Eiji Kondoh

Epigenetic regulation of CD133 and tumorigenicity of CD133+ ovarian cancer cells

The cancer stem cell hypothesis posits that malignant growth arises from a rare population of progenitor cells within a tumor that provide it with unlimited regenerative capacity. Such cells also possess increased resistance to chemotherapeutic agents. Resurgence of chemoresistant disease after primary therapy typifies epithelial ovarian cancer and may be attributable to residual cancer stem cells, or cancer-initiating cells, that survive initial treatment. As the cell surface marker CD133 identifies cancer-initiating cells in a number of other malignancies, we sought to determine the potential role of CD133+ cells in epithelial ovarian cancer. We detected CD133 on ovarian cancer cell lines, in primary cancers and on purified epithelial cells from ascitic fluid of ovarian cancer patients. We found CD133+ ovarian cancer cells generate both CD133+ and CD133− daughter cells, whereas CD133− cells divide symmetrically. CD133+ cells exhibit enhanced resistance to platinum-based therapy, drugs commonly used as first-line agents for the treatment of ovarian cancer. Sorted CD133+ ovarian cancer cells also form more aggressive tumor xenografts at a lower inoculum than their CD133− progeny. Epigenetic changes may be integral to the behavior of cancer progenitor cells and their progeny. In this regard, we found that CD133 transcription is controlled by both histone modifications and promoter methylation. Sorted CD133− ovarian cancer cells treated with DNA methyltransferase and histone deacetylase inhibitors show a synergistic increase in cell surface CD133 expression. Moreover, DNA methylation at the ovarian tissue active P2 promoter is inversely correlated with CD133 transcription. We also found that promoter methylation increases in CD133− progeny of CD133+ cells, with CD133+ cells retaining a less methylated or unmethylated state. Taken together, our results show that CD133 expression in ovarian cancer is directly regulated by epigenetic modifications and support the idea that CD133 demarcates an ovarian cancer-initiating cell population. The activity of these cells may be epigenetically detected and such cells might serve as pertinent chemotherapeutic targets for reducing disease recurrence.

Ovarian cancer tumor infiltrating T-regulatory (Treg) cells are associated with a metastatic phenotype☆

OBJECTIVE The objective of this study was to examine the clinicopathologic correlates of T-regulatory (T(reg)) cell infiltration in serous ovarian cancers and to define gene signatures associated with high T(reg)s. METHODS Tumor infiltrating T(reg) and cytotoxic T-cells (CTLs) were quantitated in 232 primary serous ovarian cancers by immunostaining for FOXP3 and CD8. Expression microarray analysis was performed in a subset of 48 advanced cancers with the highest and lowest numbers of infiltrating T(reg)s and a genomic signature was developed using binary regression. ANOVA analysis was performed to assess the most differentially expressed genes and these genes were further assessed using Ingenuity Pathway Analysis (IPA) software. RESULTS High T(reg) infiltration in ovarian cancers was associated with high grade (p<0.0001), advanced stage (p=0.004) and suboptimal debulking (p<0.04), but not with survival. In contrast, high tumor infiltrating CD8 CTL infiltration was associated with favorable survival (median survival 48.7 vs. 34.6 months, p=0.01). A microarray-based genomic signature for high tumor-infiltrating T(reg) cells had a 77% predictive accuracy using leave-one-out cross-validation. ANOVA of microarray data revealed the antigen presentation pathway as the most differentially expressed canonical pathway (p<0.00001) between cancers with high and low T(reg) cells. CONCLUSIONS These data suggest that there may be an association between increased T(reg) cell infiltration in ovarian cancers and advanced stage. Increased T(reg) infiltration is characterized by a genomic signature enriched with several immunologic pathway genes. Therapeutic strategies that reduce tumor infiltrating T(reg) cells are under investigation and may prove useful in ovarian cancers with high numbers of these cells.

Decreased Lectin-Like Oxidized LDL Receptor 1 (LOX-1) and Low Nrf2 Activation in Placenta Are Involved in Preeclampsia

CONTEXT Serum concentration of oxidized low-density lipoprotein (oxLDL) is higher in women with preeclampsia than in normal pregnant woman. Lectin-like oxLDL receptor-1 (LOX-1) is one of the scavenger receptors for oxLDL and is abundantly expressed in placenta. It is well known that oxLDL activates nuclear factor erythroid 2-related factor 2 (Nrf2), a master regulator of antioxidant and cytoprotective genes such as heme oxygenase-1 (HO-1), which play an important role in preeclampsia. However, it has yet to be elucidated whether LOX-1, along with Nrf2, participates in the pathology of preeclampsia. OBJECTIVE The objective of the study was to assess LOX-1 expression and Nrf2 activation in preeclamptic placentas and to manifest their physiological roles in preeclampsia. METHODS Expression and regulation of LOX-1, HO-1, and Nrf2 were evaluated by real-time quantitative RT-PCR and Western blotting. The functions of LOX-1 and Nrf2 were examined using an anti-LOX-1 antibody and Nrf2 activator in JAR, a choriocarcinoma cell line, and placental explants. RESULTS Both LOX-1 expression and Nrf2 activation were significantly decreased in preeclamptic placentas compared with normal controls. A significant decrease in LOX-1 mRNA was found in placental explant cultures under hypoxic conditions. Activation of Nrf2 up-regulated HO-1 in both the JAR cells and placental explants. Furthermore, oxLDL increased HO-1 mRNA, whereas the blockade of LOX-1 inhibited the increase of HO-1 mRNA in JAR cells. CONCLUSION Decreasing LOX-1 expression in preeclamptic placenta may contribute to high oxLDL concentration, low Nrf2 activation, and low HO-1 expression. These findings provide novel insights into the crucial role of LOX-1 and Nrf2 in the pathogenesis of preeclampsia.

Stress affects uterine receptivity through an ovarian-independent pathway

BACKGROUND Although stress is known to disturb natural fertility through the inhibition of the hypothalamic-pituitary-gonadal (HPG) axis, the impact of stress on infertile women who receive exogenous gonadal hormones is not well defined. This is probably due to lack of experimental models for evaluating the impacts of stress through an ovarian-independent pathway. The objective of this study was to investigate the possible impact of stress on uterine receptivity, independent of HPG axis dysfunction, using a mouse implantation model maintained with hormone supplementation. METHODS Blastocysts from donor mice were transferred into the uterine lumen of ovariectomized (OVX) Balb/c female recipient mice following supplementation with estradiol and progesterone. The recipients were divided into two groups: those exposed (stress group) or not exposed (control group) to intermittent sonic exposure prior to embryo transfer (ET). The number of implantation sites (IS) was compared between these groups. Microarray analysis was performed to elucidate stress-induced molecular alterations in uteri during the implantation period. Sequential gene expression of leukemia inhibitory factor (Lif), an estradiol-inducible gene, was also analyzed using real-time PCR. RESULTS A non-mating OVX model with satisfactory implantation rates was established. The number of IS in the stress group (n = 20) was significantly less than that in the control group (n = 18) (Mann-Whitney test, P = 0.0375). Implantation-related genes and ovarian-hormone-responsive genes were repressed in the stress group despite ovarian hormone supplementation. The expression of Lif was suppressed in the stress group. CONCLUSIONS Stress can cause decreased uterine receptivity through an ovarian-independent pathway.

Differential expression and the anti-apoptotic effect of human placental neurotrophins and their receptors

Neurotrophin (NT) is important in the survival, maintenance and differentiation of neuronal tissue, and functions in follicle maturation, tumor growth, angiogenesis and immunomodulation; however, the expression of NT and its receptors (NTR) in human placenta and their influence on fetal growth are unclear. Here we investigated the correlation of NT and NTR in human placenta with uterine environment and fetal growth. TrkB, a NTR, mRNA was expressed on decidual and villous tissue and increased with gestational age, localizing in the trophoblast layer and endothelium by immunohistochemistry. Villous TrkB mRNA was significantly increased in preeclampsia (PE) than in controls and was higher in the normotensive small for gestational age (SGA) placenta, although it was not significant. It was also significantly increased in the small twin of discordant twin pregnancies. Brain-derived neurotrophic factor (BDNF), the main ligand of TrkB, was expressed in membranous chorion and villous tissue and was significantly higher in maternal plasma in normotensive SGA and PE than in controls. TrkB mRNA expression was up-regulated on cultured villous tissue explants and on JEG-3, a choriocarcinoma cell line, by H(2)O(2) treatment. BDNF decreased apoptotic cells in H(2)O(2)-treated JEG-3, indicating that BDNF/TrkB signaling had anti-apoptotic effects against oxidative stress in JEG-3, suggesting a protective role of BDNF/TrkB in human villous tissue under unfavorable conditions in utero.

GPR54 Is a Target for Suppression of Metastasis in Endometrial Cancer

Invasion into deep myometrium and/or lymphovascular space is a well-known risk factor for endometrial cancer metastasis, resulting in poor prognosis. It is therefore clinically important to identify novel molecules that suppress tumor invasion. Reduced expression of the metastasis suppressor, kisspeptin (KISS1), and its endogenous receptor, GPR54, has been reported in several cancers, but the significance of the KISS1/GPR54 axis in endometrial cancer metastasis has not been clarified. Metastin-10 is the minimal bioactive sequence of genetic products of KISS1. Clinicopathological analysis of 92 endometrial cancers revealed overall survival is improved in cancers with high expression of GPR54 (P < 0.05) and that GPR54 expression is associated with known prognostic factors including FIGO stage, grade, and deep myometrial invasion. Through RNAi and microarray analyses, metastin-10 was predicted to suppress metastasis of GPR54-expressing endometrial cancers in vivo. Methylation analysis revealed GPR54 is epigenetically regulated. Metastin-GPR54 axis function was restored following treatment with the DNA hypomethylating agent 5-aza-DC. These data suggest that metastin-10 may be effective at inhibiting the metastatic spread of endometrial cancers in combination with demethylating agents to induce GPR54 expression. Mol Cancer Ther; 10(4); 580–90. ©2011 AACR.

Targeting slow-proliferating ovarian cancer cells.

Advanced ovarian cancer has a high rate of recurrence and mortality despite relative chemosensitivity at the time of initial treatment. Conventional chemotherapeutic agents typically target rapidly dividing cells. Disease relapse may therefore result from the survival and later emergence of latent slow‐proliferating and/or quiescent cancer cells. We sought to identify drugs that target this cell population and to investigate the influence of these cells on outcome of patients in remission from advanced ovarian cancer. Drugs with increased efficacy against slower proliferating cells were identified using correlation‐based screening of 44,657 compounds tested on the NCI‐60 panel of cancer cell lines. Validation of candidates was performed in comparison with Cisplatin or Paclitaxel and by manipulation of proliferation rates by serum deprivation. Cytostatic and cytocidal effects were evaluated using MTT assays and active caspase‐3 immunocytochemistry. Ki‐67 proliferation indices were determined for tumors from 104 patients in remission. UCN‐01 efficacy was correlated with longer doubling time among the NCI‐60 cell lines (R = 0.54, p < 0.0001) and in a panel of 24 ovarian cancer cell lines (R = 0.42, p = 0.04), whereas this was not the case for Cisplatin (R = −0.10, p = 0.65) and Paclitaxel efficacy correlated with shorter doubling time (R = −0.52, p = 0.009). Cytostatic and cytocidal effects of UCN‐01 were increased in serum‐deprived cells. A low proliferation index was associated with presence of persistent disease at second‐look surgery (p = 0.01) and poor survival (disease‐free survival, p = 0.002; overall survival, p = 0.04). These results suggest that targeting quiescent ovarian cancer cells may be a worthwhile therapeutic approach to improving survival of women with ovarian cancer.

Pyomayoma during pregnancy: A case report and review of the literature

Pyomyoma (suppurative leiomyoma) is a rare but serious complication of uterine leiomyomas. Although the management of leiomyomas during pregnancy is usually expectant, prompt surgical intervention is mandatory for pyomyoma. We present a case of pyomyoma with peritonitis that necessitated myomectomy at 21 weeks of gestation in a 28‐year‐old nullipara in which the pregnancy continued to successful delivery at 37 weeks of gestation. Perinatal and neonatal outcomes in pregnancies complicated with pyomyoma may be improved by prompt surgical intervention even in the early second trimester. A brief review of the literature regarding pyomyoma associated with pregnancy is also described.

Ileal perforation and massive intestinal haemorrhage from endometriosis in pregnancy: case report and literature review.

An increasing number of women with severe endometriosis have conceived through assisted reproductive technology, but endometriosis can cause life-threatening complications for both the mother and baby during pregnancy. We describe a case of endometriosis-induced spontaneous ileal perforation in pregnancy with massive intestinal haemorrhage that required caesarean section, right hemicolectomy and terminal ileum resection at 33 weeks of gestation. Spontaneous perforation associated with intestinal endometriosis in pregnancy is a rare complication, and only seven cases have been reported in the English literature. To the best of our knowledge, this is the first report of spontaneous ileal perforation due to endometriosis. Moreover, this is probably the first case of massive intestinal haemorrhage in pregnancy that resulted from intestinal endometriosis. These risks must be explained to couples suffering from endometriosis-related infertility prior to conception by assisted reproductive technology, and multidisciplinary management may be mandatory for women with severe endometriosis in pregnancy.

Cervical clamp with ring forceps to prevent prolapse of an intrauterine balloon in the management of postpartum hemorrhage

An intrauterine balloon tamponade is a simple but highly effective method for the management of postpartum hemorrhage. However, treatment failure can occur due to prolapse of an intrauterine balloon into the vagina. We present two cases with a successful maneuver in maintaining the intrauterine placement of the balloon by clamping the cervix with two ring forceps in the management of postpartum hemorrhage. Although the balloon was initially expelled through a dilated cervix, a cervical clamp using ring forceps prevented displacement of the balloon, and the hemorrhage ceased. Clamping the cervix with two ring forceps to retain the balloon can be a simple and readily available approach to consider when an intrauterine balloon tamponade does not work due to its expulsion.