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Background: Management of tumors has become more complex owing to tumor heterogeneity. Fewer studies have been performed on intra-tumor heterogeneity of endometrial cancer (EC) until now. Therefore, it is of great clinical value to explore the intra-tumor heterogeneity of EC based on clinical features and gene expression profiles., Methods: A total of 1688 patients with EC were screened and 114 patients were finally selected, including specimens from 84 patients with primary EC without relapse (PE) and the paired metastases (P-M) specimens, as well as specimens from 30 patients with primary EC with relapse (RPE) and the paired relapsed EC (P-RE) specimens. Microarray and RNA-seq were used to detect gene expression of EC samples. Clinicopathological characteristics and molecular data were compared between PE and P-M groups and between RPE and P-RE groups to explore the intra-tumor heterogeneity of EC., Results: The clinical intra-tumor spatial heterogeneity of pathological type, grade, ER status, and PR status between PE and P-M were 17.9%, 13.1%, 28.6%, and 28.6%, respectively. The clinical intra-tumor spatiotemporal heterogeneity of pathological type, grade, ER status, and PR status between RPE and P-RE were 16.7%, 33.3%, 25.0%, and 37.5%, respectively. Cluster analysis sorts EC samples based on progression type of lesion and their pathological type. There were differentially expressed genes between PE and P-M and between RPE and P-RE, of which gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis were mainly enriched in cell proliferation, the p53 signaling pathway, etc. CONCLUSIONS:: Clinical and molecular data showed that there was spatiotemporal heterogeneity in intra-tumor of EC, which may add to the complexity of diagnosis and therapeutics for EC. Considering the intra-tumor heterogeneity, sequential chemotherapy and precision medicine may be a more suitable treatment plan for EC.

Background: Fusion genes may play an important role in tumorigenesis, prognosis, and drug resistance; however, studies on fusion genes in endometrial cancer (EC) are rare. This study aimed to identify new fusion genes and to explore their clinical significance in EC., Methods: A total of 28 patients diagnosed with EC were enrolled in this study. RNA sequencing was used to obtain entire genomes and transcriptomes. STAR-comparison and STAR-fusion prediction were applied to predict the fusion genes. Chi-square tests and Student t tests were used to verify the clinical significance with SPSS 13.0 software., Results: New fusion genes were found, and the number of fusion genes varied from 3 to 110 among all patients with EC. The type of fusion genes varied and included messenger RNA (mRNA)-mRNA, long non-coding RNA (lncRNA)-lncRNA, and lncRNA-mRNA. There were six fusion genes with high fusion rates, namely, RP11-123O10.4-GRIP1, RP11-444D3.1-SOX5, RP11-680G10.1-GSE1, NRIP1-AF127936.7, RP11-96H19.1-RP11-446N19.1, and DPH7-PTP4A3. Further studies showed that these fusion genes are related to stage, grade, and recurrence, in which NRIP1-AF127936.7 and DPH7-PTP4A3 were found only in stage III patients with EC. DPH7-PTP4A3 was found in grades 2 and 3, and recurrent patients with EC., Conclusion: Fusion genes play an essential role in EC. Six genes that are overexpressed with high fusion rates are identified. NRIP1-AF127936.7 and DPH7-PTP4A3 might be related to stage, and DPH7-PTP4A3 be related to grade and recurrence.

Background: Tumors with different gene expression develop and progress in different ways. To deepen our understanding of the progression in endometrial cancer, and provide a useful tool for accurate diagnosis and prognosis assessment, we identified the new molecular prognostic markers in endometrial carcinoma and analyzed the relationship of them with clinical and pathological features of endometrial carcinoma., Methods: Ninety-four cases of endometrial endometrioid adenocarcinoma with complete data from the Peking University People's Hospital from 2000 to 2008 and 40 cases of normal endometrium were enrolled. Among these, 30 endometrial endometrioid adenocarcinoma samples of different International Federation of Gynecology and Obstetrics (FIGO) stage were selected for further Agilent genome-wide microarray analysis. Significance analysis of microarrays (SAM) was used to identify genes that are significantly associated with tumor progress. Immunohistochemistry was utilized to identify the genes of interest in endometrial carcinoma and normal endometrium. The relationship between the genes and the age, clinical stage, histological grade, myometrium invaded depth, lymph node metastasis status, and the expression of ER, PR, P53, and PTEN were analyzed by χ(2) test., Results: Analysis between FIGO 1988 stage I and stage III identified a 362-gene "progress signature"; 171 down-regulated and 191 up-regulated genes. Among the alterative genes, TARP (T cell receptor gamma alternate reading frame protein) and KRT5 (keratin 5) decreased 3.57 fold and 5.8 fold in FIGO stage III patients. The expression of TARP in endometrial carcinoma increased compared to normal endometrium, while that of KRT5 decreased (P < 0.05). The expression of TARP and KRT5 decreased when stage, histological grading, myometrium invaded depth increased (P < 0.05). In the cases with lymph node metastasis, the expression of TARP decreased, while the expression of KRT5 did not differ (both P < 0.05) both. The expression of P53 had a negative relationship with the expression of KRT5 (P < 0.05), but not with the expression of TARP (P > 0.05). There was no correlation between the expression of TARP and KRT5 and the expression of ER, PR, PTEN (all P > 0.05). There was no significant difference in TARP and KRT5 expression in patients aged 50 or younger and patients older than 50 (P > 0.05)., Conclusion: The expression of TARP and KRT5 was correlated with the progress of endometrial cancer and their role needs further study.

Objective: To explore the mechanism resistance of medroxyprogesterone 17-acetate(MPA) on the endometrial cancer side-population(SP) cells., Methods: (1) Ishikawa-SP cells from endometrial cancer cell lines Ishikawa were be separated by Hoechst 33342 dyeing method and flow cytometry analysis. The clone formation efficiency between Ishikawa-SP cells and Ishikawa-non-SP cells were performed by clone formation assay. Breast cancer resistance protein (BCRP) was examined by immunocytochemistry method. (2) Ishikawa, Ishikawa-SP, Ishikawa-non-SP cells were treated with various concentrations of MPA at 5, 10, 15, 20 µmol/L. After cultured for 24, 48, and 72 hours, cells growth were measured by methanethiosulfomate (MTS) assay. (3) The groups of Ishikawa, Ishikawa-SP, Ishikawa-non-SP cells incubated with MPA at the half maximal inhibitory concentration (IC50) were selected for cell apoptosis assay by using flow cytometry. After MPA treatment, the expression of caspase-3 was examined by immunocytochemistry method., Results: (1) There were few proportion of Ishikawa-SP cells in Ishikawa endometrial carcinoma, which were 2.7%. There were stronger clone formation efficiency for Ishikawa-SP cells than that for Ishikawa-non-SP cells in Ishikawa [(6.02 ± 1.17)% vs.(0.53 ± 0.20)%, P = 0.001]. And there were higher level expression of BCRP (P = 0.001) and also more resistant Taxol and radiation between Ishikawa-SP cells and Ishikawa-non-SP cells. (2) The inhibitory effect of MPA was concentration-dependent and time-dependent. (3)After MPA treatment, the apoptosis rates of Ishikawa-SP, Ishikawa-non-SP,Ishikawa were (4.01 ± 0.43) %, (9.30 ± 0.67) %, and (4.64 ± 0.18) %, respectively(P < 0.05). The level expression of caspase-3 in Ishikawa group after MPA treated were higher than that in Ishikawa-SP group., Conclusion: MPA may be inhibit the growth of endometrial cancer, Ishikawa-SP and Ishikawa-non-SP cells, while Ishikawa-SP may be more resistant to MPA than Ishikawa-non-SP, which mechanism of resistance on MPA may be related to the properties of cancer stem-like cells and cell apoptosis.

Objective: To clarify the relationship between the expression of dual specificity phosphatase-1 (DUSP1) and the prognosis of endometrioid adenocarcinoma., Methods: The expression of DUSP1 was determined by immunohistochemical staining in specimens from 81 patients with endometrial carcinoma undergoing surgical resection. The relationship between DUSP1 expression, clinicopathological factors and prognosis were further evaluated., Results: In 81 endometrioid carcinoma samples, 59 (72.84%) cases were positive while 22 negative.Except for lymph node metastasis, the expression of DUSP1 was correlated with FIGO stage, tumor grade, myometrial invasion and the expressions of estrogen receptor (ER) and progesterone receptor (PR) (P < 0.05) .Kaplan-Meier analysis showed that patients with a positive DUSP1 expression had significantly prolonged 5-year disease-free survival rates of 98.2% and 76.0% respectively (P < 0.05). And COX regression analysis revealed that the expressions of DUSP1 and PR were independent prognostic indicators of endometrioid carcinoma, the HR (hazard ratio) of DUSP1 negative expression was 21.2.Spearman analysis further showed that the expression of DUSP1 was positively correlated with PR (r = 0.256, P < 0.05)., Conclusion: DUSP1 may be a potential negative prognostic indicator for endometrioid adenocarcinoma.

Epidemiological studies have demonstrated that type 2 diabetes mellitus (T2DM) and hyperinsulinemia are associated closely with endometrial carcinoma risk, although the molecular mechanism remains unclear. Insulin receptor isoformA expression is upregulated in many cancer cells and tissues, which suggests that IR-A-mediated signaling pathways may have important implications for cancer pathogenesis. We measured the expression of insulin receptor isoforms (IR-A and IR-B in the normal endometrium tissues, the endometrial carcinoma tissues and the endometrial carcinoma cell lines. We found that the total insulin receptor (IR) and IR-A expression mRNA levels and the ratio of IR-A to total IR in endometrial carcinoma specimens were significantly higher than them in control endometrial tissue specimens(P<0.05). Further analysis indicated that the tendency was more prominently in patients with T2DM. IR-A mRNA was differentially expressed in four endometrial carcinoma cell lines (Ishikawa, KLE, RL95-2 and HEC-1-A. RL95-2 cells have a low endogenous IR-A expression, and these were used to construct a stable cell line overexpressing IR-A. We found that IR-A overexpression significantly increased cell proliferation, the proportion of cells in S phase, activation of the Akt pathway and tumorigenicity of xenografts in nude mice. In contrast, there was no significant difference in the the percentage of apoptotic cells between cells overexpressing IR-A and control cells. Moreover, levels of phosphorylated ERK1/2 protein were significantly decreased in cells overexpressing IR-A relative to controls. These findings reveal the pivotal role of IR-A in endometrial cancer carcinogenesis, and suggest that the association of elevated IR-A levels with cell proliferation and tumorigenicity may be causally linked to its effect on the proportion of cells in S phase and the activation of the Akt pathway.

Background: Early stage (FIGO stage I-II) endometrioid endometrial adenocarcinoma (EEA) is very common in clinical practice. However, patients with the early stage EEA show various clinical behaviors due to biological heterogeneity. Hence, we aimed to discover distinct classes of tumors based on gene expression profiling, and analyze whether the molecular classification correlated with the histopathological stages or other clinical parameters., Methods: Hierarchical clustering was performed for class discovery in 28 early stage EEA samples using a special cDNA microarray chip containing 492 genes designed for endometrial cancer. Correlations between clinicopathologic parameters and our classification were analyzed. And the significance analysis of microarrays (SAM) array was used to identify the signature genes according to the tumor grade and myometrial invasion., Results: Three tumor subtypes (subtypes I, II and III) were identified by hierarchical clustering, each subtype had different clinicopathological factors, such as tumor grade, myometrial invasion status, and FIGO stage. Moreover, SAM analysis showed 34 up-regulated genes in high grade tumors, and 38 up-regulated genes and 1 down-regulated in deep myometrial invasive tumors. The overlap genes between these two high-risk factors were markedly up-regulated in subtype I, but down-regulated in subtype III., Conclusion: We have identified novel molecular subtypes in early stage EEA. Differential gene signatures characterize each tumor subtype, which could be used for recognizing the tumor risk and providing a basis for further treatment stratification.

Background: Hyperinsulinemia, insulin-like growth factor (IGF)-I and -II (IGF-II) are associated with increased risk of endometrial carcinoma. Insulin receptor isoform A (IR-A) is more frequently expressed in endometrial carcinoma than in normal endometrial tissues. To better understand their roles in endometrial carcinoma, we investigated the effects of insulin, IGF-I, and IGF-II in endometrial carcinomas cells with different IR-A expression levels., Methods: To explore the role of IR-A in mediating the activity of IGF-I, IGF-II, and insulin, we investigate the cellular proliferation of endometrial carcinoma cell lines RL95-2 and RL95-2-IR-A by MTS assays. Then we examined the protein kinase Akt phosphorylation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in both cell lines by Western blotting. The effect of IGF-II and AG1024 on cell cycle progression and apoptosis was assessed by flowcytometry. To examine whether the effects of IGFs were mediated by IR-A, we blocked IGF-I receptor (IGF-IR) in both cell lines using AG1024, an IGF-IR-specific inhibitor., Results: IGF-I and IGF-II significantly enhanced proliferation of both cell lines (P < 0.05). By contrast, insulin significantly increased proliferation of RL95-2-IR-A cells only (P < 0.05). IGF-I and IGF-II significantly increased pAkt levels in RL95-2 cells and pERK1/2 levels in RL95-2-IR-A cells (all, P < 0.05). Insulin increased pERK1/2 levels in RL95-2-IR-A cells only (P < 0.05). LY294002 and PD98059 inhibited the specific signaling activities and cellular proliferation. After AG1024 pretreatment, neither IGF-I nor IGF-II affected pAkt levels in RL95-2 cells. IGF-II, but not IGF-I, increased pERK1/2 levels in RL95-2-IR-A cells. After AG1024 pretreatment, the proliferation rate and DNA content corresponding to the S phase increased and apoptosis decreased significantly in IGF-II-treated RL95-2-IR-A cells only (P < 0.05)., Conclusions: The proliferation effect of insulin is mediated by IR-A. When IR-A dominates in a cell line, IGF-II activated cell proliferation mainly through the ERK1/2 pathway. On the other hand, IGF-II activated cell proliferation mainly through the Akt pathway. IR-A can at least partly mediate the proliferative and anti-apoptotic effects of IGF-II through the ERK1/2 pathway.

Objective: To investigate the expression of insulin receptor isoforms (IR, including IR-A and IR-B) in endometrial carcinoma (EC), and explore the role of IR-A in the growth of endometrial carcinoma cells., Methods: The expression of IR isoforms were detected by reverse transcription (RT)-PCR and real-time PCR in 4 different endometrial cancer cell lines (HEC-1-A, Ishikawa, RL95-2 and KLE), with human breast cancer cell line MCF-7 cells and hepatocellular carcinoma cell line Hep-G2 as positive control and in the endometrial cancer tissue specimens of 42 cases, admitted in Peking University People's Hospital from November 2007 to July 2009, with normal endometrial tissues from 15 cases of ovarian neoplasms at the same period as controls. The relationships among the expression of IR, IR-A isoforms and the clinicopathological parameters of EC tissues were analyzed by Spearman rank correlation analysis. An eukaryotic IR-A expression plasmid was constructed and transfected into RL95-2 cells [RL95-2(IR-A)] for overexpression of IR-A in RL95-2 cells, in which the expression of IR-A originally was low. Non radioactive cell proliferation assay-MTS was used to determine the proliferation curves in the four EC cells listed above and in RL95-2 or RL95-2 (IR-A)., Results: (1) There were two isoforms of IR-A and IR-B co-expressed were detected in EC cells and EC tissues. Among four kinds of EC cell lines, the expression level of IR mRNA in RL95-2 cells was the highest, followed by Ishikawa, KLE and HEC-1-A cells, in which the relative IR mRNA expression levels were (26.54 ± 1.82)×10(-4), (15.44 ± 3.29)×10(-4), (10.14 ± 0.10)×10(-4) and (2.63 ± 0.23)×10(-4), respectively (P < 0.01). The expression level of IR-A mRNA was the highest in Ishikawa cells, followed by KLE, RL95-2 and HEC-1-A cells, with the relative expression levels were (12.07 ± 3.31)×10(-4), (4.68 ± 0.63)×10(-4), (3.03 ± 0.22)×10(-4) and(1.46 ± 0.03)×10(-4), respectively (P < 0.01). The relative expression level of IR and IR-A mRNA were 0.017 ± 0.013 and 0.011 ± 0.010 in the EC tissues, respectively, compared with 0.015 ± 0.014 and 0.010 ± 0.012 in the controls, in which there were no significant differences in the expression level of IR or IR-A mRNA between EC tissues and the control (P = 0.662, P = 0.780). The expression of IR and IR-A in EC tissues had no significant relevance with International Federation of Gynecology and Obstetrics (FIGO) stage, cell differentiation, depth of myometrial invasion, invasion of lymph-vascular space, lymph nodes metastasis, the expression status of estrogen receptor, human progesterone receptor, and PTEN gene (all P > 0.05). The expression of IR-A mRNA in EC patients with type 2 diabetes mellitus (DM) was significantly higher than that in patients without type 2 DM (P = 0.031), while there were no statistical correlation between the expression of IR mRNA and type 2 DM (P = 0.438). (2) The proliferation rates of the four kinds of EC cells was positively related with the IR-A expression ratio, with the most growth potential in Ishikawa, followed by HEC-1-A, KLE and RL95-2 cells. The overexpression of IR-A in RL95-2 (IR-A) cells showed a significant proliferation-promoting effect than that in control RL95-2 cells (P < 0.01)., Conclusion: There are two isoforms of IR-A and IR-B co-expressed in EC. The overexpression of IR-A may promote the proliferation of EC cells.

Objective: To establish the cisplatin (DDP)-resistant cell line from human endometrial cancer cell line Ishikawa and to investigate its resistant mechanism to DDP., Methods: A resistant endometrial cancer cell line ISH/DDP was established by gradually increasing dose of cisplatin and high-dose stimulation. The resistant index was estimated by 3-(4,5-dimethylthiazol-zyl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Cell growth curve, doubling time and cell cycle phase distribution were measured; drug-resistant protein of breast cancer resistance protein (BCRP), P-glycoprotein (P-gp) and glutathione-S-transferase-π (GST-π) were examined by immuocytochemistry. Results The DDP-resistant cell line ISH/DDP was established with the resistant index of 3.77. The proliferation of ISH/DDP got slow, doubling time prolonged, which were 40.1 hours, while it was 34.1 hours in Ishikawa (P<0.05); and the cell number of G0/G1 phase [(44.3±5.7)% and (39.0±10.7)%, P>0.05] and G2/M phase [(11.9±0.7)% and (5.7±2.4)%, P<0.05] decreased, while S-phase [(44.2±6.1)% and (55.3±8.4)%, P<0.05] increased compared with parent cells. The comprehensive score of the expression of BCRP in ISH/DDP was 16.3±2.0, while it was 13.4±1.5 in Ishikawa (P<0.05). The score of the expression of P-gp in ISH/DDP and Ishikawa were 15.5±1.2 and 16.1±1.0 (P>0.05), respectively. The score of the expression of GST-π in ISH/DDP and Ishikawa were 15.2±1.9 and 14.9±1.1 (P>0.05)., Conclusion: ISH/DDP cell line showed a typical resistant phenotype and biological characteristics, which may be accounted for high BCRP expression.

Objective: To explore the regulation of Bub1 mRNA expression in endometrial carcinoma cells by estrogen and paclitaxel., Methods: The high differentiated endometrial adenocarcinoma cells (Ishikawa cell line) were cultured in DMED/F12 supplemented with 10% fetal bovine serum (FBS) or phenol red-free DMED/F12 supplemented with 5% dextran-charcoal FBS (dcFBS). Firstly, the cells were stimulated by 10 nmol/L estradiol (17β-E(2)) or no hormonal stimulation as control group, and the cell proliferation was quantified at 24, 48 and 72 hours using cell counting kit-8 (CCK-8) method. Then the cells were stimulated with different concentrations of 17β-E(2) (0.1, 10, 1000 nmol/L) at different periods (5, 15, 30 minutes and 2, 4, 8, 12, 16, 24, 30 hours), the expression of Bub1 mRNA was detected by real-time quantitative PCR. Ishikawa cells were cultured with non-serum DMEM/F12 to be synchronized, and then were treated with different concentrations of paclitaxel (10, 100 nmol/L) for 8 and 24 hours. While, non-synchronized Ishikawa cells were exposed to 100 nmol/L paclitaxel for different periods (4, 8, 16, 24, 48 hours), and real-time quantitative PCR was also used to detect the expression levels of Bub1 mRNA. Data were presented as folds change relative to control group, in which values < 1 were down-regulated, and those > 1 were up regulated., Results: The proliferation rate of cells in the presence of 17β-E(2) was significantly higher than that of the control group after treated 24 hours (A value: 0.70 ± 0.08 vs. 0.86 ± 0.10, P = 0.049). Time-dependent experiments revealed that addition of 17β-E(2) could increase cell numbers during 72 hours period, while the expression level of Bub1 mRNA was decreased in Ishikawa cell. Dose-dependent experiments revealed maximal estradiol stimulation effects at 10 nmol/L (P = 0.020). After being treated with serum-free culture, Ishikawa cells were exposed to 10 nmol/L paclitaxel for 8 and 24 hours, and the expression of Bub1 mRNA decreased (0.403 ± 0.008 vs. 0.775 ± 0.144, P = 0.251). Compared to the control cells, the mRNA expression levels of Bub1 in cells treated by paclitaxel for 8 hours was significantly decreased (P = 0.009), while there was not significantly decreased at 24 hours (P = 0.396). When exposed to 100 nmol/L paclitaxel for 8 and 24 hours, the expression of Bub1 mRNA was also decreased (0.697 ± 0.017 vs. 0.850 ± 0.004, P = 0.061). Compared to the control cells, Bub1 mRNA expression was also significantly decreased (P = 0.038 and P = 0.019, respectively). While with serum free-treatment culture, when Ishikawa cells exposed to 100 nmol/L paclitxel for 4, 8, 16, 24 or 48 hours, the expression of Bub1 mRNA significantly increased (1.127 ± 0.105 vs. 1.614 ± 0.154 vs. 2.092 ± 0.179 vs. 1.381 ± 0.061 vs. 1.519 ± 0.182, P = 0.002), of which was significantly increased at 16 hours treatment., Conclusion: Bub1 expression could be regulated by estradiol and paclitaxel, in which deregulated Bub1 expression may contribute to chemotherapeutic efficacy of paclitaxel.

Background: Type I gonadotropin-releasing hormone (GnRH-I) agonists have been applied for the treatment of steroid-dependent tumors such as breast carcinoma, ovarian cancer and prostatic carcinoma. But the mechanism has not been clarified yet. There are few reports about the treatment of endometrial carcinoma using GnRH-I agonists. Type II GnRH (GnRH-II) is a new subtype of GnRH. Our aim was to investigate the effects of GnRH-I agonists and GnRH-II on estrogen receptor-negative human endometrial carcinoma cells and the effect from phosphatase and tensin homolog gene (PTEN) to them., Methods: A lentiviral vector-mediated RNAi method was used to establish a PTEN-negative HEC-1A cell clone (HEC-1A-ND). MTT and flow cytometry were used to detect the cell proliferation, cell cycle and apoptosis of HEC-1A, HEC-1A-NC and HEC-1A-ND cells after treatment with GnRH-I agonist Triptorelin (10(-11) mol/L to 10(-5) mol/L) or GnRH-II (10(-11) mol/L to 10(-5) mol/L). Western blotting was used to detect AKT and ERK1/2 activation after treatment with different concentrations of Triptorelin or GnRH-II for 30 minutes in the above mentioned three kinds of cells., Results: Triptorelin and GnRH-II induced apoptosis and inhibited proliferation of HEC-1A, HEC-1A-ND and HEC-1A-NC in a dose-dependent manner. This effect was augmented in HEC-1A-ND cells in which PTEN gene was knocked-down. Furthermore, Triptorelin and GnRH-II inhibited the AKT and ERK activity in HEC-1A-ND cells., Conclusions: Triptorelin and GnRH-II can promote apoptosis rate and inhibit cell proliferation of estrogen receptor-negative endometrial carcinoma cells in a dose-dependent manner. PTEN gene can inhibit the effects of Triptorelin or GnRH-II on human endometrial carcinoma cells.

Objective: To study the effect of gonadotropin-releasing hormone-I (GnRH-I) agonist triptorelin and gonadotropin-releasing hormone-II (GnRH-II) on human endometrial carcinoma with different states of PTEN., Methods: The endometrial carcinoma cells (Ishikawa, Ishikawa-PTEN, and Ishikawa-neo) were treated with different concentrations of triptorelin (10(-11) to 10(-5) mol/L) or GnRH-II (10(-11) to 10(-5) mol/L). Thirty min later, serine/threonine protein kinase (Akt) and extracellular signal-regulated kinase (ERK) 1/2 activation were detected using western blot method. 48 h later, the cell proliferation, cell cycle and apoptosis were detected using methyl thiazolyl tetrazolium (MTT) and flow cytometry. 17beta-estradiol(17beta-E2, 10(-8) mol/L) or the specific estrogen receptor (ER) antagonist, ICI182780I (10(-6) mol/L) was added. After using the two drugs: triptorelin or GnRH-II, the above parameters were detected again., Results: After treated with different concentrations (10(-11), 10(-9), 10(-7), 10(-5) mol/L) of triptorelin and GnRH-II, the cell growth was slowed, the percentage of G0/G1 phase cells increased, the percentage of G2/M and S phase cells decreased and the apoptosis rate increased in a dose-dependent manner (P < 0.01, P < 0.05). These changes were more obvious in Ishikawa. The apoptosis rate induced by GnRH-II was higher than that by the same concentration of triptorelin in the three cell lines. Triptorelin and GnRH-II inhibited the Akt and ERK1/2 activity in the endometrial carcinoma cells. 17beta-E2 counteracted the effect of triptorelin and GnRH-II on the endometrial carcinoma cells (P < 0.01, P < 0.05)., Conclusion: Triptorelin and GnRH-II can promote apoptosis rate of endometrial carcinoma cells and inhibit cell proliferation in a dose-dependent manner which may be caused by ERK1/2 and Akt activity inhibition, and is related to the status of PTEN and could be offset by 17beta-E2.

Objective: To study the nongenomic effect on mitogen-activated kinase (MAPK) signal transduction of 17beta-estradiol at plasma membrane in endometrial cancer cell Ishikawa., Methods: Estradiol-bovine serum albumin conjugates (E(2)-BSA) was added to Ishikawa cells at different time points. Western blot was used to detect the protein expression of phosphorylated extracellular signal-regulated kinase (ERK) 1/2. Confocal was used to verify if the phosphorylation of ERK1/2 was initiated at the membrane., Results: Phosphorylation of ERK1/2 was elicited after 5 minute exposure to 1 x 10(-7) mol/L E(2)-BSA. It was induced to a maximum following 60 minute exposure and descended after 2 hours. The expression levels of phosphorylated ERK1/2 were 0.52, 0.98, and 0.09, respectively. Confocal proved that E(2)-BSA combined with membrane receptors after 5 minutes and elevated the phosphorylation of ERK1/2., Conclusion: Membrane actions of estrogen can activate the rapid signaling cascades of MAPK in endometrial carcinoma cells.

Objective: The aim of study was to investigate the proliferative effects of stromal cell-derived factor-1alpha (SDF-1alpha) on endometrial carcinomas cell lines with different estrogen receptors (ER) and PTEN protein profiles., Methods: MTT assays was used to detect the proliferation of HEC-1A and Ishikawa cells, and Western blotted analysis was used to detect activation of Akt and ERK1/2 in both cell lines after exposure to various concentrations of SDF-1alpha, MAPK-specific inhibitor PD98059 or PI3K-specific inhibitor LY294002., Results: Low concentrations of SDF-1alpha (50 ng/ml) induced proliferation in both cell lines. ERK1/2 was significantly activated for more than 2 h by SDF-1alpha at 20 ng/ml in HEC-1A cells, but not in Ishikawa cells. In contrast, Akt was significantly activated for over 2 h in Ishikawa cells but remained unchanged in HEC-1A cells. High concentrations of SDF-1alpha activated Akt and ERK1/2 pathways in both cell lines in a dose-dependent manner, which was primarily inhibited by LY294002 for pAkt and by PD98059 for pERK 1/2., Conclusions: SDF-1alpha could stimulate the cell proliferation of endometrial carcinoma with different expression status of ER and PTEN in vitro, likely through the activation of both Akt and ERK1/2 signaling pathways.

Objective: To explore the roles of estrogen receptor-related receptor (ERR) alpha and estrogen receptor alpha in endometrial carcinoma and their clinical values., Methods: Thirty-five cases of endometrial carcinoma were examined by immunohistochemistry. Clinicopathologic features including FIGO stage, histological grade, myometrial invasion and nodal metastasis were reviewed., Results: The expression rate of ERalpha in patients with FIGO stage I endometrial carcinoma was more than that in stage II-IV(P = 0.005). The expression rate of ERRalpha in endometrial carcinoma patients at FIGO stage I was lower than that at stages II-IV(P = 0.007). The percentage of patients at FIGO stageI or with G1-2 or myometrial invasion < 1/2 in ERalpha (+) and ERRalpha (-) groups was higher than that in ERalpha (-) and ERRalpha (+) groups(P = 0.000, P = 0.031 and P = 0.022)., Conclusion: ERalpha may be a biomarker of good prognosis while ERRalpha may serve as a biomarker of poor prognosis in endometrial carcinoma. The combinative measure of ERalpha and ERRalpha may improve the prognostic value.

Objective: To investigate the expressions of SDF-1 and CXCR4 in human endometrial carcinoma tissues., Methods: SDF-1 and CXCR4 protein expression levels were detected by immunohistochemical staining in 26 cases of atypic hyperplasia endometrium,simple hyperplasia endometrium and normal endometrium and 44 cases of endometrial carcinoma tissues., Results: Both SDF-1 mRNA and CXCR4 mRNA were expressed in Ishikawa, while no expression of SDF-1 mRNA was detected in HEC-1A. The rates of CXCR4 and SDF-1 protein expressions were 100% in glandular cells of human normal endometrial tissues, while 90.9% and 93.2% in glandular cells of endometrial carcinoma tissues. These immunoreactivities of SDF-1 and CXCR4 were significantly low in endometrial carcinoma tissues as compared with those in normal endometrium; and the intensities of immunohistochemical staining significantly decreasesd in immunoreactivity of SDF-1 and CXCR4 in low grade as compared with that in hight grade., Conclusion: SDF-1 and CXCR4 were expressed differently in endometrial cell lines, human normal endometrial tissues,and endometrial carcinoma tissues. As compared with normal endometrium ,these were significantly low levels expression of SDF-1 and CXCR4 in endometrial carcinoma tissues.

Background & Objective: Estrogen receptor-related receptor alpha (ERRalpha), a member of the subfamily of orphan nuclear receptors, could compete with estrogen receptor alpha (ERalpha) to bind the same target genes and interfere in ER signal pathway. Therefore, it might be associated to the tumorigenesis of endometrial carcinoma. This study was to explore the regulatory effect of 17beta-estradiol (17beta-E(2)) on ERRalpha expression, and to elucidate the relationship between ERRalpha and ER signal pathway in endometrial carcinoma cell lines., Methods: ERalpha-positive cell line Ishikawa and ERalpha-negative cell line HEC-IA were treated with different concentrations of 17beta-E(2) (1x10(-10) mol/L, 1x10(-8) mol/L, and 1x10(-6) mol/L) for 24 h and 48 h, respectively. The levels of ERRalpha mRNA and protein were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. 17beta-E(2) (1x10(-8) mol/L) and complete ER inhibitor ICI182,780 (1x10(-6) mol/L) were given concomitantly to observe the change of ERRalpha expression., Results: The levels of ERRalpha mRNA and protein in Ishikawa cells were down-regulated after stimulated for 24 h and 48 h by different concentrations of 17beta-E(2). The maximal effect was observed at the concentration of 1x10(-8) mol/L. When 17beta-E(2) and ICI182,780 were given simultaneously to Ishikawa cells, this down-regulation was blocked. However, the level of ERRalpha mRNA, not protein, in HEC-IA cells was up-regulated after stimulated by different concentrations of 17beta-E(2) for 24 h. After stimulated by 17beta-E(2) for 48 h, the level of ERRalpha protein was up-regulated, which could not be blocked by ICI182,780., Conclusions: 17beta-E(2) can down-regulate the expression of ERRalpha in Ishikawa cells, which is mediated by ERalpha. 17beta-E(2) can up-regulate the expression of ERRalpha in HEC-IA cells, but this regulation cannot be blocked by ICI182,780.

Cellular response to estrogen is mediated both by estrogen receptor (ER) binding to estrogen response element (ERE) and by non-nuclear actions like activation of signal transducing pathways. The main aims are to study if PI3K/Akt signaling pathway can be activated by 17beta-estradiol (E2) via non-nuclear action and to investigate the relationship of the action of E2 and ER in endometrial cancer cells expressing with different status of ER. The levels of phosphorylated Akt (Ser473) (P-Akt) and total Akt were examined by western blot and Akt kinase activity was measured in cells after stimulation with 1 microM E2 at different time points. Inhibitory role of LY294002 on activation of Akt induced by E2 and its estrogen antagonist, ICI182780 were also tested. P-Akt/Akt was used as a measure of activation of Akt. We found that maximum P-Akt/Akt and Akt kinase activity took place at 30 min in Ishikawa cells and 15 min in HEC-1A cells and the activation persisted for at least 2 h after stimulation with 1 microM E2. The activation of Akt elicited gradually with increasing doses of E2. PI3K inhibitor, LY294002, stopped the activating Akt in a dose-dependent manner and 50 microM LY294002 completely blocked the activation of Akt induced by E2. ICI182780 could block the activation of PI3K/Akt in ER-positive Ishikawa cells but not in HEC-1A cells with poor-expressed ER. This study demonstrated that E2 is able to promptly activate PI3K/Akt signal pathway in Ishikawa cells in an ER-dependent manner and ER-independent in HEC-1A cells. Blockage of PI3K/Akt cascade may become a potential and effective way to control endometrial carcinoma, especially in ER-negative cancers, which show no response to endocrinal therapy.

Background: Mutation or deletion in the phosphatase and tensin homologue deleted on chromosome ten (PTEN) gene has been identified as an important cause of endometrial carcinoma; stromal cell derived factor-1alpha (SDF-1alpha) exerts growth-promoting effects on endometrial cancer cells through activation of the PI-3 kinase/Akt pathway and downstream effectors such as extracellular-responsive kinase (ERK). In this study, a plasmid containing the PTEN gene was transfected into Ishikawa cells to investigate the difference in growth and signal transduction between Ishikawa-PTEN and Ishikawa cells after SDF-1alpha stimulation, and to study mechanisms of the involvement of PTEN protein in endometrial carcinoma development., Methods: Ishikawa cells were transfected with a plasmid (pLXSN-PTEN) containing the PTEN gene and a plasmid (pLXSN-EGFP) with enhanced green fluorescent protein (EGFP). Cells were then screened to obtain Ishikawa-PTEN cells and Ishikawa-neo cells that can both stably express PTEN protein and EGFP. Expression of PTEN protein, phosphorylation levels of AKT and ERK (pAKT and pERK) and growth differences in Ishikawa-PTEN, Ishikawa-neo and Ishikawa cells before and after SDF-1alpha stimulation were then determined by Western blots and MTT assays., Results: Western blot analysis showed that Ishikawa cells produced PTEN after transfection with the PTEN gene. At 15 minutes after SDF-1alpha stimulation, the pAKT level of Ishikawa-PTEN cells was lower than that of Ishikawa-neo cells and Ishikawa cells. There was no significant difference in pERK levels among the three cell lines. The positive effect of SDF-1alpha on Ishikawa-PTEN cells growth was markedly less than the effect on Ishikawa-neo and Ishikawa cells. However, in the absence of SDF-1alpha stimulation (baseline), the pAKT level in Ishikawa-PTEN cells was less than that in Ishikawa cells. There was a significant difference in growth between the Ishikawa-PTEN cells and the Ishikawa-neo cells., Conclusions: PTEN gene transfection can regulate the level of pAKT but not pERK in Ishikawa-PTEN cells. PTEN protein may suppress the growth-promoting effect of SDF-1alpha on endometrial carcinoma by inhibiting the PI-3K/AKT signal transduction pathway.

Background & Objectives: The machanism of signal transduction of insulin-like growth factor (IGF-1) in endometrial carcinoma is still unknow. The objective of this paper was to study extracellular signal-regulated kinase(ERK) activation in endometrial carcinoma cell line Ishikawa under the stimulation of IGF-1, and to elucidate the role of suppressor encoprotein phosphatase and tensin homologue(PTEN) in activation of ERK., Methods: Retrovirus vector of PTEN and PTEN (G129E) was constructed. Ishikawa was transfected in vitro. Expressionstable cell line was screened by G418. Western blot was applied to examine PTEN expression in Ishikawa cells after transfection. Optimal concentration and time of IGF-1 and 17-beta-estrodial which activated ERK in Ishikawa-PTEN and Ishikawa-PTEN(G129E) cells were detected. Western blot was applied to examine ERK activation under the stimulation of 17-beta-estrodial in NIH3T3 fibroblasts after transient transfection of pCXN2hER alpha and pCXN2hER beta., Results: IGF-1 activated ERK cascades(mainly ERK2) in Ishikawa cells. There was no obvious difference in ERK activation among different doses of IGF-1 (20, 40, and 80 micrograms/L), But the maximal activations of ERK2 took place at 5 min after stimulation with IGF-1. The activation of ERK2 was inhibited obviously by PTEN at 30 min, 2 h, and 24 h. There was no obvious difference in ERK activation between Ishikawa-PTEN(G129E) and Ishikawa-EGFP. 17-beta-estrodial activated ERK cascades (mainly ERK2) in Ishikawa cells. The activation of ERK was increased with increasing of concentration. The maximal activations of ERK2 took place at 5 min after stimulation with 17-beta-estrodial. The activation of ERK2 was inhibited obviously by PTEN, not by PTEN(G129E). 17-beta-estrodial activated ERKs cascades in NIH3T3 fibroblasts after transient transduction of pCXN2h-ER alpha., Conclusions: 17-beta-estrodial and IGF-1 activated ERK cascades in Ishikawa cells. Lipid phosphatase of PTEN had an inhibitory role in the activation of ERK under the stimulation of 17-beta-estrodial and IGF-1.