Normal Endometrial Epithelial Cells Research Articles

Abstract 4768: Paired box 2 (PAX2) is involved in carcinogenesis and regulated by DNA methylation in endometrial cancer

Abstract Objective: PAX2 is recognized as a significant factor in the development of urogenital system. It is expressed in multiple tumors and is essential in tumor cell survival. This study aimed to investigate the effect, expression, methylation of PAX2 in endometrial cancer, and the possible regulative mechanism of DNA methylation related PAX2 expression. Methods: PAX2 expression in endometrial cancer, hyperplasia and normal tissues was detected by immunohistochemistry, and its expression in cell lines was measured by real-time PCR, western blot and immunocytochemistry. The role of PAX2 on cell viability and mobility was measured by CCK-8, invasion and migration assays. Bisulfite sequencing and MassARRAY were used to quantify the DNA methylation level of PAX2 promoter in endometrial cells and tissues. Deletion analysis of PAX2 promoter was performed by luciferase reporter gene to analyze its promoter activity. Transcription factors (TFs) combined with PAX2 promoter were forecasted by TF search database and the regulatory function of these TFs were measured by detecting the expression of PAX2 mRNA and protein following the knock down of these TFs. Results: PAX2 was over-expressed in endometrial cancer tissues compared to hyperplasia and normal endometrium. Compare to normal endometrial epithelial cells, endometrial cancer cell lines exhibited moderate to high PAX2 expression. Knocking down PAX2 by siRNA reduced cell viability, invasion and migration, while PAX2 over-expression exhibited opposite role. The methylation level of PAX2 promoter was higher than normal counterparts in both cancer tissues and cells, which was positively related to PAX2 expression. After 5-Aza-CdR treatment, the PAX2 mRNA and protein was down-regulated along with the methylation level of PAX2 promoter. Deletion analysis confirmed that a repressive transcriptional regulatory region of PAX2 promoter coincided with the hypermethylated region found in MassARRAY analysis. The binding sites of myeloid zinc finger 1 (MZF1) and the early growth responsive gene 1(EGR-1) were found in the repressive transcriptional regulatory region of PAX2 promoter. Knocking down MZF1 or EGR-1 upregulated both the mRNA and protein level of PAX2 after 5-Aza-CdR treatment, which indicated that MZF1 and EGR-1 may act as repressive transcription factors in the DNA methylation related PAX2 transcription. Conclusion: PAX2 is involved in carcinogenesis of endometrial cancer by stimulating cell growth and promoting cell motility. Overexpression of PAX2 in endometrial cancer may be regulated by promoter hypermethylation via repressive transcription factors such as MZF1 or EGR-1. Citation Format: Nan Jia, Jieyu Wang, Qing Li, Xiang Tao, Keqin Hua, Yinhua Yu, Jan P. Baak, Kwong Kwok Wong, Weiwei Feng. Paired box 2 (PAX2) is involved in carcinogenesis and regulated by DNA methylation in endometrial cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4768. doi:10.1158/1538-7445.AM2015-4768

Metformin regulates stromal-epithelial cells communication via Wnt2/β-catenin signaling in endometriosis

In previous studies, we found that endometriotic stromal cells lose the ability to regulate cell survival signaling in endometriotic epithelial cells. Here, we invested the effect of Metformin on the stromal-epithelial cells crosstalk in endometriosis and explored the pathway that might be involved. We found that ectopic endometriotic stromal cells (ESC) expressed and secreted higher Wnt2 protein compared with normal endometrial stromal cells (NSC). Conditioned medium (CM) from ESC supplemented with Wnt2 antibody significantly inhibited the growth of normal endometrial epithelial cells (NEC), while CM from ESC per se showed no significant effect on the growth of NEC. Metformin decreased the expression and secretion of Wnt2 in ESC. CM from Metformin-pretreated ESC significantly inhibited the growth of NEC. In conclusion, Wnt2/β-catenin signaling was involved in stromal-epithelial cells interaction in endometriosis. Metformin might regulate the stroma-epithelium communication via Wnt2-mediated signaling in endometriosis.

Clinical significance of Sam68 expression in endometrial carcinoma.

Sam68 (Src-associated in mitosis of 68 kDa) is a substrate for tyrosine kinase c-Src during mitosis. The nuclear protein level has been found to be associated with progression and prognosis in various human malignant tumors. The aim of this study is to investigate the clinical value of Sam68 in endometrial carcinoma (EC). Sam68 expression was confirmed by real-time PCR, Western blot, and immunofluorescent assay in primary normal endometrial epithelial cells, endometrial carcinoma cell lines, as well as seven pairs of EC and matched adjacent noncancerous endometrial tissues. Moreover, the protein level of Sam68 was evaluated by immunohistochemistry in a cohort of surgical specimens derived from 131 patients including primary endometrial carcinoma (n = 95), endometrial atypical hyperplasia (precancerous lesions, n = 26), and normal endometria (n = 10). In endometrial cancer cell lines, RNA interfering approach was employed to downregulate Sam68 expression to determine its role in proliferation. Clinicopathological relevance and prognostic associations were examined by statistical analyses. Compared with normal endometrial and endometrial atypical hyperplasia tissues, Sam68 significantly elevated in endometrial cancer samples (P < 0.01), which was negative or low in 37 cases (38.9 %) and high in 58 cases (61.1 %). The high expression of Sam68 was associated with histological grade (P < 0.001), FIGO stage (P = 0.039), and myometrial invasion (P = 0.002). Kaplan-Meier analysis demonstrated that overexpression of Sam68 correlated with shorter overall survival. It is confirmed by univariate and multivariate analysis (P < 0.001 and P = 0.048, respectively). Additionally, we found that Sam68 was highly expressed at both the transcriptional and translational levels in endometrial cancer cell lines (Ishikawa, HEC-1B, AN3CA, KLE, and RL95-2) and siRNA knockdown of Sam68 remarkably inhibited cellular proliferation in in vitro models. Sam68 may be useful prognostic marker for EC, and it plays an important role in promoting the cellular proliferation. Further investigation of Sam68 as a potential therapeutic target for EC patients could be of interest.

Abstract 3326: Parallel EMT pathways mediated by epidermal growth factor, EpCAM and mesenchymal cadherins in benign endometriotic lesions and endometrial cancer

Abstract The endometrium is the major target of oncogenesis in the uterus. Endometrial tissue undergoes several cycles of disintegration and repair during women's reproductive years. The involvement of repair mechanisms that include transforming growth factor beta (TGF-beta) pathway may entail epithelial msenchymal transition (EMT) known to be involved in tissue repair. This suggests that the EMT program is inherent in endometrial epithelial cells and may be subverted during the development of invasive disease, whether benign such as endometriosis or malignant such as endometroid endometrial carcinoma. We therefore examined candidate mechanistic modulators of EMT in normal and malignant endometrial epithelial cells as well as endometrial carcinoma samples using cell signaling, gene expression and epigenetic regulation methodologies. Our evidence strikingly show that although both TGF-beta and epidermal growth factor (EGF) stimulated primary normal endometrial epithelial cell invasiveness, EGF was the main inducer of EMT in these cells. EGF also strongly induced EMT in endometrial cancer cells. In normal and malignant cells, Raf-1/MAPK mediated EGF actions. Interestingly, EGF stimulated epithelial adhesion molecule (EpCAM) cleavage and internalization of its intracellular domain EpICD into the nucleus to activate EMT-related genes such as mesenchymal cadherins. This was associated with loss of the epithelial marker E-cadherin and upregulation of its negative regulator Snail1. The involvement of EMT in aggressive endometrial malignancy was further confirmed in a permissive transcriptional epigenetic profile of mesenchymal cadherins in endometrial cancers with poor survival compared to a silenced epigenetic signature in less aggressive tumors. This epigenetic regulation including DNA methylation and histone modification was mediated by EpCAM, as shown in EpCAM-knockdown endometrial cancer cells, suggesting that EpCAM binding to target promoters maintains an open transcriptional conformation. Combined, our data suggest that while plasticity is required for endometrial regeneration during the menstrual cycles, committed epigenetic signatures may alter the path of normal tissue repair to aberrant growth associated with benign tumors or malignant growth. In concert, activation by cell signaling such as EGF/EpCAM may determine the extent of invasiveness and disease progression. Citation Format: Ya-Ting Hsu, Joseph Liu, Peter A. Binkley, Robert S. Schenken, Rajshwar R. Tekmal, Tim H.-M. Huang, Nameer B. Kirma. Parallel EMT pathways mediated by epidermal growth factor, EpCAM and mesenchymal cadherins in benign endometriotic lesions and endometrial cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3326. doi:10.1158/1538-7445.AM2014-3326

Cell Line Contamination and Misidentification

Geneva Foundation for Medical Education and Research, Versoix, SwitzerlandDear Editors:RE: Cell line contamination and misidentificationIn order to further knowledge in the area of reproductivebiology, endocrinology, and immunology in a meaningfulmanner, researchers should be aware of cell line contaminationand misidentification, which is commonplace [1, 2]. Last yearKorch et al. [3] reported that this is the case for a number ofwidely used endometrial and ovarian cell lines.I, therefore, wish to highlight two issues:1. The American Type Culture Collection (ATCC) Recom-mendations. The ATCC has recently published a Standard forhuman cell line authentication, recommending short tandemrepeat (STR) profiling for authentication of human cell lines toaddress this problem [4].2. The paucity of endometrial epithelial cell lines available.Korch et al. identified EEC1 cells as being Ishikawa cells,MCF-7 cells or a mixture of both; hTERT EEC cells,previously thought to be normal endometrial epithelial cells,as being MCF-7 breast cancer epithelial cells; and HES cells asHeLa cervical cancer cells.It is advisable to alert researchers in training to theimportance of not just authenticating new cell lines uponreceipt but taking all necessary technical precautions in order toprevent cell line contamination as well as evaluating cell linesfor phenotypic or functional traits, which can be altered withdifferentiation and increasing passages [5].Yours sincerely,Geraldine O. Canny Ph.D.REFERENCES

MicroRNA-182 Promotes Tumor Cell Growth by Targeting Transcription Elongation Factor A-like 7 in Endometrial Carcinoma

Background/Aims: Endometrial carcinoma (EC) is the most common gynecological malignancy among women worldwide. Despite its prevalence, the molecular mechanisms underlying endometrial carcinogenesis are poorly understood. The purpose of this study was to examine the role of microRNA-182 and its target gene transcription elongation factor A-like 7 (TCEAL7) in EC. Methods: The expression of miR-182 in human normal endometrial epithelial cells (NEEC) and in three human endometrial carcinoma cell lines (HEC-1B, RL95-2 and AN3CA) was measured by qRT-PCR, and the mRNA and protein expression of TCEAL7 were assessed in the same three endometrial carcinoma cell lines and NEEC by qRT-PCR and western blotting, respectively. Subsequently, the target of miR-182 was predicted by bioinformatics and confirmed using a luciferase assay. Cell proliferation and colony formation of RL95-2 cells were examined by MTT assay and crystal violet staining, respectively. The expression of NFκB-p65, c-Myc and cyclin D1 proteins was determined by Western blot analysis. Results: MiR-182 was significantly upregulated and TCEAL7 was downregulated in EC cell lines compared to NEEC. We showed that MiR-182 binds directly to a conserved 8 bp sequence in the 3'-UTR of TCEAL7, and inhibition of miR-182 upregulated TCEAL7 mRNA and protein expression to levels comparable to those induced by lentiviral-mediated overexpression of TCEAL7. MiR-182 inhibition decreased cell proliferation and colony formation ability, downregulated the expression of the pro-proliferative genes c-Myc and cyclin D1, and inhibited NFκB activation, and these effects were mimicked by TCEAL7 overexpression. Conclusions: miR-182 acts as an oncogenic miRNA in EC, promoting cell proliferation by targeting the tumor suppressor gene TCEAL7 and modulating the activity of its downstream effectors c-Myc, cyclin D1 and NFκB.

The Role of Progesterone and Its Receptor on Cyclin G1 Expression in Endometrial Carcinoma Cells

Cyclin G1 protein is expressed in normal endometrial epithelial cells in a progesterone-dependent manner. It is an important mediator of the inhibiting effect of progesterone on cell proliferation. Moreover, the expression of cyclin G1 is also found to be decreased in human endometrial carcinoma cells (ECCs). To study the mechanism of decrease in the expression levels of cyclin G1, 3 ECC cell lines, Ishikawa, HEC-1-B, and KLE cells were treated with progesterone (P(4)). The KLE cells, in which progesterone receptor (PR) expression was absent, were transfected with PR-expressing plasmid before treatment with P(4). The results showed that cyclin G1 expression increased in Ishikawa and HEC-1-B cells after treatment with P(4), additionally the cell proliferation was suppressed but not in KLE cells. When the PR-expressing plasmid was transfected into KLE cells, the effect of P(4) was restored. Our data suggest that the deficiency of progesterone and its receptors is an important cause of the decreased expression of cyclin G1 in endometrial carcinoma, which may account for carcinogenesis and development of endometrial carcinomas.

Re-establishment of gap junctional intercellular communication (GJIC) between human endometrial carcinomas by prostaglandin E2

Reduced intercellular communication via gap junctions is correlated with carcinogenesis. Gap junctional intercellular communication (GJIC), between normal human endometrial epithelial cells is enhanced when endometrial stromal cells were present in culture. This enhancement of GJIC between normal epithelial cells also occurs when they are cultured in medium conditioned by stromal cells. This observation indicated that a soluble compound (or compounds) produced and secreted by stromal cells mediates GJIC in epithelial cells. Previous studies have shown that endometrial stromal cells release prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) under physiological conditions. When we evaluated the response of normal endometrial epithelial cells to various concentrations of PGE2, we found enhanced GJIC with 1nM PGE2. This is a smaller increase in GJIC than that induced by medium conditioned by stromal cells. When the extracellular concentration of PGE2 was measured after incubation with stromal cells, it was found to be similar to the concentrations showing maximal GJIC between the normal epithelial cells. When indomethacin was used to inhibit prostaglandin synthesis by stromal cells, GJIC was reduced but not eliminated between normal endometrial epithelial cells. These observations suggest that although PGE2 secreted by stromal cells is an important mediator of GJIC between the epithelial cells, it is not the sole mediator. Transformed endometrial epithelial cells did not demonstrate GJIC even in the presence of stromal cells. However, we were able to re-establish GJIC in transformed epithelial cells when we added PGE2 to the cells. Our findings show that PGE2 may serve as an intercellular mediator between stromal and epithelial cells that regulates GJIC in normal and malignant epithelial cells. This suggests that maintenance of GJIC by preserving or replacing PGE2 secretion by endometrial stromal cells may have the potential to suppress carcinogenesis in endometrial epithelial cells.

Estrogen and Progesterone Regulate p27kip1 Levels via the Ubiquitin-Proteasome System: Pathogenic and Therapeutic Implications for Endometrial Cancer

The levels of proteins that control the cell cycle are regulated by ubiquitin-mediated degradation via the ubiquitin-proteasome system (UPS) by substrate-specific E3 ubiquitin ligases. The cyclin-dependent kinase inhibitor, p27kip1 (p27), that blocks the cell cycle in G1, is ubiquitylated by the E3 ligase SCF-Skp2/Cks1 for degradation by the UPS. In turn, Skp2 and Cks1 are ubiquitylated by the E3 ligase complex APC/Cdh1 for destruction thereby maintaining abundant levels of nuclear p27. We previously showed that perpetual proteasomal degradation of p27 is an early event in Type I endometrial carcinogenesis (ECA), an estrogen (E2)-induced cancer. The present studies demonstrate that E2 stimulates growth of ECA cell lines and normal primary endometrial epithelial cells (EECs) and induces MAPK-ERK1/2-dependent phosphorylation of p27 on Thr187, a prerequisite for p27 ubiquitylation by nuclear SCF-Skp2/Cks1 and subsequent degradation. In addition, E2 decreases the E3 ligase [APC]Cdh1 leaving Skp2 and Cks1 intact to cause p27 degradation. Furthermore, knocking-down Skp2 prevents E2-induced p27 degradation and growth stimulation suggesting that the pathogenesis of E2-induced ECA is dependent on Skp2-mediated degradation of p27. Conversely, progesterone (Pg) as an inhibitor of endometrial proliferation increases nuclear p27 and Cdh1 in primary EECs and ECA cells. Pg, also increases Cdh1 binding to APC to form the active E3ligase. Knocking-down Cdh1 obviates Pg-induced stabilization of p27 and growth inhibition. Notably, neither E2 nor Pg affected transcription of Cdh1, Skp2, Cks1 nor p27. These studies provide new insights into hormone regulation of cell proliferation through the UPS. The data implicates that preventing nuclear p27 degradation by blocking Skp2/Cks1-mediated degradation of p27 or increasing Cdh1 to mediate degradation of Skp2-Cks1 are potential strategies for the prevention and treatment of ECA.

The Role of DJ-1 in the Pathogenesis of Endometriosis

BackgroundEndometriosis is an estrogen-dependent disease causing pelvic pain and infertility in 10% of reproductive-aged women. Despite a long history of the disease the pathogenesis of endometriosis is poorly understood. It is known that the expression of several proteins is either up or down regulated during endometriosis, but their precise role remains to be determined. DJ-1 is one such protein that is upregulated in eutopic endometrium of women having endometriosis suggesting that DJ-1 may be involved in the pathogenesis of endometriosis.Methodology and Principal FindingsThe role of DJ-1 in the pathogenesis of endometriosis was investigated. For this purpose the influence of DJ-1 on endometrial cell survival, attachment, proliferation, migration, and invasion either by overexpressing DJ-1 in normal endometrial cells or by knocking down DJ-1 expression in endometriotic cells using siRNA was investigated. The results indicated that DJ-1 protects endometrial cells from oxidative stress mediated apoptosis. Overexpression of DJ-1 in normal endometrial epithelial cells increases the adhesion on collagen type IV. However, no significant difference was observed incase of stromal cells. It was further demonstrated that DJ-1 regulates cell proliferation, migration, and invasion in normal endometrial and endometriotic epithelial cells whereas in the case of normal endometrial and endometriotic stromal cells, it regulates cell proliferation and invasion but not migration. Furthermore, the present study also indicated that DJ-1 regulates these cellular processes by modulating PI3K/Akt pathway by interacting and negatively regulating PTEN.ConclusionsAbnormally high levels of DJ-1 expression may be involved in endometriosis, possibly by stimulating endometrial cell survival, proliferation, migration, and invasion.

Abstract 4596: Restoring hypoxia-inducible factor-1α in MCF-7 breast cancer cells in vitro by preventing the excessive degradation induced by high oxygen unmasks strong estradiol induction of vascular endothelial growth factor expression

Abstract Vascular endothelial growth factor (VEGF) is the most potent inducer of increased microvascular permeability known and the key mediator of angiogenesis, pivotal processes in development, reproduction, and tumor growth. VEGF expression is rapidly and strongly induced in normal uterine endometrial epithelial cells in vivo by 17β-estradiol (E2; Kazi et al. 2009 Biol Reprod 81:378). Recent studies indicate that E2-induced epithelial cell proliferation in the mammary gland is also dependent on VEGF. VEGF is also strongly up-regulated by E2 in rat mammary tumors in vivo (Nakamura et al. 1996 Endocrinology 137:5589). By contrast, numerous studies have found that E2 has little or no effect on VEGF expression by breast cancer cells in vitro. We have recently shown that the E2 induction of VEGF is mediated by both estrogen receptor α (ERα) and hypoxia-inducible factor 1α (HIF-1α; Kazi et al. 2005 Mol Endocrinol 19:2006; Molitoris et al. 2009 Endocrinology 150:5405). Cultured cells, however, usually lack HIF-1α due to excessive degradation caused by the abnormally high oxygen level (20%) to which they are exposed. To test the hypothesis that restoring HIF-1α would restore the ability of E2 to strongly induce VEGF expression in breast cancer cells, we treated MCF-7 cells with cobalt chloride (CoCl2; 12.5-100 µM), which prevents oxygen-induced HIF-1α degradation. HIF-1α was very low to undetectable in untreated MCF-7 cells grown in 20% O2, but increased in a dose-dependent manner in response to CoCl2. Although high doses of CoCl2 alone significantly stimulated VEGF mRNA expression, 12.5 µM did not do so. A modest increase in VEGF expression was also seen when MCF-7 cells were treated with E2 alone (∼ 2.5-fold). When cells were co-treated with E2 and 12.5 µM CoCl2, however, a much larger increase in VEGF expression (∼ 6-fold), similar to that induced by E2 in vivo, was seen. This finding is consistent with the hypothesis that HIF-1α is required for maximal E2 induction of VEGF expression in carcinoma cells. Interestingly, when cells were grown for 4 days before treatment, high levels of HIF-1α protein were present in untreated cells. The tendency for MCF-7 cells to express HIF-1α as they become crowded in vitro, even in the presence of high oxygen levels, may explain the ability of E2 alone to induce some VEGF expression in vitro as observed here and in a few previous studies. In conclusion, these results confirm that HIF-1 plays an essential role in E2-induced VEGF expression. The rapid induction of VEGF expression by E2 and the increased microvascular permeability that results may play a critical role in tumor growth by exposing cancer cells to circulating IGF-1, which synergizes with E2 to induce mitosis [Supported by NICHD SCCPIR U54 HD36207 and R01 HD047275]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4596.

Abstract 3885: Mechanisms that mediate the increase in p27kip1 by progestins: Therapeutic implications for endometrial carcinoma

Abstract The steroid hormones, estrogen (E2) and progestins (Pg) regulate growth of the endometrium. E2 stimulates and Pg inhibits endometrial epithelial cell (EEC) proliferation. The importance of these hormones in controlling EEC growth is underscored by E2-induction of type I endometrial cancer (ECA; 85% of all ECAs), and by the therapeutic effect of Pg on causing regression of both endometrial hyperplasia and ECA. The cyclin-dependent kinase inhibitor, p27kip1 (p27) arrests cells in G1 phase of the cell cycle by blocking Cdk2 activity. We previously showed that nuclear p27 is lost in the glands of ECA tissue due to a high rate of p27 degradation via the ubiquitin-proteasome pathway. In vitro, using primary EECs derived from hysterectomies, we also showed that E2 induces proteasomal degradation of p27. Therefore, together with the lack of nuclear p27 in endometrial hyperplasia tissue, p27 degradation appears to be an early critical molecular event involved in the pathogenesis of E2-induced ECA. In contrast and importantly, we show in vitro that 100 nM Pg markedly increases p27 in primary normal EECs and ECA cells. Moreover, the PR antagonist, RU486, completely blocks the increase in nuclear p27 and sequesters p27 in the cytoplasm in EECs suggesting that Pg might act by enabling nuclear-cytoplasmic shuttling, which is abrogated by RU486. In addition, we propose that the therapeutic effect of Pg on endometrial hyperplasia and ECA is through Pg-induction and nuclear localization of p27. Indeed we show that patients responding to Pg [Megace®] therapy with histological regression of disease demonstrate a concomitant increase in nuclear p27. To understand the mechanisms involved in upregulation of p27 by Pg, we investigated mRNA and protein regulation of p27 by Pg in EECs. By real-time PCR, Pg does not increase mRNA transcription of p27 (time points=0.5 to 12 hr). However, Pg induced Cdh1 protein, the E3 ligase for Cks1 and thus, decreased Cks1, a rate limiting co-factor that mediates proteasomal degradation of p27, thereby simultaneously increasing nuclear p27. These effects were reversed by RU486 and the decrease in Cks1 was reversed by the proteasome inhibitor, lactacystin. Thus, we show that through the PR, Pg increases p27 by preventing its degradation by Cks1 through Pg-induced upregulation of Cdh1. Finally, whereas Pg induced growth inhibition of EECs by 25%, the addition of normal stromal cells (NSFs) in co-culture further mediated growth inhibition of EECs by 75% over untreated controls. In contrast, co-cultures of stromal cells derived from ECA tissue (CAFs) did not have this effect. Similarly, conditioned media from CAFs, but not NSFs stimulated EEC growth with a concomitant decrease in p27 and increase in Cks1. These studies reveal mechanisms of how Pg acts as a therapeutic agent to control endometrial growth through increased p27 nuclear localization involving participation by normal adjacent stroma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3885.

Synergistic anti-neoplastic effect of AG1478 in combination with cisplatin or paclitaxel on human endometrial and ovarian cancer cells

AG1478, a potent inhibitor of epidermal growth factor receptor (EGFR), facilitates the induction of cell death in combination with a variety of cytotoxic and chemotherapeutic agents in certain human tumor cell lines. The purpose of this study was to elucidate the effect of AG1478 on three endometrial cancer and two ovarian cancer cell lines as compared to normal human endometrial epithelial cells. Cells were treated with various concentrations of AG1478 alone or in combination with the chemotherapeutic drugs cisplatin or paclitaxel, and the effect of AG1478 on cell growth, the cell cycle and apoptosis was investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed that all the cancer cell lines were sensitive to the growth-inhibitory effect of AG1478. Normal endometrial epithelial cells remained viable after treatment with AG1478 at the same doses as those which induced growth inhibition in the endometrial and ovarian cancer cells. Synergistic anti-neoplastic effects were obtained with a combination of AG1478 and cytostatic drugs. Cell-cycle analysis indicated that exposure to these drugs decreased the proportion of cells in the S-phase and increased the proportion in the sub G0/G1 fractions of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and by loss of mitochondrial transmembrane potential. These results suggest that AG1478 alone or in combination with chemotherapeutic drugs may be a novel therapeutic option for the treatment of endometrial and ovarian cancer.

PK11195 enhances chemosensitivity to cisplatin and paclitaxel in human endometrial and ovarian cancer cells

The potential anticancer agent 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a translocator protein ligand, facilitates the induction of cell death by a variety of cytotoxic and chemotherapeutic agents in various human tumor cell lines. The purpose of this study was to elucidate the effect of PK11195 on three endometrial cancer cell lines, two ovarian cancer cell lines and normal human endometrial epithelial cells. Endometrial and ovarian cancer cells were treated with various concentrations of PK11195 alone or in combination with chemotherapeutic drugs (cisplatin, paclitaxel), and its effect on cell growth, the cell cycle, apoptosis and related measurements was investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed that all endometrial and ovarian cancer cell lines were sensitive to the growth-inhibitory effect of PK11195, although normal endometrial epithelial cells were viable after treatment with the same doses of PK11195 that induced growth inhibition in endometrial and ovarian cancer cells. Synergistic anti-neoplastic effects were obtained by a combination of PK11195 with cytostatic drugs. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with altered expression of genes related to apoptosis. These results suggest that PK11195 alone or in combination with chemotherapeutic drugs might be a new therapeutic option for the treatment of endometrial and ovarian cancers.

RETRACTED: Erucylphosphocholine shows a strong anti-growth activity in human endometrial and ovarian cancer cells

A membrane-targeted, lipophilic ether lipid of synthetic phospholipid analog, erucylphosphocholine (ErPC) induces apoptosis in some lines of human tumor cells. We investigated the effect of ErPC on three endometrial cancer cell lines, two ovarian cancer cell lines, and normal human endometrial epithelial cells. Endometrial and ovarian cancer cells were treated with various concentrations of ErPC, and its effect on cell growth, cell cycle, apoptosis, and related measurements was investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that all endometrial and ovarian cancer cell lines were sensitive to the growth-inhibitory effect of ErPC, although normal endometrial epithelial cells were viable after treatment with the same doses of ErPC that induced growth inhibition in endometrial and ovarian cancer cells. Cell cycle analysis indicated that their exposure to ErPC decreased the proportion of cells in the S-phase and increased the proportion in the G2/M phases of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with altered expression of genes related to cell growth, malignant phenotype, and apoptosis. These results suggest that the anticancer activity of ErPC may occur with higher sensitivity of cancer cells compared with normal healthy cells, when using low concentration, rising hopes that ErPC may become a useful adjuvant therapy for endometrial and ovarian cancers.

Bufalin induces growth inhibition, cell cycle arrest and apoptosis in human endometrial and ovarian cancer cells

Bufalin is a traditional Chinese medicine and it induces apoptosis in certain human tumor cell lines. We investigated the effect of bufalin on three endometrial cancer cell lines, two ovarian cancer cell lines, and on normal human endometrial epithelial cells. Endometrial and ovarian cancer cells were treated with various concentrations of bufalin, and its effect on cell growth, cell cycle, apoptosis, and related measurements was investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that all endometrial and ovarian cancer cell lines were sensitive to the growth-inhibitory effect of bufalin, although normal endometrial epithelial cells were viable after treatment with the same doses of bufalin that induced growth inhibition in endometrial and ovarian cancer cells. Cell cycle analysis indicated that their exposure to bufalin decreased the proportion of cells in the S-phase and increased the proportion in the G0/G1 phases of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with the altered expression of genes related to cell cycle and apoptosis. These results suggest that bufalin may become a useful adjuvant therapy for endometrial and ovarian cancers with minimal side effects.

Utility of thin‐layer preparations in endometrial cytology: Immunocytochemical expression of PTEN, beta‐catenin and p53 for benign endometrial lesions

This article focuses on the characteristic features of morphology and molecular biology of PTEN, beta-catenin, and p53 immunocytochemistry in normal endometrium (proliferative, secretory, and atrophic) and endometrial glandular and stromal breakdown (EGBD) using thin-layer specimens. During a 6-month period, 120 endometrial samples were collected directly using the Uterobrush and a thin-layer specimen was prepared. Immunocytochemical expression of PTEN, beta-catenin, and p53 were investigated using 30 cases each of proliferative endometrium (PE), secretory endometrium (SE), atrophic endometrium (AE), and EGBD.PTEN expression of normal endometrial glandular epithelial cells changes with the hormonal status; PE produce very high expression, SE creates attenuation or disappearance of PTEN expression and AE diminished more in comparison with SE. PTEN expression of EGBD showed a tendency towards attenuation compared with PE, but showed high expression in comparison with SE and AE. As for the immunoreactivity of beta-catenin, in all phases (PE, SE, AE, and EGBD), it was observed in the cytoplasm of glandular epithelial cells, but not nuclei, and showed strong membranous staining. As for the p53 immunoreactivity, p53 positivity was not observed in the glandular epithelial cells in all phases (PE, SE, AE, and EGBD) with the exception of some metaplastic cells. The presence of p53 immunoreactivity of a weak, low ratio in metaplastic cells was unexpected. In the current study, the expression manner of PTEN, beta-catenin, and p53 immunocytochemistry was observed in the normal endometrium (PE, SE, and AE) and EGBD.

K252a is highly effective in suppressing the growth of human endometrial cancer cells, but has little effect on normal human endometrial epithelial cells

The furanosylated indolocarbazole K252a belongs to a family of microbial alkaloids that also includes staurosporine, which is known to inhibit proliferation, stimulate apoptosis and induce the cell cycle arrest of cancer cells. To elucidate the involvement of K252a in endometrial cancer, we investigated the effects of K252a on three endometrial cancer cell lines. Endometrial cancer cells were treated with K252a and its effect on cell growth, cell cycle and related measurements was assessed. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed that the endometrial cancer cell lines were sensitive to the growth inhibitory effect of K252a, although normal endometrial epithelial cells were viable after treatment with the same doses of K252a that induced the growth inhibition of endometrial cancer cells. Cell cycle analysis indicated that their exposure to K252a decreased the proportion of cells in the S phase and increased the proportion of cells in the G0/G1 phase of the cell cycle. TUNEL assays demonstrated that K252a induced apoptosis. This occurred in concert with an altered expression of p21WAF1 and bcl-2 proteins related to the G0/G1 phase of the cell cycle and apoptosis. These results raise the possibility that K252a may prove to be particularly effective in the treatment of endometrial cancers.

β-Hydroxyisovalerylshikonin has a profound anti-growth activity in human endometrial and ovarian cancer cells

Objectives β-Hydroxyisovalerylshikonin (β-HIVS), a compound isolated from the traditional oriental medicinal herb Lithospermum radix, is an ATP non-competitive inhibitor of protein-tyrosine kinases, such as v-Src and EGFR, and it induces apoptosis in some lines of human tumor cells. We investigated the effect of β-HIVS on three endometrial cancer cell lines, two ovarian cancer cell lines, and normal human endometrial epithelial cells. Methods Endometrial and ovarian cancer cells were treated with various concentrations of β-HIVS, and its effect on cell growth, cell cycle, apoptosis, and related measurements was investigated. Results The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that all endometrial and ovarian cancer cell lines were sensitive to the growth-inhibitory effect of β-HIVS, although normal endometrial epithelial cells were viable after treatment with the same doses of β-HIVS that induced growth inhibition in endometrial and ovarian cancer cells. Cell-cycle analysis indicated that their exposure to β-HIVS decreased the proportion of cells in the S phase and increased the proportion in the G0/G1 phases of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with altered expression of genes related to cell growth, malignant phenotype, and apoptosis. Conclusions These results suggest that the anticancer activity of β-HIVS may occur with higher sensitivity of cancer cells compared with normal healthy cells, when using low concentration, rising hopes that β-HIVS may become a useful adjuvant therapy for endometrial and ovarian cancers.

Human Endometrial and Ovarian Cancer Cells: Histone Deacetylase Inhibitors Exhibit Antiproliferative Activity, Potently Induce Cell Cycle Arrest, and Stimulate Apoptosis

Because epigenetic alterations are believed to be involved in the repression of tumor suppressor genes and promotion of tumorigenesis in endometrial cancers and ovarian cancers, novel compounds endowed with a histone deacetylase (HDAC) inhibitory activity are an attractive therapeutic approach. Clonogenic assay in soft agar and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed that many endometrial and ovarian cancer cell lines were sensitive to the growth inhibitory effect of HDAC inhibitors (HDACIs), although normal endometrial epithelial cells were viable after the treatment with the same doses of HDACIs that induced growth inhibition of endometrial and ovarian cancer cells. Cell cycle analysis indicated that their exposure to HDACIs decreased the proportion of cells in the S-phase and increased the proportion in the G0/G1 phases and/or G2/M phases of the cell cycle. Induction of apoptosis was confirmed by TUNEL assay, annexin V staining of externalized phosphatidylserine, and loss of the transmembrane potential of mitochondria. This induction occurred in concert with altered expression of genes related to cell growth, malignant phenotype, and apoptosis. In nude mice experiments, valproic acid significantly inhibited human endometrial and ovarian tumor growth without toxic side-effects. Although there are few clinical trials on these cancers, some clinical trials showed that HDACIs in well tolerated doses have significant antitumoral activities in another cancers. These results raise the possibility that HDACIs may prove particularly effective in the treatment of endometrial cancers and ovarian cancers.