Increased TET1 Expression in Inflammatory Microenvironment of Hyperinsulinemia Enhances the Response of Endometrial Cancer to Estrogen by Epigenetic Modulation of GPER.

Background: Insulin resistance (IR) has been well studied in the initiation and development of endometrial endometrioid carcinoma (EEC). As yet, it has been largely neglected for estrogen sensitivity in local endometrium in hyperinsulinemia-induced systemic microenvironment. The aim of this study was to investigate the role of insulin in regulating estrogen sensitivity and explore the potential mechanisms in insulin-driven inflammatory microenvironment. Methods: We first investigated the effect of insulin on estradiol-driven endometrial cancer cells proliferation in vitro to address the roles of insulin in modulating estrogen sensitivity. Then GPER, ERα and TET1 in EEC samples with or without insulin resistance were screened by immunohistochemistry to confirm whether insulin resistance regulates estrogen receptors. Further mechanism analysis was carried out to address whether TET1 was mediated epigenetic modulation of GPER in insulin-induced microenvironment. Results: Insulin enhanced estradiol-driven endometrial cancer cells proliferation by up-regulating G-protein-coupled estrogen receptor (GPER) expression, but not ERα or ERß. Immunohistochemistry of EEC tissues showed that GPER expression was greatly increased in endometrial tissues from EEC subjects with insulin resistance and was positively correlated with Ten-eleven-translocation 1 (TET1) expression. Mechanistically, insulin up-regulates TET1 expression, and the latter, an important DNA hydroxymethylase, could up-regulate GPER expression through epigenetic modulation. Conclusion: This study identified TET1 as the upstream regulator of GPER expression and provides a possible mechanism that insulin-induced positive regulation of estrogen sensitivity in endometrial cancer cells. Increasing expression of GPER through TET1-mediated epigenetic modulation may emerge as the main regulator to enhance the response of endometrial cancer to estrogen in insulin-driven inflammatory microenvironment.

TET1-GPER-PI3K/AKT pathway is involved in insulin-driven endometrial cancer cell proliferation.

Large amount of clinical evidence has demonstrated that insulin resistance is closely related to oncogenesis of endometrial cancer (EC). Despite recent studies showed the up-regulatory role of insulin in G protein-coupled estrogen receptor (GPER/GPR30) expression, GPER expression was not decreased compared to control when insulin receptor was blocked even in insulin treatment. The purpose of this study was to explore the possible mechanism by which insulin up-regulates GPER that drives EC cell proliferation. For this purpose, we first investigated the GPER expression in tissues of endometrial lesions, further explored the effect of GPER on EC cell proliferation in insulin resistance context. Then we analyzed the role of Ten-Eleven Translocation 1 (TET1) in insulin-induced GEPR expression and EC cell proliferation. The results showed that GPER was highly expressed in endometrial atypical hyperplasia and EC tissues. Mechanistically, insulin up-regulated TET1 expression and the latter played an important role in up-regulating GPER expression and activating PI3K/AKT signaling pathway. TET1 mediated GPER up-regulation was another mechanism that insulin promotes EC cell proliferation.

Management of type II unruptured cesarean scar pregnancy: Comparison of gestational mass excision and uterine artery embolization combined with methotrexate.

OBJECTIVE: This research was carried out to investigate the effectiveness, rationality, and safety of laparotomy management compared with uterine artery embolization (UAE) combined with methotrexate (MTX) for the treatment of deep implantation cesarean scar pregnancy (CSP II). MATERIALS AND METHODS: Data from 29 patients seen between June 2008 and February 2012 were retrospectively analyzed. The patients were divided into the surgery group and the UAE combined with MTX group according to the treatment they received. We compared the clinical characteristics and treatment outcomes between the two groups. RESULTS: The patients' clinical characteristics did not differ between the surgery group and the UAE combined with MTX group. However, the mean blood loss was decreased in the surgery group compared with the UAE combined with MTX group (90 ± 4.5 mL vs. 286 ± 5.2 mL, p < 0.05). No patients required blood transfusion in the surgery group, whereas two patients in the UAE combined with MTX group received blood transfusions. The length of time for the serum beta human chorionic gonadotropin (ß-HCG) level to normalize, the time required for the disappearance of the gestational mass, and the duration of hospital stay were significantly less in the surgery group than in the UAE combined with MTX group (13.7 ± 1.0 days vs. 40.7 ± 1.7 days, 7.1 ± 1.3 days vs. 135.4 ± 6.7 days, and 11.0 ± 1.2 days vs. 41.4 ± 3.2 days, respectively; p < 0.01). Although the treatment success rate did not differ significantly between the two groups, the success rate was 100% for the surgery group and 73% for the UAE combined with MTX group. CONCLUSION: Surgical treatment can remove gestational masses and allow wound repair. Moreover, laparotomy is available in almost all hospitals. Thus, surgery can be an effective and reasonable treatment for CSP II.

Network motifs in the transcriptional regulation network of cervical carcinoma cells respond to EGF.

PURPOSE: Cervical carcinoma is the second most prevalent and the fifth most deadly malignancy seen in women worldwide. Dysregulated activation of EGF ErbB system has been implicated in diverse types of human cancer; however, it is elusive how it is regulated in human cervical cancer cells. We herein aimed to explore the mechanisms of cervical carcinoma response to epidermal growth factor (EGF), with a view of the pathways activated by EGF. METHODS: Using the GSE6783 affymetrix microarray data accessible from gene expression omnibus database, we first identified the differentially expressed genes between EGF-stimulated and -unstimulated samples. Then we constructed a regulation network and identified the network motifs. We also performed biological process and pathway enrichment analyses to functionally classify the genes in the regulation network. RESULTS: A total of 11 network motifs were identified in the regulation network. EGF treatment could increase the risk of cancer via dysregulation of cancer-related pathways and immune response pathways. CONCLUSIONS: Network motif analysis is useful in mining the useful information underlying the network. We hope our work could serve as a basis for further experimentation.

Oestrogen receptor α mediates 17ß-estradiol enhancement of ovarian cancer cell motility through up-regulation of survivin expression.

OBJECTIVE: To determine the role of oestrogen receptor α (ERα) in the regulation of survivin expression by 17ß-estradiol (E(2)) in ovarian cancer cells and to evaluate the mechanism of E(2) action on ovarian cancer cell migration. METHODS: We performed RT-PCR and Western blot analysis to assess the expression of ERα in the ovarian cancer cell lines NIH:OVCAR-3 and SKOV-3. Full-length ERα cDNA was reintroduced into SKOV-3 cells through stable transfection. After treatment with E(2), with or without pre-incubation of anti-oestrogen compound ICI 182780, RT-PCR and Western blot analysis were performed to detect survivin expression at the mRNA and protein levels. RNA interference (RNAi) was used to inhibit the expression of survivin in SKOV-3 cells. Wound healing-induced migration and Matrigel invasion experiments were performed to determine the motility of ovarian cancer cells. RT-PCR and gelatin zymography were used to detect the expression and activity of MMP-9 in SKOV-3 cells. RESULTS: A stably transfected clone with over-expression of ERα, SKOV-α, was isolated. Exogenous or endogenous expression of ERα in SKOV-3 or NIH:OVCAR-3 cells resulted in a significant up-regulation of survivin in the presence of E(2). Pre-treatment with ICI 182780 attenuated the up-regulation of survivin by E(2). Previous data from our laboratory showed that E(2) enhanced the motility of ovarian cancer cells. RNAi strongly inhibited survivin expression in SKOV-3 cells. Knock-down of survivin expression reduced the migration and invasion of SKOV-3 cells, which correlated with down-regulation of MMP9 mRNA expression and activity. CONCLUSIONS: ERα may be responsible for the up-regulation of survivin after E(2) treatment in ovarian cancer cells. The mechanism of oestrogen-promoted ovarian cancer metastasis may due to the up-regulation of survivin conducted through the ERα signalling pathway.

Regulation of epithelial-mesenchymal transition by homeobox gene DLX4 in JEG-3 trophoblast cells: a role in preeclampsia.

The pathogenesis of preeclampsia is unclear but is thought to be related to shallow trophoblast invasion. An invasive phenotype is acquired by trophoblasts through the process of epithelial-mesenchymal transition (EMT). We proposed that EMT in trophoblasts is deregulated in preeclampsia. The homeobox gene DLX4 plays an important role in epithelial-mesenchymal interactions during embryonic and placental development. To elucidate the role of DLX4 in trophoblast EMT and preeclampsia, we investigated the expression of DLX4 in preeclampsia-affected placentas and the effect of DLX4 on EMT in trophoblast-derived JEG-3 cells. DLX4 expression was downregulated in preeclampsia-affected placentas and hypoxic JEG-3 cells. Knockdown of DLX4 by RNA interference (RNAi) inhibited the motility and invasion ability of JEG-3 cells, decreased the expression of E-cadherin, and upregulated the expression of the E-cadherin repressor Snail. Our findings suggest that decreased expression of DLX4 leads to the pathogenesis of preeclampsia by inhibiting EMT in trophoblasts and provides new insight into the pathophysiological mechanism of preeclampsia.

[Survey on menopausal age and menstruation span in women in Pudong district of Shanghai].

OBJECTIVE: To investigate natural spontaneous menopausal age, menstruation span and their relationship with menarche age and parity in Pudong district of Shanghai. METHODS: From Jan 2007 to Jul 2008, 15 083 spontaneous menopause women undergoing cervical cancer screening were enrolled in this study. The questionnaire included menarche age, parity, spontaneous menopausal age and menstruation span. Those women were divided into four groups based on age, which were group of 56 - 60, 61 - 65, 66 - 70 and more than 70.Analysis of variance (ANOVA) was used for comparing difference between menopausal age and menstruation span. Multiple factor regressions was used to analyze the relationship between menarche age, parity and menopausal age and menstruation span. RESULTS: (1) Spontaneous menopausal age: the minimum was 29 years old, the maximum was 61 years old, and the mean age was (50.6 ± 3.7) years old. The mean spontaneous menopause age were (50.9 ± 3.4), (50.7 ± 3.7), (50.0 ± 4.1), (49.6 ± 4.0) years in groups of 56 - 60, 61 - 65, 66 - 70 and more than 70 years. With the increasing age range in four groups, the increasing trends of menopausal age were observed, which the difference of 1.36 year was shown between groups of 56 - 60 and more than 70 years. (2) Menstruation span: the mean of menstruation span was (34.3 ± 4.1) years, which the minimal age of 12 years and maximal age of 48 years were recorded. (34.6 ± 3.8), (34.3 ± 4.1), (33.9 ± 4.6), (33.2 ± 4.5) were observed in groups of 56 - 60, 61 - 65, 66 - 70 and more than 70 years. With the increasing age range in four groups, the increasing trends of menstruation span were observed, which the difference of 1.41 year was shown between groups of 56 - 60 and more than 70 years. (3) The impact of menarche age on menopausal age and menstruation span: there was no correlation between menarche age and menopausal age (r = 0.02); however, menstruation span was found to be negatively correlated with the menarche age (r = -0.43). (4) The impact of parity on menopausal age and menstruation span: the mean menopausal age of women who had 1 - 2 deliveries was significantly higher than those had no delivery or more than 3 deliveries (P < 0.05). However, there was no difference in menopausal age between women with 1 and 2 deliveries or between women without delivery and more than 3 deliveries (P > 0.05). Menstruation span of women with 1 delivery was significantly longer that those with more than 1 delivery (P < 0.05), similarly, women with 2 deliveries had longer menstruation span than women without delivery or more than 3 deliveries (P < 0.05). There were no difference in menstruation span between women with more than 3 deliveries and without delivery (P > 0.05). (5) Multifactor regression analysis for menstruation span: menarche age was correlated with menstruation span negatively (r = -0.97, P < 0.001). There was significantly different menstruation span between group of 61 - 65, 66 - 70 or more than 70 years and group of 56 - 60 (r = -0.18, P = 0.020; r = -0.78, P < 0.001 and r = -1.23, P < 0.001). Menstruation span in women with 1 - 2 deliveries was significantly longer than that of women without delivery or more than 3 deliveries. (6) Multifactor logistic analysis of menopausal age: there was no association between menarche age and menopausal age, however, significant differences were found in mean menopausal age between different groups, which show that menopausal age of group 56 - 60 years was significant higher than the other groups, including age-group 61 - 65 years, 66 - 70 years and over 70 years (r = -0.18, P = 0.020; r = -0.78, P < 0.001; r = -1.23, P < 0.001). Menopausal age in women with 1 - 2 deliveries was significantly higher than those of women without delivery or with more than 3 deliveries, however, no difference between women with 1 and 2 deliveries or between women without deliveries and more than 3 deliveries was observed. CONCLUSION: (1) Menopausal age and menstruation span exhibited increasing trends in Pudong district of Shanghai. (2) Menarche age and parity were the important factors influencing menopausal age and menstruation span. (3) With younger age of menarche, the menstruation span become longer.(4) Deliveries of 1 - 2 times can significantly delay the menopause and prolong menstruation span, however, the multiple deliveries (≥ 3 times) had no significant impact on menopausal age and menstruation span.

[Expression of glyoxalase I and its effect on cell proliferation and apoptosis in endometrial carcinoma].

OBJECTIVE: To examine the expressions of glyoxalase I (GLO-I) in endometrial cancer tissues and cell lines and to investigate the roles of GLO-I on proliferation and apoptosis in endometrial cancer cells. METHODS: Immunohistochemistry, western blot and RT-PCR were used to investigate the expressions of GLO-I protein and mRNA in endometrial cancer tissues and Ishikawa cell lines;enzyme activity of GLO-I in normal endometrium, endometrial cancer and paraneoplastic tissue samples was detected with spectrophotometer;proliferation and apoptosis of Ishikawa cell before and after RNA interference (RNAi) procedure were detected by the methyl thiazolyl tetrazolium (MTT) and flow cytometry, respectively. RESULTS: (1) There were significant differences of GLO-I expression between normal endometrium (0/19) and endometrial cancer tissues (76%, 22/29); these were also significant differences of enzyme activity of GLO-I among normal endometrium, paraneoplastic and endometrial cancer tissues (1.1, 0.8 vs 92.3 IU/mg; P < 0.01). Enzyme activity of GLO-I in fresh normal endometrium and paraneoplastic tissues was weak, while that of fresh endometrial cancer tissues was as high as 92.3 IU/mg in average. (2) The expression of GLO-I mRNA in Ishikawa cell transfected with GLO-I siRNA was significantly lower than that in negative group (0.25 ± 0.06 vs 0.93 ± 0.10, P < 0.01), and the similar results that in the expression of GLO-I protein (0.38 ± 0.06 vs 0.94 ± 0.13, P < 0.01). (3) Proliferation in Ishikawa cell was significantly inhibited after silencing RNA expression of GLO-I (P = 0.028). The apoptosis rate of cells transfected with GLO-I siRNA was significantly higher than that of negative control group and blank control group [(6.7 ± 0.8) % vs (1.2 ± 0.4)%, (1.4 ± 0.4)%; P < 0.01]. CONCLUSION: The expression and enzyme activity of GLO-I is significantly increased in endometrial cancer, which could promote abnormal proliferation and inhibit apoptosis in endometrial cancer cells.

Detection of TERC amplification in cervical epithelial cells for the diagnosis of high-grade cervical lesions and invasive cancer: a multicenter study in China.

Because the activation of telomerase is a relatively early event in the progression of cervical carcinogenesis, the expression of the human telomerase RNA gene, TERC, has the potential to serve as a biomarker for both the diagnosis and prognosis of cervical neoplasias. In total, 83 research centers participated in the study, and 7786 patients were enrolled. TERC amplification was detected using a dual-color fluorescence in situ hybridization (FISH) probe set, and these results were compared with cytological and histological results, testing for high-risk human papillomavirus (HPV) DNA (n = 2316 for the HPV DNA test), as well as patient age. TERC amplification was found to be increased in more advanced cases of cervical carcinogenesis. Moreover, a Youden's index value and the area under the receiver operating characteristic (ROC) curve were also calculated for samples with TERC amplification and found to be higher than the same values calculated for both cytology and high-risk HPV analyses of the same samples. With regard to cytological ASCUS and LSIL findings, the combination of HPV + TERC testing showed the potential to provide effective triaging to detect CIN2(+). Therefore, TERC amplification represents a valuable genetic biomarker, which in combination with an evaluation of cytology or HPV testing, can achieve higher sensitivity and specificity in distinguishing high-grade cervical lesions and invasive cancers from low-grade lesions compared with conventional methods.

Angiogenesis, vasculogenesis, and vasculogenic mimicry in ovarian cancer.

Neovascularization is essential for tumor growth and metastasis. An adequate vasculature feeds tumor growth and enhances the potential of metastasis. For many years, tumor vessels were thought to be lined exclusively by endothelial cells (ECs). However, therapeutic benefits from the promising antiangiogenic strategy targeting genetically stable ECs are frequently limited by the development of resistance, implying an oversimplified view of tumor vasculature. In fact, latest studies have revealed that in addition to ECs, other cells including bone marrow-derived and plastic tumor cells do contribute to tumor vascularization, which is also indicated in ovarian cancer, the most lethal gynecologic malignancy characterized by widespread metastases within the peritoneal cavity upon diagnosis. Given the principle that tumor progression and metastasis are dependent on a persistent blood supply, it is logical that the capability of generating neovessels through diverse mechanisms of ovarian cancer is associated with its malignant potential. This review will discuss the diverse origins of ovarian cancer vascular cells and emphasize their clinical relevance (in the hope of providing insight into the prognostic assessment of women at risk for aggressive disease behavior) and alternative targets for therapeutic intervention.

[Establishment of sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo].

OBJECTIVE: To establish a sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo. METHODS: Human embryonic stem were (hESCs) were cultured on the mouse embryo fibroblasts and then were induced to differentiate to form three-dimensional EB. The hEBs were cultured in media containing various angiogenesis-related factors: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), endostatin, angiostatin, and platelet factor (PF)-4 of different concentrations for 3 days to observe the sprouting of the hEBs. 3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate labeled acetylated low density lipoprotein (Dil-AcLDL) was added onto the hEBs foe 4 h Immunofluorescence assay was used to observe if Dil-AcLDL was absorbed and if CD31 was expressed so as to determine the existence of embryonic endothelial cells in the sprouting structures. The ideal culturing condition was analyzed. RESULTS: The differentiated EBs formed sprouting structures in the collagen I matrix containing VEGF and FGF. The sprouts among individual EBs were able to link to each other and form vascular network-like structures. In the presence of VEGF and FGF, the sprouts branching from the EBs assimilated Dil-AcLDL, expressed CD31 and formed a 3-dimensional cylindrical organization. The concentrations of growth factors ideally stimulating sprouting growth were 100 ng/ml of VEGF and 50 ng/ml of FGF. The networks among the EBs were abolished by the angiostatin, endostatin, and PF4. CONCLUSION: The sprouting from hEBs accumulates embryonic endothelial cells and the sprouting network-like structures are indeed endothelial in nature. Inducing of sprouting EBs is an ideal model that mimics early embryonic vasculogenesis in humans.

Silencing of IQGAP1 by shRNA inhibits the invasion of ovarian carcinoma HO-8910PM cells in vitro.

BACKGROUND: IQGAP1 is a scaffolding protein and overexpressed in many human tumors, including ovarian cancer. However, the contribution of IQGAP1 to invasive properties of ovarian cancer cells remains unknown. Here, we investigated the effect of IQGAP1-specific short hairpin RNA (shRNA) expressing plasmids on metastatic potential of ovarian cancer HO-8910PM cells. METHODS: We used RT-PCR and Western blot analysis to characterize expression of IQGAP1 in three human ovarian cancer-derived cell lines SK-OV-3, HO-8910 and HO-8910PM. We then determined whether expression of endogenous IQGAP1 correlated with invasive and migratory ability by using an in vitro Matrigel assay and cell migration assay. We further knocked down IQGAP1 using shRNA expressing plasmids controlled by U1 promoter in HO-8910PM cells and examined the proliferation activity, invasive and migration potential of IQGAP1 shRNA transfectants using MTT assay, in vitro Matrigel-coated invasion assay and migration assay. RESULTS: IQGAP1 expression level seemed to be closely associated with the enhanced invasion and migration in ovarian cancer cell lines. Levels of both IQGAP1 mRNA and protein were significantly reduced in HO-8910PM cells transfected with plasmid-based IQGAP1-specific shRNAs. RNAi-mediated knockdown of IQGAP1 expression in HO-8910PM cells resulted in a significant decrease in cell invasion and migration. CONCLUSION: Our findings support the hypothesis that IQGAP1 promotes tumor progression and identify IQGAP1 as a potential therapeutic strategy for ovarian cancer and some other tumors with over-expression of the IQGAP1 gene.

[Effects of follicle stimulating hormone on proliferation, apoptosis, migration and invasion of ovarian carcinoma cells: an in vitro experiment].

OBJECTIVE: To investigate the effects of follicle stimulating hormone (FSH) on the proliferation, apoptosis, migration and invasion of ovarian cancer cells. METHODS: Ovarian cancer cells of the lines SKOV-3 and ES-2 were cultured, and treated by FSH of the concentrations of 10, 20, 40, 80, and 160 mU/ml for 48 h or 24 h respectively. The cells without FSH treatment were used as control cells. The proliferative effects of the cells were detected by MTT colorimetry. The apoptosis and cell cycle were examined by flow cytometry. The matrix metalloproteinases-2 (MMP-2) protein levels in the supernatant were determined by zymography. The cytoplasm levels of MMP-2 protein in cells were tested by Western blotting. RT-PCR was used to detect the expression of MMP-2 mRNA in cells. The migration and invasion of the cells were examined. RESULTS: The a values of the SKOV-3 treated with FSH of the concentrations of 10 - 160 mU/ml were all significantly higher than those without FSH treatment (all P < 0.01). The apoptosis rates of the SKOV-3 treated with FSH of the concentrations 10 - 160 mU/ml were (0.94 +/- 0.06)%, (0.71 +/- 0.03)%, (0.22 +/- 0.02)%, (0.32 +/- 0.02)%, and (0.55 +/- 0.05)% respectively, all significantly lower than those without FSH treatment [(1.30 +/- 0.10)%, all P < 0.01]. After treatment with FSH of the concentrations 40 to 160 mU/ml the percentages of the SKOV-3 at the stage G(0)/G(1) gradually decreased and the cells at the stage S gradually increased compared with the control groups (all P < 0.05). The MMP-2 mRNA and protein expression levels of the SKOV-3 increased with the concentration increase of FSH (P < 0.05 or P < 0.01). Boyden chamber invasive assay showed that the numbers of the SKOV-3 that penetrated the basement membrane were (157 +/- 20)/hp (x200), significantly higher than those of the control groups [(27 +/- 9)/hp, P < 0.01]. Scarification test showed that the distance between scratches of the FSH-treated SKOV-3 cells was significantly shorter than that of the control group (P < 0.01). FSH also induced similar results in ES-2 cells. CONCLUSION: FSH induces the proliferation, migration, and invasion and suppresses the apoptosis of ovarian cancer cells.

[Influence of long-term treatment with MPA on the biological character of endometrial carcinoma Ishikawa cell].

The study was undertaken to investigate the effect of long-term treatment with MPA on the growth and invasiveness of endometrial carcinoma Ishikawa cells and the expression of the subtype of estrogen receptor and progesterone receptor. The human endometrial carcinoma Ishikawa cells were continually exposed to 2.5 micromol/L step-wise increase of MPA. MTT assay, flow cytometry and Matrigel invasion assay were used to detect the effect of MPA on the growth, cell cycle distribution and the invasion capability of the cells respectively. RT-PCR was performed to detect the changes of CyclinD1, MMP2 and MMP9 over different time treatment of MPA. Immunocytochemistry was used to examine the expression of estrogen receptor subtype and progesterone receptor subtype. Endometrial carcinoma Ishikawa cells became resistant to the inhibitory effect of MPA over ten months MPA treatment. The cells resistant to the growth inhibitory effect were named progestin-resistant Ishikawa cells and the cells which the progestin-resistant originated from were named the parent Ishikawa cells. The effects of MPA shifted from growth and invasiveness inhibitory effects on the parent Ishikawa cells to stimulatory effect on the progestin-resistant Ishikawa cells. Consistent with the phenomena, the treatment with MPA on Ishikawa cells resulted in statistically significant decreases of CyclinD1, MMP2 and MMP9 gene expression, reversely, the treatment with MPA on the progestin-resistant Ishikawa cell resulted in statistically significant increases of CyclinD1, MMP2, MMP9 gene expression. Immunocytochemical analysis showed reduced ERalpha and PR-B expression and increased ERbeta expression in the progestin-resistant Ishikawa cells compared with parental Ishikawa cells. From the experiment, it was concluded that the prolonged treatment with MPA on Ishikawa cell could give rise of the resistant effect of MPA, the effect of MPA on the growth and invasiveness capability of endometrial carcinoma shifted from inhibitory to stimulatory. The imbalance of ER subtype and PR subtype might contribute to the mechanisms involved in the progesterone resistance over long-term MPA treatment.

[Intermediate-conductance-Ca2+-activated K+ channels are overexpressed in endometrial cancer and involved in regulating proliferation of endometrial cancer cells].

OBJECTIVE: To study the expression of intermediate-conductance-Ca(2+)-activated K(+) (IKCa1) channels in endometrial cancer and its role in regulating proliferation of endometrial cancer cells. METHODS: Western blot and RT-PCR were used to examine the expression of IKCa1 channels in 13 normal endometrial specimens and 25 endometrial cancer specimens; and RNA interference (RNAi), [(3)H] thymidine incorporation, and inhibitor of IKCa1 channel were used to explore the role of IKCa1 channels in regulation of proliferation of endometrial cancer cells HEC-1A. RESULTS: The expression rate and level of IKCa1 mRNA in endometrial carcinoma (84%, 0.89 +/- 0.52) were higher than in normal endometria (8%, 0.14 +/- 0.12; P < 0.01). The expression rate and level of IKCa1 protein in endometrial carcinomas (80%, 1.18 +/- 0.41) were higher than in normal endometria (15%, 0.71 +/- 0.26; P < 0.01). Clotrimazole, an inhibitor of IKCa1 channels known to suppress the function of the channels, caused a both time- and dose-dependent decrease in cell number of HEC-1A cell. Western blot analysis revealed that the IKCa1 level in whole lysates of the cells transfected with target-IKCa1 small interference RNA (siRNA) was (48.27 +/- 9.07)% of that found in the cells transfected with non-silencing RNA; [(3)H] thymidine incorporation in HEC-1A cells transfected with target-IKCa1 siRNA was also reduced, siRNA inhibited HEC-1A cell proliferation, compared with the cells transfected with non-silencing RNA (P < 0.05). CONCLUSION: The expression of IKCa1 channels may be closely related to the proliferation of endometrial cancer, and down regulation of its expression may suppress its development.

Characteristics and differentiated mechanism of vascular endothelial cells-like derived from epithelial ovarian cancer cells induced by hypoxia.

A few highly aggressive and malignant tumor cells could acquire identities by turning on genes expressed by endothelial cells and recruit blood vessels to sustain tumor growth. Hypoxia was reported recently to play an essential role in these events. These 'plastic' tumor-cell phenotypes and the exact mechanism driving transendothelial differentiation by hypoxia-inducible factor (HIF)-1alpha is unclear. In this study, epithelial ovarian carcinoma cells were exposed to hypoxia and the tumor cells were transformed into endothelial cells-like (ECs-like). Typical endothelial features such as cell markers and uptaking of acetylated low density lipoprotein were identified constantly. Small interference RNA was used to block the expression of HIF-1alpha. Analysis revealed that hypoxia promotes transendothelial differentiation through stimulating HIF-1-dependent transcriptional expression of vascular endothelial growth factor (VEGF), VEGF receptor-2 (Flk-1) and P53, and through decreasing HIF-1-independent transcriptional expression of Cyclin D1. These results demonstrate that ECs-like derived from epithelial ovarian cancer cells are similar to endothelial progenitor cells rather than endothelial cells. HIF-1alpha is crucial but not unique in alternation of tumor cells towards ECs-like.

[Effects of estrogen receptor beta on proliferation of ovarian clear cell adenocarcinoma ES-2 in vitro and in vivo].

OBJECTIVE: To investigate the involvement of estrogen receptor (ER) beta in the proliferation of ovarian clear cell adenocarcinoma by restoring ERbeta expression in a cell line ES-2. METHODS: A plasmid with full length ERbeta cDNA, pRSV-ERbeta and its negative vector control pRSV were introduced into ES-2. The cells transfected were named according to the plasmids: ES-pRSV, ES-pRSV-ERbeta. RT-PCR and western blot were used to detect the expression of ERbeta in ES-2, ES-pRSV and ES-pRSV-ERbeta cells. The growth activities of cells in vitro were detected by methyl thiazolyl tetrazolium (MTT) assay, and in vivo growth in nude mice was also observed. Flow cytometry was performed to show the change of cell cycles. RESULTS: The ES-pRSV-ERbeta cells were identified with ERbeta mRNA and protein expression. The growth activities of ES-pRSV-ERbeta were inhibited in vitro. In MTT analysis, the values of ES-2, ES-pRSV, and ES-pRSV-ERbeta cells were 0.78 +/- 0.05, 0.81 +/- 0.06, and 0.53 +/- 0.07 (the third was lower compared with the former two, P < 0.01). In vivo, the volume of transplants of ES-pRSV-ERbeta cells in mice, (2868 +/- 879) mm(3) was smaller than that in ES-2, (3603 +/- 724) mm(3), and in ES-pRSV, (3913 +/- 624) mm(3) (P < 0.05). The S phase ratios of the cell cycle of ES-2, ES-pRSV, and ES-pRSV-ERbeta were (37 +/- 9)%, (39 +/- 10)%, and (20 +/- 5)% (P < 0.05). CONCLUSIONS: ERbeta may play an important role in the proliferation, and DNA synthesis of ES-2. The evidence indicates ERbeta may be an inhibitor in the initiation and development of ovarian clear cell adenocarcinoma.

The reinforcement of invasion in epithelial ovarian cancer cells by 17 beta-Estradiol is associated with up-regulation of Snail.

OBJECTIVE: The transcription factor Snail, which is implicated in the triggering of epithelial-mesenchymal transitions (EMT), plays an important role in adhesion, invasion and metastasis of tumor cells. In the present study, we assessed 17beta-Estradiol (E2)'s effect on Snail, E-cadherin and MMP-2 expression of epithelial ovarian cancer cell line ES-2 and SKOV3. Then we induced Snail gene silencing by RNA interference to explore the effect of E2 on E-cadherin and MMP-2 expression when Snail gene expression was blocked. METHODS: Treated by 10(-8) M E2, Snail, E-cadherin and MMP-2 mRNA expression of the cells was measured by RT-PCR; Snail, MMP-2 protein expression was detected by IHC; and MMP-2 activity was determined by Zymography. E-cadherin protein level was measured by Western blot. We constructed the small interfering dsRNA expression vector (pRNAT-U6.1/Neo-Snail) targeting Snail gene, as well as a negative control vector (pRNAT-U6.1/Neo-Neg). Then the cells were transiently transfected with the vectors. Western blot and zymography were conducted to determine E-cadherin protein level and matrix metalloproteinase activity of the cells transfected with pRNAT-U6.1/Neo-Snail or pRNAT-U6.1/Neo-Neg after treated with E2 for 24 h. RESULTS: The expression of ER alpha mRNA and protein was negative in ES-2 cells and positive in SKOV3 cells, and ER beta expression was positive in both cell lines. 10(-8) mol/l E2 elevated expression of Snail and MMP-2 mRNA and protein in both ES-2 and SKOV3 cells, and reduced expression of E-cadherin mRNA and protein in SKOV3 cells. While in the RNAi group transfected with the small interfering dsRNA expression vector (pRNAT-U6.1/Neo-Snail) targeting Snail gene, E2 treatment did not have a significant effect on MMP-2 activity or E-cadherin protein in ES-2 and SKOV3 cells. CONCLUSIONS: 17beta-Estradiol increased Snail expression in both ER alpha-negative ES-2 cells and ER alpha-positive SKOV3 cells independent of the existence of ER alpha. The increase of MMP-2 expression in ES-2 and SKOV3 cells and decrease of E-cadherin expression in SKOV3 cells induced by E2 were associated with up-regulation of Snail.

[Analysis of 16 patients with early cervical cancer treated by laparoscopic vaginal radical trachelectomy].

OBJECTIVE: To evaluate the therapeutic efficacies of preserving fertility treatment in patients with early cervical cancer. METHODS: Sixteen patients with early cervical cancer treated by laparoscopic vaginal radical trachelectomy and pre- or postoperative chemotherapy were analyzed retrospectively, focusing on the treatment indication and management of high risk patients. RESULTS: The median age was 29 years (range 26 to 34 years). Eleven were nulligravida and 4 multipara. All patients had a desire to maintain fertility. For clinical stage, 2 were stage Ia2, 13 stage Ib1 and 1 stage Ib2. Fifteen patients had squamous cell carcinoma and 1 had adenosquamous cell carcinoma. Mean operative time was 3 hours and 12 minutes, and mean blood loss was 320 ml. There were no intra- or postoperative complications. With mean follow-up time of 13 months, one patient had recurrence (6%), and no one became pregnant. CONCLUSIONS: It is possible to preserve fertility in the treatment of patients with early cervical cancer, but treatment indication should be considered carefully. The management of high risk patients should be investigated extensively.