical data, pathological findings, and pretreatment pelvic MRI findings were retrospectively reviewed. Thirty-two tumors were pure NEC and 30 mixed with other histotypes. The NECs were small cell type (41), large cell type (18), or a mixture of both (3). Conclusions Homogeneous lesion texture with obvious restricted diffusion throughout the tumor are features suggestive of cervical NEC. Our findings show that MRI is reliable for T staging of cervical NEC.

The circadian rhythm, which is necessary for reproduction, is controlled by clock genes. In the mouse uterus, the oscillation of the circadian clock gene has been observed. The transcription of the core clock gene period (Per) and cryptochrome (Cry) is activated by the heterodimer of the transcription factor circadian locomotor output cycles kaput (Clock) and brain and muscle Arnt-like protein-1 (Bmal1). By binding to E-box sequences in the promoters of Per1/2 and Cry1/2 genes, the CLOCK-BMAL1 heterodimer promotes the transcription of these genes. Per1/2 and Cry1/2 form a complex with the Clock/Bmal1 heterodimer and inactivate its transcriptional activities. Endometrial BMAL1 expression levels are lower in human recurrent-miscarriage sufferers. Additionally, it was shown that the presence of BMAL1-depleted decidual cells prevents trophoblast invasion, highlighting the importance of the endometrial clock throughout pregnancy. It is widely known that hormone synthesis is disturbed and sterility develops in Bmal1-deficient mice. Recently, we discovered that animals with uterus-specific Bmal1 loss also had poor placental development, and these mice also had intrauterine fetal death. Furthermore, it was shown that time-restricted feeding controlled the uterine clock’s circadian rhythm. The uterine clock system may be a possibility for pregnancy complications, according to these results. We summarize the most recent research on the close connection between the circadian clock and reproduction in this review.

Although direct adhesion of cancer cells to the mesothelial cell layer is considered to be a key step for peritoneal invasion of ovarian cancer cell masses (OCM), we recently identified a different strategy for the peritoneal invasion of OCM. In 6 out of 20 cases of ovarian carcinoma, extraperitoneal growth of the OCM was observed along with the neovascularization of feeding vessels, which connect the intraperitoneal host stroma and extraperitoneal lesions through the intact mesothelial cell layer. As an early step, the OCMs anchor in the extraperitoneal fibrin networks and then induce the migration of CD34‐positive and vascular endothelial growth factor A (VEGF‐A)‐positive endothelial cells, constructing extraperitoneal vascular networks around the OCM. During the extraperitoneal growth of OCM, podoplanin‐positive and α smooth muscle actin (αSMA)‐positive cancer‐associated fibroblasts (CAF) appears. In more advanced lesions, the boundary line of mesothelial cells disappears around the insertion areas of feeding vessels and then extraperitoneal and intraperitoneal stroma are integrated, enabling the OCM to invade the host stroma, being associated with CAF. In addition, tissue factors (TF) are strongly detected around these peritoneal implantation sites and their levels in ascites were higher than that in blood. These findings demonstrate the presence of neovascularization around fibrin net‐anchored OCMs on the outer side of the intact peritoneal surface, suggesting a novel strategy for peritoneal invasion of ovarian cancer and TF‐targeted intraperitoneal anti‐cancer treatment. We observed and propose a novel strategy for peritoneal implantation of ovarian cancer. The strategy includes the preinvasive growth of fibrin‐anchored cancer cells along with neovascularization on the outer side of the intact peritoneal surface.

Recently, it was demonstrated that the expression of BMAL1 was decreased in the endometrium of women suffering from recurrent spontaneous abortion. To investigate the pathological roles of uterine clock genes during pregnancy, we produced conditional deletion of uterine Bmal1 (cKO) mice and found that cKO mice could receive embryo implantation but not sustain pregnancy. Gene ontology analysis of microarray suggested that uterine NK (uNK) cell function was suppressed in cKO mice. Histological examination revealed the poor formation of maternal vascular spaces in the placenta. In contrast to WT mice, uNK cells in the spongiotrophoblast layer, where maternal uNK cells are directly in contact with fetal trophoblast, hardly expressed an immunosuppressive NK marker, CD161, in cKO mice. By progesterone supplementation, pregnancy could be sustained until the end of pregnancy in some cKO mice. Although this treatment did not improve the structural abnormalities of the placenta, it recruited CD161-positive NK cells into the spongiotrophoblast layer in cKO mice. These findings indicate that the uterine clock system may be critical for pregnancy maintenance after embryo implantation.

Background Aberrant expression of P-cadherin has been reported in various cancers, and has been attracting attention as a target for cancer treatment. Ovarian cancer, the leading cause of death among gynecologic malignancies, is classified into four histological subtypes: serous, mucinous, endometrioid, and clear cell, and each has distinct biological behavior. Although a negative survival impact in serous ovarian cancer patients and some functional role in peritoneal dissemination have been reported, differences of P-cadherin expression in histological subtypes and the proportion and distribution of positive cells remain to be investigated. The aims of this study were to clarify the histological and distributional profiles of P-cadherin expression in ovarian cancer for development of target-therapy in near future. Methods A total of 162 primary, 60 metastatic, and 8 recurrent tumors (all cases from 162 ovarian cancer patients) were enrolled in the study. Immunohistochemistry was performed for P-cadherin expression. Associations with clinicopathological characteristics and survival were analyzed. Results P-cadherin expression showed a strong correlation with the FIGO stage, histological subtypes, positive peritoneal dissemination (P P P = 0.050). P-cadherin was intensely and broadly expressed in mucinous, endometrioid, and serous subtypes (P P Conclusions Mucinous, endometrioid, and serous ovarian cancer patients accompanied with peritoneal disseminations are the most potent candidates for P-cadherin targeted drug delivery strategies. P-cadherin-targeted therapy may benefit and improve survival of poor-prognosis populations.

Activation-induced cytidine deaminase (AID, Aicda) is a master gene regulating class switching of immunoglobulin genes. In this study, we investigated the significance of AID expression in the ovary. Immunohistological study and RT-PCR showed that AID was expressed in murine granulosa cells and oocytes. However, using the Aicda-Cre/Rosa-tdRFP reporter mouse, its transcriptional history in oocytes was not detected, suggesting that AID mRNA in oocytes has an exogenous origin. Microarray and qPCR validation revealed that mRNA expressions of growth differentiation factor-9 (GDF-9) in oocytes and stem cell factor (SCF) in granulosa cells were significantly decreased in AID-knockout mice compared with wild-type mice. A 6-h incubation of primary granuloma cells markedly reduced AID expression, whereas it was maintained by recombinant GDF-9. In contrast, SCF expression was induced by more than threefold, whereas GDF-9 completely inhibited its increase. In the presence of GDF-9, knockdown of AID by siRNA further decreased SCF expression. However, in AID-suppressed granulosa cells and ovarian tissues of AID-knockout mice, there were no differences in the methylation of SCF and GDF-9. These findings suggest that AID is a novel candidate that regulates cross-talk between oocytes and granulosa cells through a GDF-9 and SCF feedback system, probably in a methylation-independent manner.

Mucinous tumors are a common ovarian cystic tumor, having a characteristic finding on MR imaging, the so-called stained glass appearance. We report a rare case of a solidified mucinous tumor of the ovary, which showed unique findings on MR images. The tumor demonstrated hypointensity on T2-weighted images, not stained glass appearance, mimicking ovarian fibrous tumor such as thecoma-fibroma. Histopathological examination confirmed solidified contents of the tumor with numerous septa and diagnosed mucinous borderline tumor/atypical proliferative tumor. Keywords: Mucinous tumor, Ovary, Stained glass appearance, MRI

The invasion of an extravillous trophoblast (EVT) into maternal decidual tissues, especially towards maternal spiral arteries, is an essential process in the human placental formation and subsequent normal fetal development. However, the precise regulatory mechanisms to induce EVT invasion towards arteries and/or to protect EVT from further invasion are not well understood. We found that a chemokine receptor, CCR1, was specifically expressed on EVT migrating towards maternal arteries. Using EVT isolated from a primary villous explant culture, RANTES, which is one of the ligands for CCR1, was shown to enhance EVT invasion. Furthermore, we observed that the platelets were deposited among intravascular EVT and platelet-derived soluble factors, which contained RANTES, enhanced EVT invasion. On the one hand, dipeptidyl peptidase IV (DPPIV), which can metabolize RANTES on the cell surface, was expressed on non-invading EVT and was demonstrated to suppress EVT invasion. In contrast, laeverin/aminopeptidase Q, which is specifically expressed on EVT, was shown to induce EVT invasion. Also, CD9 which is a cell surface marker of platelets and a regulator of integrin function was expressed on EVT and gene knockdown of the CD9 molecule enhanced EVT invasion. These findings suggest that the chemokine-chemokine receptor, chemokine-peptidase, and CD9-integrin systems play important roles in the regulation of EVT invasion during early human placental formation.

Objectives To evaluate the clinical performance of novel detection kits for 14 high-risk human papillomavirus (hrHPV) types with the BD Onclarity HPV Assay (Onclarity; Becton Dickinson, Sparks, MD). Methods Two cervical specimens from 144 women were obtained and placed in BD SurePath Collection Vials. The first specimen was used for cervical cytology and digene HC2 High-Risk HPV DNA Test (HC2; Qiagen, Germantown, MD), and the second specimen was used for Onclarity and Roche Cobas 4800 HPV (Cobas; Roche Molecular Systems, Pleasanton, CA). Other HPV genotyping kits were used for specimens identified as positive by Onclarity or Cobas. Results Fifty-three of 144 specimens were positive by Onclarity. Overall agreement rates of Onclarity with HC2 and Cobas were 93.8% and 94.4%, respectively. The sensitivity and specificity for cervical intraepithelial neoplasia type 2 or higher of Onclarity were similar to HC2 and Cobas. Conclusion The results showed that the clinical performance of Onclarity was equivalent to HC2 and Cobas.

Circulating tumor cells (CTC) are newly discovered biomarkers of cancers. Although many systems detect CTC, a gold standard has not yet been established. We analyzed CTC in uterine cervical cancer patients using an advanced version of conditionally replicative adenovirus targeting telomerase-positive cells, which was enabled to infect coxsackievirus-adenovirus receptor-negative cells and to reduce false-positive signals in myeloid cells. Blood samples from cervical cancer patients were hemolyzed and infected with the virus and then labeled with fluorescent anti-CD45 and anti-pan cytokeratin antibodies. GFP (+)/CD45 (-) cells were isolated and subjected to whole-genome amplification followed by polymerase chain reaction analysis of human papillomavirus (HPV) DNA. CTC were detected in 6 of 23 patients with cervical cancers (26.0%). Expression of CTC did not correlate with the stage of cancer or other clinicopathological factors. In 5 of the 6 CTC-positive cases, the same subtype of HPV DNA as that of the corresponding primary lesion was detected, indicating that the CTC originated from HPV-infected cancer cells. These CTC were all negative for cytokeratins. The CTC detected by our system were genetically confirmed. CTC derived from uterine cervical cancers had lost epithelial characteristics, indicating that epithelial marker-dependent systems do not have the capacity to detect these cells in cervical cancer patients.

A 65-year-old woman received chemotherapy using taxane and carboplatin prior and following optimal debulking surgery for ovarian cancer stage IV. Five months later, intra-abdominal recurrence was diagnosed, and second-line chemotherapy using nogitecan and bevacizumab was administered. After five courses, the patient presented with a symptom of subileus and subsequent intestinal perforation occurred. An emergent surgery revealed two perforation sites and longitudinally extended ulcerative lesions in the ileum. Pathologically, although metastatic sites were not observed in the submucus layer just beneath the ulcers, there were a number of vascular endothelial growth factor (VEGF)-C-positive cancer cell invasion sites along with marked edema and an increase of the lymphatic endothelial cell marker 'podoplanin'-positive cells in subserous regions. Since bevacizumab is able to inhibit VEGF-A, but not VEGF-C, and induce compensatory increase in VEGF-C production, these findings suggest that the local disturbance of lymphatic circulation in the subserous regions by VEGF-C-producing cancer cells is a possible risk factor for the development of intestinal ulceration and perforation during bevacizumab therapy.

Positron emission tomography (PET) with fluorodeoxyglucose F18 (18 F-FDG) is useful for detecting malignancies, but benign lesions occasionally have false-positive 18 F-FDG uptake. Here, we report the cases of five postmenopausal women with solid ovarian tumors suspected to be ovarian cancer on magnetic resonance imaging and 18 F-FDG uptake. Mean age of the five patients was 57 years (range, 53-65 years). Average early standardized uptake value (SUV) of 18 F-FDG was 5.76 (range, 2.2-12.0) and delayed SUV was 6.56 (range, 2.4-13.8). In all five patients, frozen section diagnosis at surgery was thecoma, and bilateral salpingo-oophorectomy was performed. On immunohistochemistry, immunoreactive glucose transporter 5 (GLUT5) expression was detected in thecoma tissues. This case shows that thecoma sometimes has positive 18 F-FDG uptake on positron emission tomography-computed tomography (PET-CT), indicating the need for caution regarding false-positive PET-CT in patients with benign solid ovarian tumor.

It is frequently difficult to distinguish multiple primary carcinomas from single primary carcinoma with metastasis. Here, we report a case of synchronous endometrioid adenocarcinomas that independently occurred in the uterine cervix and corpus. A 47-year-old woman complaining of genital bleeding was preoperatively diagnosed with cervical adenocarcinoma with an endometrial lesion. On surgical treatment, two separate malignant lesions bearing endometrioid adenocarcinoma were identified in the uterine cervix and cavity. Although both lesions expressed the same type of human papillomavirus (HPV) gene, type 16, microscopic continuity was not observed. Furthermore, we detected a critical difference in PTEN mutation between the tumors and finally diagnosed this case as multiple primary cancers. This is the first report to show multiple primary endometrioid adenocarcinomas simultaneously arising in the uterine cervix and corpus. Considering the rarity of this case, the coexistence of HPV suggests its possible involvement in the carcinogenesis of the endometrioid adenocarcinomas.

Recent reports showed that neoadjuvant chemotherapy (NAC) has been successfully applied to treat advanced uterine cervical cancers during pregnancy. However, its side effects on the fetus remain unclear. Here, we report a 33-year-old primipara who underwent four courses of NAC therapy, paclitaxel and cisplatin, from 17 to 27 weeks of gestation due to uterine cervical cancer stage IB2. At 31 weeks of gestation, cesarean section and radical hysterectomy were performed, and a female baby weighing 1446 g was born. Although pre- and postnatal courses were uneventful, neonatal erythroderma over the entire body was observed just after delivery. The pathological diagnosis was ichthyosiform erythroderma, which was later demonstrated to be keratitis-ichthyosis-deafness syndrome, by exome sequencing analysis. Although her skin disorder was consistent with keratitis-ichthyosis-deafness syndrome, the skin condition gradually improved after delivery. These findings suggest that NAC therapy during pregnancy might cause or exacerbate systemic skin lesions in the fetus/neonate.

The current report describes a case of extragenital gestational choriocarcinoma in the kidney. A 36-year-old woman with a history of two deliveries of male babies visited the present hospital due to secondary amenorrhea following a positive urinary pregnancy test. Despite a high serum level of human chorionic gonadotropin, at 51,800 mIU/ml, diagnostic imaging methods and pathological examination did not detect any conceptus in the genital tract. Positron emission tomography-computed tomography detected 18F-FDG-positive tumors in the left kidney and right lung. A fine needle biopsy of the renal lesion pathologically revealed the presence of choriocarcinoma and a subsequent polymerase chain reaction analysis verified the presence of a Y-chromosome-specific the sex-determining region Y (SRY) gene, diagnosed as extragenital gestational choriocarcinoma. Clinically, 10 cycles of EMA/CO chemotherapy were administered and an optimal response was obtained. In conclusion, this is the first report of the diagnosis of extragenital gestational choriocarcinoma by the detection of the SRY gene with PCR using biopsy samples.

Problem We previously proposed that platelets promote re-epithelialization during menstruation. As cell movement is one of the important cell behaviors in the process of tissue remodeling, we examined the effects of platelets on endometrial epithelial cell invasion. Method of Study The platelets were isolated from healthy women. Using a human endometrial epithelial cell-derived immortalized cell line, EM-E6/E7/hTERT cells, we examined the effects of platelets and platelet-derived condition media with or without microparticles on the morphological and invasive properties of EM-E6/E7/hTERT cells. Results Platelets and microparticle-containing conditioned media inhibited Matrigel invasion by EM-E6/E7/hTERT cells along with an increase in cortical ring formation, whereas microparticle-depleted conditioned media promoted their invasion without any significant changes of cortical ring formation. Conclusion These results support our previous proposal and newly suggest the dual roles of platelets: platelet-derived soluble factors that promote cell movement in the distant area, and microparticles that induce re-epithelialization by endometrial epithelial cells in the proximal area.

Background: Recently developed detection system for circulating tumour cells (CTCs) using a telomerase-specific replicative adenovirus generated nonspecific green fluorescent protein (GFP) signals because of the co-presence of white blood cells (WBCs) nonspecifically infected by viruses. Here, we established a unique detection system for CTCs that completely excludes nonspecific signals. Methods: Blood obtained from the patients was subjected to haemolytic processes to eliminate red blood cells. The cell pellets were then infected with OBP-401, fixed, incubated with fluorescence-labelled anti-CD45 antibody to mark white blood WBCs, and examined on slides under a microscope. Results: Preparatory experiments with cancer cells artificially added to healthy donor samples confirmed that CD45 labelling could distinguish GFP-positive cancer cells from WBCs. In 53 patients with gynaecological cancers, CTCs were detected in 21 patients (39.6%) when CD45-positive cells were excluded as WBCs among GFP-positive cells. No CTCs were detected in samples from healthy volunteers. There was no significant correlation between CTC counts and known clinicopathological factors. The CTCs rapidly vanished after surgery or chemotherapy in most patients whose treatments were effective. In contrast, the persistence of CTCs even after treatments was tightly associated with poor response to the treatments (P

When transvaginal removal of ovarian cysts is performed successfully, the procedure compares favorably with laparoscopy in terms of invasiveness. However, the approach into peritoneal cavity has been laborious. The objective was to evaluate feasibility of an ultrasound-guided culdotomy using a newly developed umbrella needle. New culdotomy was performed on 36 patients with ovarian cysts. Each cyst was directly punctured by the needle from vagina under ultrasound guidance. The vaginal walls on both sides of the needle were incised with an electric scalpel. Through the wound, cyst was exteriorized and enucleated. Preoperative characteristics of patients, outcome, operating time, blood loss, complications, and cyst histology were analyzed. Culdotomy was performed successfully in all cases. Operating time was less than 10 minutes and blood loss was less than 10 mL. There were no culdotomy-associated complications. Culdotomy assisted by ultrasound imaging and an umbrella needle is a simple, safe, and reliable method for vaginal ovarian cystectomy. © 2011 The Author(s).

Purpose: Although the KRAS mutation is one of critical genetic alterations in endometrial carcinogenesis, the downstream targets are not known. Experimental Design: In this study, we investigated the molecular targets of KRAS signals, using tumorigenic cells with oncogenic KRAS mutation established from telomerase reverse transcriptase (TERT)-immortalized endometrial epithelial cells. Results: We first confirmed that the RAF-ERK pathway, but not the PI3K-Akt pathway, was activated in KRAS tumorigenic cells. However, the introduction of constitutively active MAP/ERK kinase into immortalized cells to mimic RAF-ERK activation failed to obtain tumorigenic phenotypes, indicating the existence of other carcinogenic pathways triggered by KRAS. Recent evidence suggestive of linkage with KRAS signals prompted us to examine the involvement of NF-κB in endometrial carcinogenesis. We found that the DNA-binding activity of NF-κB was markedly elevated in KRAS tumorigenic cells compared with TERT-immortalized cells. Furthermore, the ability of NF-κB to activate the target gene promoters significantly increased in KRAS tumorigenic cells. Introduction of a mutant IκB that is resistant to degradation and thereby enhances the inhibitory effect on NF-κB largely abrogated the transformed phenotypes of KRAS tumorigenic cells. Thus, oncogenic KRAS signals contributed to the tumorigenic phenotypes of endometrial cells by activating the transcription function of NF-κB. Conclusions: These findings clearly show that NF-κB activation is a novel target of oncogenic KRAS in endometrial carcinogenesis, implying the potential utility of NF-κB inhibitors for endometrial cancer chemoprevention, especially with KRAS mutation. Clin Cancer Res; 17(6); 1341–50. ©2011 AACR.

Background: Disabled phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase/extracellular signal-regulated kinase signalling is involved in endometrial carcinogenesis, and there is evidence that expression of epidermal growth factor receptor (EGFR) family members has a role in such intracellular signalling pathways. This study analysed the prognostic impact of EGFR family expression in endometrial cancer in relation to PI3K–AKT and MAPK–ERK signalling, as well as drug sensitivity. Methods and results: Immunohistochemical analysis using 63 surgical specimens of endometrioid-type endometrial cancers revealed that EGFR, human epidermal growth factor receptor (HER)-2 and HER-4 were expressed in 25 (39.7%) of 63, 26 (41.3%) of 63 and 31 (49.2%) of 63 tumours, respectively. Gene amplification of HER-2 was observed in 2 of 26 patients with high HER-2 expression. Kaplan–Meier analysis revealed that high HER-2 expression was a factor that negatively influenced the progression-free and overall survival rate (P

In humans, progesterone production of the corpus luteum during pregnancy is maintained by human chorionic gonadotropin (HCG) secreted by the implanting embryo. In addition to this endocrine system, accumulating evidence suggests that circulating immune cells play an important role in the embryo-maternal cross talk through blood circulation. Peripheral blood mononuclear cells (PBMC) derived from women in early pregnancy enhanced progesterone production by human luteal cells and trophoblast invasion in vitro. Spleen cells derived from mice during early pregnancy induced endometrial differentiation and embryo implantation in vivo. Furthermore, recombinant HCG stimulated human PBMC to produce chemokines through lectin-glycan interaction, promoting trophoblast invasion. Consequently, we proposed that the maternal immune system undergoes functional changes by recognizing developing embryos from the early stage of pregnancy and assists embryo implantation in cooperation with the endocrine system. On the other hand, chemokines secreted from the locally deposited platelets induced neovascularization during corpus luteum formation and promoted extravillous trophoblast invasion during placental formation, reconstructing maternal endometrial spiral arteries. These findings suggest that circulating blood cells including PBMC and platelets positively contribute to embryo-maternal cross talk and maternal tissue remodeling around the implantation period.

がん進展制御研究所, Deregulated signaling via the phosphatidylinositol 3-kinase (PI3K) pathway is common in many types of cancer, but its clinicopathological significance in endometrial cancer remains unclear. In the present study, we examined the status of the PI3K signaling pathway, especially in relation to PTEN and PIK3CA status, in endometrioid-type endometrial cancer. The immunohistochemical analysis revealed a high level of phosphorylated (p)-AKT expression, which is a hallmark of activated PI3K signaling, in approximately 60% of endometrial cancers. There was no correlation between p-AKT expression and clinicopathological characteristics, such as International Federation of Gynecology and Obstetrics stage, tumor grade, and myometrial invasion. Unexpectedly, a high level of p-AKT expression occurred independently of the presence of PTEN or PIK3CA mutations. Furthermore, p-AKT expression did not correlate with the expression of potential downstream targets, including p-mTOR and p-FOXO1/3a. In turn, p-AKT expression was strongly associated with extracellular-regulated kinase 1/2 expression (P = 0.0031), which is representative of the activated RAS-MAP kinase pathway. Kaplan-Meier analysis suggested that low p-AKT expression was associated with low rates of relapse-free survival, although the difference was not statistically significant, indicating that AKT activation does not confer worse prognosis. The present study demonstrates the presence of complex signaling pathways that might mask the conventional tumorigenic PTEN-PI3K-AKT-mTOR pathway, and strongly suggests a close association between the extracellular-regulated kinase and PI3K pathways in this tumor type. © 2007 Japanese Cancer Association.

Purpose: Tamoxifen is an anti-estrogenic drug that is widely used for endocrine-dependent breast cancer as adjuvant hormonal therapy, and its use has been reported to be frequently associated with high levels of serum estradiol. Since the population of premenopausal women receiving tamoxifen therapy is growing in Japan, we retrospectively analyzed the incidence of ovarian hyperstimulation by tamoxifen therapy in Japanese women. Methods: Eleven patients who received surgical therapy for endocrine-dependent breast cancer and showed high values of serum estradiol during post-operative tamoxifen therapy were recruited in this study and evaluated by examining the serum concentration of follicular stimulating hormone (FSH) and follicular development. Results: The mean age, serum concentrations of estradiol and FSH, and follicular diameter were 41.3years old, 1015.8pg/mL, 11.8mIU/mL, and 3.47cm, respectively. In 6 cases, multiple follicular development was observed, while the other cases showed single follicular development with a mean serum estradiol level of 848.6pg/mL and follicular diameter of 4.46cm. There was no significant difference in age or FSH concentration between the two groups. The mean periods from the start of the single administration of tamoxifen to the initial detection of a high estradiol concentration was 716.5days. Conclusions: These findings indicate that tamoxifen could stimulate the ovarian function even after 2-year treatment. Since single and multiple follicular developments with large sizes were observed, dual mechanisms through the inhibition of both negative and positive feedback to the hypothalamic-pituitary-axis can be proposed to explain the adverse effects of tamoxifen on ovarian function. © 2015, Yamazaki et al.

Objectives Nerve-sparing techniques to avoid bladder dysfunction in abdominal radical hysterectomy have been established during the past two decades, and they have been applied to radical trachelectomy. Although trachelectomy retains the uterine corpus, no report mentions the preservation of uterine branches of pelvic nerves. The aim of the present study was to introduce and discuss our unique concept for preserving them. Study design and results Four cases with FIGO stage Ia2-Ib1 cervical cancer, in which preservation of uterine branches of the pelvic nerves was attempted, are presented. Operative procedures basically followed the previously reported standard approaches for nerve-sparing radical hysterectomy or trachelectomy, except for some points. Before resection of the sacrouterine ligament, the hypogastric nerve was first identified and translocated laterally. Subsequently, the uterine branches of the pelvic nerve were identified as a continuation of the hypogastric nerve and could be scooped with forceps by detachment of the surrounding connective tissues. Further detachment toward the uterine corpus enabled them to be completely separated from the cervix. This separation was extended up to the level of the junction of the upper and lower branches of the uterine artery. Thereafter, standard resection of the parametrium and paracolpium was performed, followed by cervical resection when it was confirmed that the isolated uterine branches of the pelvic nerves were safely translocated and preserved. There were no recurrences of cancer in these patients. Conclusions Uterine branches of autonomic nerves can be safely preserved, and the procedure may be considered one of the nerve-sparing techniques for radical abdominal trachelectomy, which may hopefully improve the reproductive outcomes of this operation, although it needs to be evaluated with more patients.

Mouse models show that progressive shortening of telomeres with ageing causes chromosomal instability, which can lead to the initiation of cancer. However, it is unclear what roles telomere shortening plays in human carcinogenesis. The present study has investigated the involvement of telomere dynamics in uterine carcinogenesis. Using telomere-FISH (telo-FISH) assays, telomere lengths in premalignant and malignant cervical and endometrial lesions were measured and compared with chromosomal arm loss or gain. Telo-FISH signals were visualized with Cy3-labelled telomere-specific probes and presented as telomere intensity (TI). Early-stage cervical intraepithelial neoplasias (CINs), especially CIN2, had significantly shorter telomeres than corresponding normal squamous epithelia (p = 0.019), together with increased rates of chromosomal arm loss/gain (p < 0.001). Cervical cancers had relatively short telomeres, but they also showed greater heterogeneity than other sampled tissues, including those with long telomeres. In contrast, there was no significant difference between the telomere length of normal endometrium and of endometrial hyperplasia and endometrial cancer. There was no significant difference in the rate of chromosomal arm loss/gain between normal endometrium and endometrial hyperplasia. These findings suggest that progressive shortening of telomeres occurs in CIN, in association with chromosomal instability, which may play critical roles in cervical carcinogenesis. In contrast, endometrial hyperplasias have relatively stable telomeres without widespread chromosome alteration, implying that endometrial carcinogenesis involves mechanisms distinct from those of cervical carcinogenesis, possibly including microsatellite instability.

Epithelial-mesenchymal transition (EMT) is critical not only for morphogenesis during embryonic development but also the conversion of early-stage tumors into infiltrative phenotypes. The present study examines the expression of Twist, a highly conserved bHLH transcription factor that is known to promote EMT, and evaluated its prognostic significance in endometrial cancers. Tissue specimens from 70 patients with endometrial cancer who underwent primary surgery were immunohistochemically evaluated for Twist expression. A semiquantitative scoring system was developed based on the intensity and extent of cancer cells with Twist expression. Thirty-six patients (51%) exhibited high Twist expression and 34 (49%) had low expression. Most cases exhibited both cytoplasmic and nuclear staining mainly observed in cancer foci and, preferentially, at the margin but, in some cases, the stromal cells located adjacent to cancer foci as well. Among various clinical variables, high Twist expression was significantly associated with deep myometrial invasion (>1/2) (P = .012) and concurrent with decreased E-cadherin expression (P < .001), a hallmark of EMT. In univariate survival analyses, both myometrial invasion and Twist expression influenced overall survival, but Cox multivariate analyses revealed that only Twist was an independent predictor of patient survival (hazard ratio, 5.12; P = .023). Thus, our data implies that high Twist expression is a potential novel prognostic factor for disease survival of endometrial cancer. Furthermore, the present study implicates Twist as a potential therapeutic target for this tumor type.

We studied mismatch repair deficiency and PTEN (phosphatase and tensin homologue deleted on chromosome 10) mutations in endometrial cancers and hyperplasias in a Japanese population. Methylation-sensitive restriction enzyme polymerase chain reaction revealed MLH1 hypermethylation in 21 (38%) of 56 endometrial cancers. Sequencing analysis revealed PTEN mutations in 22 patients with cancer (39%) in exons 5 and 8. A PTEN frameshift mutation was associated significantly with MLH1 hypermethylation (P = .01) and a highly positive phenotype with microsatellite instability (P < .001) but not with a PTEN missense mutation. In hyperplasia, MLH1 hypermethylation was similarly observed (11/27 [41%]), but the PTEN mutation was less frequent (5/27 [19%]), observed only in atypical hyperplasias; among the 5 patients with a PTEN mutation, the 2 patients with frameshift mutations had MLH1 hypermethylation, but the 3 patients with missense mutations had unmethylated MLH1. These findings indicate that MLH1 hypermethylation is an early event frequently occurring in hyperplasia without atypia, whereas the PTEN mutation occurs later, mostly in atypical hyperplasia, possibly caused by MLH1 hypermethylation.

Although histological grade and muscular invasion are related to the malignant behaviors of endometrial endometrioid carcinoma, lymphatic and/or distant metastases are unexpectedly encountered, even in patients in the low-risk group. To re-evaluate additional reliable parameters to predict the risk of progression, we examined the immunohistochemical expression profiles of p53 and estrogen receptor (ER) β proteins. Patients with endometrial endometrioid carcinoma who underwent surgical treatment at our hospital (n = 154) were recruited to this study, and the significance of the relationships between the incidence of regional lymph node metastasis and/or postoperative recurrence and clinical or experimental parameters was evaluated. By multivariate analysis, we found that histological grades, detection of immunoreactive p53 (positive rates more than 10%, p53-stained), and high expression of ERβ (high-ERβ) were independently associated with metastasis and/or recurrence. Among these parameters, the sensitivity and negative predictive values of high-ERβ were very high (up to 100%). In the population with high-ERβ, the positive rates of metastasis and/or recurrence were 61.1% in the p53-stained group and 21.9% in the p53-non-stained (negative) group. Furthermore, the positive rate in the group showing myometrial invasion of more than 1/2 and showing both p53-stained and high-ERβ was 80%. The disease-free survival of patients who were double-positive for p53-stained and high-ERβ was significantly shorter than that in other patients. In summary, our findings showed that increases in ERβ and p53 immunoreactivity were significantly correlated with the incidence of metastasis and/or recurrence in endometrial endometrioid carcinoma, suggesting that double-positivity for p53-stained and high-ERβ may provide a promising clinical indicator to predict the risk of progression.

A reliable method of transvaginal access is needed for natural orifice transluminal endoscopic surgery. We introduce a new culdotomy procedure, using a technique for the creation of space in the cul-de-sac, transvaginal ultrasound, and a newly developed umbrella Hakko needle. An artificially developed, saline-containing space in the cul-de-sac was punctured by an umbrella Hakko needle from the vagina under the guidance of ultrasound. The vaginal walls on both sides of the needle were incised with an electric scalpel. In five cases with benign gynecological tumors, culdotomy was successfully performed. Operating time was less than 10 minutes and blood loss was less than 10 mL. There were no culdotomy-associated complications. This procedure, named Culdotomy FourS Two U, is a simple, safe, and reliable method for entry into the cul-de-sac in transvaginal gynecological surgeries and may have future applications in transluminal endoscopic surgery through the vagina.

Objective To investigate the impact of estrogen contained in oral contraceptives (OCs) on the action of progestin on ovarian endometrioma epithelial cells. Design Experimental in vitro study and immunohistochemical analysis. Setting University hospital. Patient(s) Patients who underwent surgery due to ovarian endometrioma. Intervention(s) Not applicable. Main Outcome Measure(s) Telomerase-immortalized epithelial cells derived from ovarian endometrioma were treated with norethindorone (NET; 80 nmol/L) or levonorgestrel (LNG; 20 nmol/L) with or without 17β-ethynylestradiol (EE; 0.6 nmol/L) for 96 hours, and the cell growth was monitored. Estrogen receptor (ER) α, progesterone receptor (PR) A, and PRB expressions in clinical samples of ovarian endometrioma epithelial cells were analyzed with the use of immunohistochemistry. Result(s) NET or LNG effectively suppressed cell growth, and addition of EE significantly enhanced the growth suppression. This EE-mediated enhancement of cell growth suppression was observed only in cells that expressed ERα and therefore was ERα dependent. Western blot analysis revealed that expression of PRB was significantly induced by the addition of EE. Immunohistochemical analysis confirmed that ERα expression and PRB expression are significantly correlated, indicating that progestin-sensitive cells with PRB expression are predisposed to react with estrogen stimulation. Conclusion(s) These findings suggest that EE contained in OCs plays a supportive role in progestin-induced growth inhibition of ovarian endometrioma epithelial cells. In the absence of estrogen priming, concurrent estrogen action was essential for rapid induction of PR to achieve maximal progestin effect.

A small subset of cells with CD133 expression is thought to have increased chemoresistance and tumorigenicity, features of cancer stem cells (CSCs); the molecular mechanisms by which these properties arise remain unclear. We characterized CD133+ endometrial cancer cells based on microarray analyses of Ishikawa cells. Of the genes upregulated in CD133+ cells compared with CD133- cells, we noted several key factors involved in the aggressive behavior of cells, including ABCG2 and matrix metalloproteinase (MMP). Flow cytometric analyses identified a side-cell population (SP) with CSC features in Ishikawa cells, and they were found to be more enriched in CD133+ cells than CD133- cells. In particular, CD133+/SP cells exhibited higher proliferative and colony‑forming activity than CD133+/non-SP cells. Matrigel invasion assay revealed that CD133+ cells have enhanced invasive capacity with elevated MT1-MMP expression. siRNA‑based knockdown of MT1-MMP largely abolished the invasive capacity of CD133+ cells, but not CD133- cells due to low levels of constitutive MT1-MMP1 expression. These findings demonstrate that increased chemoresistance and tumorigenic potential of CD133+ cells are at least partly attributed to an enriched SP fraction as well as increased MMP-1 expression. These results will be of assistance in the establishment of molecular target therapy to CSCs in endometrial cancer.

Progestin inhibits the growth of normal and cancerous endometria via the progesterone receptor (PR), but the distinct functions and signalings of PR subtypes have not been fully understood. The aim of the present study was to dissect the key pathways of progestin to inhibit endometrial epithelial growth. Immortalized endometrial epithelial cells (EM-E6/E7/TERT) with stable PRA or PRB expression were established and used for the experiments. In vitro growth inhibition by progestin was mainly observed in EM-E6/E7/TERT cells with PRB rather than those with PRA. RT-PCR assay confirmed that FOXO1, a key gene for progestin action, was up-regulated by progestin in a PRB-dependent manner. cDNA microarray analysis identified IGFBP-1, which contains FOXO1 binding sites on its promoter, to be induced by medroxyprogesterone acetate (MPA) in EM-E6/E7/TERT cells with PRB but not with PRA. siRNA knockdown of FOXO1 disturbed the induction of IGFBP-1 by MPA, while IGFBP-1 knockdown showed no effect on MPA-induced FOXO1 expression, indicating that FOXO1 is an upstream regulator of IGFBP-1. Luciferase reporter assays showed that MPA activated the IGFBP-1 promoter, which was cancelled by FOXO1 knockdown. Chromatin immunoprecipitation assay confirmed the in vivo binding of FOXO1 to the core promoter of IGFBP-1. IGFBP-1 knockdown significantly attenuated the growth inhibitory effects of MPA. The FOXO1/IGFBP-1 axis is essential for PRB-dependent growth inhibition of endometrial epithelial cells, offering a potential therapeutic clue to enhance the progestin effect.

Endometriosis is a common gynaecological disorder associated with dysmenorrhoea, pelvic pain and subfertility and is a leading cause of disability and loss of productivity in women of reproductive age (Olive and Schwartz, 1993). Numerous studies have attempted to dissect the biology of endometriosis. These studies mainly use in vitro culture of stromal cells rather than culture of epithelial cells from endometriotic tissues, because the former cells are more easily and stably cultured for much longer periods than the latter cells (Noble et al, 1997; Gurates et al, 2002). In fact, it is difficult to culture endometriotic epithelial cells in vitro, because these cells lose their proliferative capacity during ongoing cultivation of primary cultures over several days. The inability of endometriotic epithelial cells to survive in vitro is an obstacle to gaining a better understanding of the biology of this disease. In particular, malignant change of endometriosis, especially of ovarian endometrioma, for which epithelial cells are exclusively responsible, has lately attracted considerable clinical attention (Kurman and Craig, 1972; McMeekin et al, 1995; Ness, 2003; Oral et al, 2003). There is therefore an urgent need to establish a stable system for the culture of endometriotic epithelial cells that can be used for research not only into the biology of endometriosis but also into its carcinogenesis. There are two major barriers within epithelial cells that inhibit cell division under usual culture conditions: premature senescence and telomere-dependent senescence (Kiyono et al, 1998). The former is observed during early passage in primary culture and is caused by the activation of Rb that leads to cell cycle arrest, whereas the latter is found at a later stage of culture and is caused by telomere shortening after a considerable number of cell divisions. Inhibition of both Rb function and telomere shortening is therefore required for long-term culture of epithelial cells. In previous studies, we successfully established a stable system for the culture of primary endometrial epithelial cells, in which the human papillomavirus type 16 E6/E7 genes and the human telomerase reverse transcriptase (hTERT) were introduced to inhibit Rb functions and to activate telomerase, respectively (Kyo et al, 2003). These immortal cells were not transformed but retained the original characteristics of endometrial epithelial cells, such as steroid responsiveness. Subsequent studies have demonstrated that overexpression of cyclin D1 or cdk4, instead of HPV E6/E7, effectively inhibited Rb activity and might be an alternative method of overcoming premature senescence in primary epithelial cells of other origins (Ramirez et al, 2004; Sasaki et al, 2009). In the present study, we sought to generate a stable culture of epithelial cells isolated from the ovarian endometriomas by the introduction of various genetic elements. These cells were successfully immortalised without generation of transformed phenotypes and were responsive to progestin and oestrogen. These cells are thus potentially useful as an experimental model for analysis of the mechanisms of steroid hormone functions as well as of carcinogenesis arising from ovarian endometrioma.

Purpose and experimental design: Despite the therapeutic utility of progestin in invasive and preinvasive endometrial neoplasias, the molecular mechanisms through which it exerts inhibitory effects on endometrial epithelial growth are largely unknown. The aim of the study was to clarify the molecular mechanisms of progestin action to endometrial epithelial cells using originally established in vitro and in vivo treatment models for immortalized and transformed endometrial epithelial cell lines that express progesterone receptor. Results: In this model, progestin effectively inhibited the cell growth, inducing G0/G1 arrest rather than apoptosis without p21/WAF-1 induction. Using DNA microarray analysis, we identified 24 genes whose expression increased more than 10-fold on progestin treatment. Of these genes, we paid special attention to forkhead box transcription factor FOXO1, known as a key gene for endometrial decidualization. Progestin markedly induced FOXO1 gene expression mainly in the nuclei in vitro and in vivo. This induction was not due to the canonical activation of FOXO1 via protein dephosphorylation but due to FOXO1 promoter activation and mRNA induction. siRNA inhibition of FOXO1 significantly attenuated the effects of progestin to inhibit endometrial epithelial cell growth. Disrupting Akt activity by the introduction of the dominant negative form of Akt increased nuclear FOXO1 accumulation and enhanced the effect of progestin. Conclusion: These findings suggest that FOXO1 is a direct target of progestin, implicating novel molecular mechanisms of progestin to eradicate endometrial neoplasia. Clin Cancer Res; 17(3); 525–37. ©2010 AACR.

Tumor-initiating cells are known to be the major source of tumor propagation and might be an attractive therapeutic target. The present study dissected the roles of CD133 as a tumor-initiating cell marker in endometrial cancer and investigated the prognostic impact of this marker expression. Flow cytometry using 6 endometrial cancer cell lines revealed that the frequency of CD133(+) cells varied widely among the cell types and that Ishikawa and MFE280 cells contained significantly higher ratio (10%-20%) of such cells; therefore, these were subjected to the subsequent analyses. Sorted CD133(+) cells showed more aggressive proliferative potential in vitro and more increased tumorigenicity in nude or NOD/SCID mice than CD133(-) cells and generated both CD133(+) and CD133(-) cells. Furthermore, they showed apparent resistance to cisplatin- or paclitaxel-induced cytotoxicity compared with CD133(-) cells. CD133(+) cells had a greater S-phase fraction than CD133(-) cells, and the serum starvation that induced G0/G1 accumulation decreased the population of CD133(+) cells. Finally, we immunohistochemically analyzed the CD133 expression in endometrial cancer specimens from 62 patients. CD133 expression was not significantly associated with any of the clinicopathologic characteristic of tumors. However, the Kaplan-Meier analysis revealed that tumors with high CD133 expression showed worse overall survival (P = .023, log-rank test) than those with low CD133 expression; and the Cox regression hazard model found that high CD133 expression was an independent prognostic factor (P = .045). Thus, the present study demonstrates that CD133 is not only a tumor-initiating cell marker but also a critical prognostic marker in endometrial cancer.

The present study evaluated the efficacy of an electrosurgical bipolar vessel sealing system for radical abdominal hysterectomy (RAH). A total of 85 consecutive patients who underwent RAH with pelvic lymphadenectomy for cervical cancer or endometrial cancer were analyzed; in 18 patients, the LigaSure system (Valleylab, Boulder, Colo) was used to secure the vascular pedicles, and in the remaining 67 patients, clamps were used, and the pedicles suture ligated. Compared with the conventional suture group, the patients in the LigaSure group had a significantly shorter operation time (mean, LigaSure 242.8 ± 36.1 minutes vs conventional, 349.1 ± 82.6) minutes; P < 0.001) and lower blood loss (mean, 583.1 ± 287.6 mL vs 999.0 ± 524.2 mL; P < 0.005). Only 1 (0.06%) of the 18 patients in the LigaSure group was transfused, whereas 27 (40.2%) of the 67 patients in the conventional suture group were transfused. Because transfusion requirements may be affected by the surgeon's bias, we compared blood loss among untransfused patients and found that the LigaSure group still had significantly lower blood loss than the conventional suture group (mean, 550.9 ± 233.1 mL vs 745.49.0 ± 230.4 mL; P < 0.01). Hemoglobin level reduction after surgery in untransfused patients was significantly lower in the LigaSure group than in the conventional suture group (mean, 2.31 ± 2.22 mg/dL vs 3.22 ± 1.11 mg/dL; P < 0.05). These findings indicate that the LigaSure vessel sealing system is useful to reduce blood loss and shorten operating time at RAH.

Despite tremendous development in chemotherapy for ovarian cancer over the past few decades, the prognosis of advanced cases with massive peritoneal dissemination is still unsatisfactory, and novel treatment modalities that can combine with chemotherapy are urgently needed. We recently developed virotherapy for solid tumors using telomerase-specific replication-selective adenoviruses (Telomelysin: OBP-301), in which the human telomerase reverse transcriptase (hTERT) gene promoter has been inserted to direct tumor-specific E1 gene expression. In this study, we investigated the anti-tumor effects of OBP-301, combined with cisplatin (CDDP), on ovarian cancer cells. In vitro treatment of SKOV3 cells with OBP-301 at a multiplicity of infection (MOI) of 0.01-100 induced significant cell death in a dose-dependent manner, with moderate cytotoxicity at an MOI of 1-10 and maximal cytotoxicity at an MOI of 100. In contrast, OBP-301 treatment of normal human cells showed no significant cell death at an MOI of 1-10 and exhibited modest cytotoxicity at an MOI of 100. The effects of low-dose CDDP at 0.5-1 microM, which induced only 20% cell death, were significantly augmented by combination with OBP-301 at an MOI of 1-10, finally achieving 40% cell death. Such enhancement of CDDP sensitivity was also observed in CDDP-resistant ovarian cancer cells. The combinatorial effects were further tested using a xenograft mouse model of SKOV3 with peritoneal dissemination. After intraperitoneal administration of OBP-301, we confirmed that injected OBP-301 fused with the green fluorescent protein (GFP) gene (OBP-401) was preferentially localized to peritoneal disseminations, as determined by fluorescence imaging. Treatment of mice with CDDP at low dose (0.5 mg kg(-1)) had modest effects, showing a 10% decrease in disseminations, whereas combination with intraperitoneal administration of OBP-301 at an MOI of 10 led to enhanced effects, achieving an approximately 80% decrease in disseminations. Kaplan-Meier analysis showed improved overall survival of mice treated with CDDP plus OBP-301 compared with CDDP alone. These findings support the therapeutic potential of intraperitoneal administration of OBP-301 to sensitize ovarian cancer cells to CDDP.