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.

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.

[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.

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.

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 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.

[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.

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.

Glycogen synthase kinase-3beta positively regulates the proliferation of human ovarian cancer cells.

Although glycogen synthase kinase-3 (GSK-3) might act as a tumor suppressor since its inhibition is expected to mimic the activation of Wnt-signaling pathway, GSK-3beta may contribute to NF-kappaB activation in cancer cells leading to increased cancer cell proliferation and survival. Here we report that GSK-3beta activity was involved in the proliferation of human ovarian cancer cell both in vitro and in vivo. Inhibition of GSK-3 activity by pharmacological inhibitors suppressed proliferation of the ovarian cancer cells. Overexpressing constitutively active form of GSK-3beta induced entry into the S phase, increased cyclin D1 expression and facilitated the proliferation of ovarian cancer cells. Furthermore, GSK-3 inhibition prevented the formation of the tumor in nude mice generated by the inoculation of human ovarian cancer cells. Our findings thus suggest that GSK-3beta activity is important for the proliferation of ovarian cancer cells, implicating this kinase as a potential therapeutic target in ovarian cancer.

[Glycogen synthase kinase-3beta (GSK-3beta) promotes proliferation of ovarian cancer cells in vitro].

OBJECTIVE: To investigate the effect of glycogen synthase kinase-3beta (GSK-3beta) on the proliferation of human ovarian cancer cells. METHODS: Two human ovarian cancer cell lines SKOV3 and ES-2 were analysed for the expression of GSK-3beta and phosphorylated GSK-3beta (pGSK-3beta) by Western blot analysis. Cell growth curve analysis done by cell count was used to investigate the effect of GSK-3beta inhibitors on the growth of SKOV3 and ES-2 cells. Four plasmids, namely, GSK-3betaS9A, GID5-6, GID5-6LP and the control vector, were cotransfected respectively with the green fluorescent protein (GFP) into SKOV3 cells by electroporation, and then BrdU incorporation assay was adopted to analyse the role of GSK-3beta activity in the proliferation of ovarian cancer cells. After transfection, G418 was added to the medium to select those stably transfected cells, which were used to investigate the long term effect of GSK-3beta activity change on the proliferation of ovarian cancer cells by colony formation assay. RESULTS: Both SKOV3 and ES-2 cells expressed GSK-3beta, though the expression level of pGSK-3beta was lower in SKOV3 than in ES-2 cells. GSK-3beta inhibitors attenuated the growth of SKOV3 and ES-2 cells. Transfection with GSK-3betaS9A to upregulate the GSK-3beta activity resulted in the increase of BrdU incorporation in SKOV3 cells compared with that in the control vector. On the contrary, transfection with GID5-6 to downregulate GSK-3beta activity decreased the BrdU incorporation in SKOV3 cells, compared with that in GID5-6LP, which is a control vector of GID5-6. Stable transfection with GSK-3betaS9A increased the colony number while stable transfection with GID5-6 decreased the colony number, compared with each control vector. CONCLUSION: GSK-3beta can promote the proliferation of ovarian cancer cells. Inhibition of GSK-3 p may become a potential theraputic

Comparison of efficacy and toxicity profile between intraperitoneal and intravenous topotecan in human ovarian cancer xenografts.

OBJECTIVE: To compare the therapeutic and toxic profile of topotecan given intraperitoneally with intravenously in human ovarian cancer xenografted into athymic nude mice. METHOD: Eighty female Balb-c/nu-nu mice were randomized assigned into eight groups (n=10). Xenografts resulted from intramesentery injection of cultured human ovarian cancer cells SKOV3 in athymic mice. Onset of intraperitoneal treatment with either topotecan or cisplatin (7.5 mg/kg) was on day 7. Animals scheduled for topotecan i.p. received intraperitoneal application of topotecan (1.5 mg/kg x 2, 3.0 mg/kg x 2, 6.0 mg/kg x 2 or 10.0 mg/kg x 1). Animals scheduled for topotecan i.v. received intravenous administration of topotecan (6.0 mg/kg x 2 or 10.0 mg/kg x 1). Two weeks after drug application animals were killed. Tumor growth inhibition were assessed and compared with untreated mice and cisplatin intraperitoneally administered mice. Acute toxicity was determined by loss of body weight. Cell cycle division and apoptosis after drug administration was determined by flow cytometric analysis. RESULTS: In a panel of ten tumour xenografts, intraperitoneal topotecan was significantly more effective than intravenous administration. The toxicity profile suggested a better tolerability in terms of weight loss after intraperitoneal administration than cisplatin control. Topotecan 10.0 mg/kg i.p. per day (1 day) schedule was an optimal treatment for ovarian cancer and well tolerated by mice with no signs of acute toxicity. Topotecan and cisplatin induce cells G0-G1 arrest and apparent apoptosis. No significant difference among mice treated with topotecan intraperitoneally or intravenously or cisplatin was observed in term of apoptosis and cell cycle perturbation. CONCLUSION: The results may have implications for the future design of clinical studies on intraperitoneal application of topotecan. It suggests that apoptosis and cell cycle perturbation play an limited role in the mechanism of topotecan administration.

[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.

[Influence of estrogen and progestin on nm23-H1 expression in epithelial ovarian cancer cell lines via activation of phosphorylation signaling].

OBJECTIVE: To assess the estrogen and progestin's effect on protein expression of metastasis repression gene nm23-H1 via regulation of phosphorylation signaling in epithelial ovarian cancer cell line ES-2. METHODS: Ovarian clear cell adenocarcinom cell line ES-2 was treated by different doses of 17beta-estradiol (estrogen), medroxyprogestogen (progestin) and dimethyl sulfoxide (control group), and then the following experiments were conducted. (1) Change of the cell migration capacity after treatment with estrogen and progestin for 24 and 48 hours was measured by in vitro wound healing assay. (2) Transwell experiments were used to detect the ability of cell invasion, which was also used to inhibit the phosphorylation of protein kinase B (AKT) pathway with estrogen and progestin. (3) Change of nm23-H1, AKT and phosphorylated protein kinase B (pAKT) protein level of ES-2 cells after treated with estrogen and progestin was detected by western blot. (4) Cells were transfected with the small interfering RNA (siRNA) expression vector targeting AKT. The efficiency of cells transfected was calculated according to the number of cells with green fluorescent produced by cells transfected. Change of nm23-H1 expression was assessed. RESULTS: ES-2 cells treated with estrogen, progestin or vehicle all migrated into the wound surface after 24 and 48 hours. The migration of the cells treated with estrogen was (1.39 +/- 0.08) mm, significantly elevated (P = 0.029), and that of the cells treated with progestin was (1.96 +/- 0.07) mm, significantly decreased compared with cells treated with vehicle (P = 0.014). The cells were cultured in transwell after 24 hours. The invasion of the cells treated with estrogen was 119 +/- 13, significantly elevated (P = 0.015), and that of the cells treated with progestin was 78 +/- 8, significantly decreased compared with cells treated with vehicle (P = 0.006). Western blot results showed that 17beta-estradiol decreased nm23-H1 expression, and both effects were dose and time dependent (P = 0.020, P = 0.001). Progestin increased nm23-H1 expression in ES-2 cells, and both effects were dose and time dependent (P = 0.003, P = 0.002). 17beta-Estradiol elevated the expression of pAKT, which was also dose and time dependent (P = 0.001, P = 0.007), while progestin repressed pAKT expression which was also dose and time dependent (P = 0.012, P = 0.039). When cells were transfected with the siRNA expression vector targeting AKT, the effects of estrogen and progestin on nm23-H1 expression were both attenuated. CONCLUSIONS: Estrogen downregulates the expression of nm23-H1 via activation of the phosphorylation signaling, thus participated in the regulation of invasion and metastasis of epithelial ovarian cancer cells. Progestin upregulates the expression of nm23-H1, and might repress invasion and metastasis of tumor cells.

[Study on mRNA expression of the human novel gene NM23-H1B in ovarian tumor].

OBJECTIVE: To study the expression of the human novel gene NM23-H1B in ovarian cancer. METHODS: Forty-eight samples from patients with ovarian tumor at different clinical stages and 8 from normal ovaries were examined for NM23-H1B mRNA expression by using RT-PCR, northern blot and in situ hybridization. RESULTS: All samples expressed NM23-H1B mRNA through RT-PCR, while the level of expression in ovarian tumor was higher than that of normal ovary. The level of expression in early stage (stage I and II) cancer was higher than in advanced (stage III and IV) cancer. The results of northern blot showed that NM23-H1B was over expressed in ovarian cancer while low expressed in normal ovary or low malignant potential (LMP) ovarian cancer. In early stage carcinoma, the expression level was related with the differentiation of tumor cell. Well-differentiated cancer expressed NM23-H1B mRNA at comparatively higher level. The result of in situ hybridization showed that positive expression rate of NM23-H1B mRNA in ovarian cancer (100%, 40/40) was significantly higher than that in normal ovary (0/8) or LMP ovarian cancer (2/8). CONCLUSION: The novel gene NM23-H1B is related to ovarian cancer.

[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.

[Evaluation of laparoscopic surgery in the early stage-malignant tumor of ovary with lower risk].

OBJECTIVE: To evaluate the laparoscopic operation for early ovarian malignant tumor with low risk. METHODS: Ten patients with ovarian malignant tumor who underwent laparoscopic total hysterectomy, pelvic lymph nodes dissection, bilateral adnexectomy, ovarian aortic and vein high ligation, omentectomy, and additional appendectomy. Eleven patients with the same diagnosis who underwent operation by laparotomy were served as control group. The operation time, intraoperative blood loss, number of pelvic lymph nodes excised, and postoperative recovery were analyzed retrospectively. RESULTS: Frozen section method during operation proved the diagnosis of ovarian malignant tumor and cytological examination proved a negative result of the peritoneal irrigation liquid. The operation time was 298 min +/- 60 min for the laparoscopy group and 182 min +/- 43 min for the laparotomy group (P < 0.05). The intraoperative blood loss was 280 ml +/- 156 ml for the laparoscopy group and 346 ml +/- 170 ml for the laparotomy group (P < 0.05). The number of pelvic lymph node resected was 25 +/- 5 and 27 +/- 7 for the laparoscopy group and laparotomy group respectively (P > 0.05). The postoperative illness rate was 20.0% and 72.7% for the laparoscopy group and laparotomy group respectively (P < 0.01). Seven patients and 1 case in the laparoscopy group and laparotomy group left their beds 48 hours after operation (P < 0.05). The right obtuator nerve was injured and was sutured on 1 patient in the laparoscopy group. CONCLUSION: The whole procedure of total hysterectomy, bilateral adnexectomy, pelvic lymph node dissection, ovarian aortic and vein high ligation, omentectomy, and additional appendectomy may be performed under laparoscope in the treatment of early stage ovarian malignant tumor with lower risk. The laparoscopic operation has the advantage of less intraoperative bleeding, less morbidity and rapid recovery.

[A multicenter randomized clinical trial of weekly paclitaxel and carboplatin vs three weeks paclitaxel and carboplatin as first line chemotherapy in epithelial ovarian cancer].

OBJECTIVE: To compare the efficacy of combination regiments of taxol given weekly plus carboplatin and taxol given every three weeks plus carboplatin. To observe the toxicity of the two regiments. To observe the two-year survival rate in the two groups. METHODS: Total 125 eligible patients in 13 centers of CGOG were entered into the two arms of this randomized clinical trial, of whom 51 were entered into weekly taxol group and 74 entered into 3 weeks taxol group. RESULTS: 81.6% (102/125) of patients had satisfactory decreasing of CA125 level after optimal cytoreductive surgery and chemotherapy. 86.3% (44/51) of patients is in weekly group and 78.4% (58/74) of patients in three weeks group (P > 0.05). Relapse frequency is 29.7% in every three weeks group and 19.6% in weekly group (P > 0.05). Median interval to relapse is 15.7 months in every three weeks group and 13.6 months in weekly group (P > 0.05). One-year survival rate is 95.2% in every three weeks and 93.9% in weekly group (P > 0.05). Two-year survival rate is 78.7% in every three weeks and 85.3% in weekly group (P > 0.05). Grade III and IV myelosuooression is 45.9% in three weeks group and, 27.5% in weekly group (P < 0.05). CONCLUSION: (1) The two regiments had equal efficacy. (2) Myelosuppression was less frequency in the weekly group than in every three weeks group. (3) Weekly taxol therapy has mild toxicity and is more suitable for the old and feeble patients. Weekly taxol therapy can be conveniently administered in outpatients department.