Ishikawa Endometrial Adenocarcinoma Cells Research Articles

Cardiac Glycoside Oleandrin Suppresses EMT Ability in Endometrial Carcinoma Cells.

Endometrial carcinoma is one of the most common types of cancer among women. The progression of cancer occurs via the Epithelial- Mesenchymal Transition (EMT) pathway. Cells lose their epithelial properties and become mobile. For this reason, the EMT process is one of the most important step to be targeted in cancer treatment. Oleandrin is a cardiac glycoside and its use is limited due to its narrow therapeutic index. In this study, we aimed to evaluate effects of lower level Oleandrin doses on EMT process in endometrial carcinoma. Oleandrin was administrated to Ishikawa endometrial adenocarcinoma cells at different doses and times. IC50 dose was determined by XTT proliferation test. Expression analysis of EMT-related genes was then performed by qRT-PCR. Invasion and colony formation abilities of cells were examined microscopically. Finally, the migration analysis of cancer cells was determined by the Wound Healing Assay. The IC50 dose of Oleandrin applied to Ishikawa cells was determined as 75.3 nM at the 48 h. According to qRT-PCR analysis, expression levels of ZEB1, FN1, ITGB1, VIM, SMAD2, SNAI1, SNAI2, SNAI3, and TGFB3 genes significantly decreased, but TIMP2, TIMP3, ITGAV and GSK3B genes significantly increased. In addition, Oleandrin significantly reduced colony formation and invasion of Ishikawa cells. According to the Wound Healing analysis, the migratory abilities of the Oleandrin-treated cells were reduced compared to the control. Low dose Oleandrin suppresses the EMT pathway in Ishikawa cells. It has been shown that Oleandrin significantly suppresses the cell's colony formation, invasion and migration ability both in gene expression analyzes and microscopically.

Blocking PARP activity with the inhibitor veliparib enhances radiotherapy sensitivity in endometrial carcinoma.

ObjectiveOur study aimed to investigate the potential clinical utility of a poly(ADP‐ribose) polymerase (PARP) inhibitor, veliparib (ABT‐888), as a radiosensitizer in the medication of endometrial carcinoma (EC).MethodsHuman Ishikawa endometrial adenocarcinoma cells were treated with veliparib, radiotherapy (RT), or combination treatment. The viabilities, radiosensitivity enhancement ratio (sensitizer enhancement ratio (SER), and apoptosis of Ishikawa cells were, respectively, evaluated by Cell Counting Kit‐8 (CCK‐8), colony formation experiment, and flow cytometry. The tumor growth was assessed by xenograft mice models. Western blot assay investigated the expression of DNA damage and apoptosis‐related proteins in vivo and in vitro.ResultsCell Counting Kit‐8 revealed that the 10% inhibition concentration (IC10) and 50% inhibition concentration (IC50) values of veliparib‐treated Ishikawa cells were 1.7 and 133.5 µM, respectively. The SER of veliparib combined with RT was 1.229 in vitro. Flow cytometry analysis results indicated that the apoptosis rate of the veliparib + RT group was markedly higher than that of the RT group in vitro (p < 0.05). Furthermore, in vivo data revealed that veliparib + RT treatment significantly decreased tumor growth compared with single treatments of veliparib or RT and with the control group (p < 0.05). Then western blot confirmed the levels of anti‐phospho‐histone (γH2AX), caspase‐3, and B‐cell lymphoma 2 (Bcl‐2) associated protein X (Bax) were significantly higher in the veliparib + RT group, while the level of Bcl‐2 was lower compared with that of the RT group (p < 0.05), both in vivo and in vitro.ConclusionOur results indicate that veliparib in combination with RT markedly improved the therapeutic efficiency in human endometrial carcinoma.

Manipulating Estrogenic/Anti-Estrogenic Activity of Triphenylethylenes towards Development of Novel Anti-Neoplastic SERMs.

Selective estrogen receptor modulators (SERMs) act as estrogen receptor (ERα) agonists or antagonists depending on the target issue. Tamoxifen (TAM) (a non-steroidal triphenylethylene derivative) was the first SERM approved as anti-estrogen for the treatment of metastatic breast cancer. On the hunt for novel SERMs with potential growth inhibitory activity on breast cancer cell lines yet no potential to induce endometrial carcinoma, we designed and synthesized 28 novel TAM analogs. The novel analogs bear a triphenylethylene scaffold. Modifications on rings A, B, and C aim to attenuate estrogenic/anti-estrogenic activities of the novel compounds so they can potentially inhibit breast cancer and provide positive, beneficial estrogenic effects on other tissues with no risk of developing endometrial hyperplasia. Compound 12 (E/Z-1-(2-{4-[1-(4-Chloro-phenyl)-2-(4-methoxy-phenyl)-propenyl]-phenoxy}-ethyl)-piperidine) showed an appreciable relative ERα agonistic activity in a yeast estrogen screen (YES) assay. It successfully inhibited the growth of the MCF-7 cell line with GI50 = 0.6 µM, and it was approximately three times more potent than TAM. It showed no potential estrogenicity on Ishikawa endometrial adenocarcinoma cell line via assaying alkaline phosphatase (AlkP) activity. Compound 12 was tested in vivo to assess its estrogenic properties in an uterotrophic assay in an ovariectomized rat model. Compared to TAM, it induced less increase in wet uterine wet weight and showed no uterotrophic effect. Compound 12 is a promising candidate for further development due to its inhibition activity on MCF-7 proliferation with moderate AlkP activity and no potential uterotrophic effects. The in vitro estrogenic activity encourages further investigations toward potential beneficial properties in cardiovascular, bone, and brain tissues.

Toros Göknarı (Abies cilicica (Antoine &amp; Kotschy) Carrière) ’ndan Elde Edilen Reçinenin In vitro Antioksidan ve Sitotoksik Aktivitelerinin Belirlenmesi

In this study, the antioxidant and cytotoxic activities of resin obtained from the Cilician Fir plant were evaluated. According to data, the in vitro antioxidant activity of the resin has changed depending on the radical scavenging activity method. This resin has antioxidant activity according to 2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging assay. The in vitro cytotoxic activity of the resin was investigated against a panel of human cancer cells (MDA-MB-231, Hep G2, PC-3, U-87, MCF-7, HT-29) with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for 48 h. Normal human lung fibroblast cells (WI-38) were used as healthy cells. The results indicated that the in vitro cytotoxic activity of the resin depends on the cell line type and concentration of the resin. According to the IC50 values, the resin has the most cytotoxic activity on endometrial adenocarcinoma cancer cells (IC50=8.94 ± 0.03 µg mL-1) compared to other cancer cells. The results also indicated that Ishikawa endometrial adenocarcinoma cells, which have Selectivity Index (SI) value &amp;gt;2, have the most sensitivity against the resin. This study provides the first evidence that the resin inhibits the different cancer cells' growth.

Electroporation of Ishikawa cells: analysis by flow cytometry.

Electroporation facilitates loading of cells with molecules and substances that are normally membrane impermeable. Flow cytometry is used in this study to examine the effects of the application of electroporation-level monopolar electric field pulses of varying electrical field strength on Ishikawa endometrial adenocarcinoma cells. Analysis of the fluorescence versus forward scatter plots corroborates the well-recognised threshold and cell size dependence characteristics of electroporation, but also shows the progression of cell lysis and generation of particulate material. Two 500 µs monopolar rectangular pulses ranging from 1.0 × 105 to 2.5 × 105 V/m were used to electroporate the cells. Electroporation yields (fraction of viable cells exhibiting significant propidium iodide uptake) ranged from 0 to 97%, with viability ranging between 78 and 34% over the electric field strength range tested. The higher electric field strength pulses not only reduced cell viability, but also generated a substantial amount of sub-cellular sized particulate material indicating cells have been physically disrupted enough to create these particles.

The cigarette smoke components induced the cell proliferation and epithelial to mesenchymal transition via production of reactive oxygen species in endometrial adenocarcinoma cells

Cigarette smoke (CS) causes about 480,000 deaths each year worldwide and is well-known to have harmful effects on the human body, leading to heart disease, stroke, lung cancer, and cardiovascular problems. In the present study, the effects of acrylonitrile (AN), benzo(a)pyrene (B(a)P), formaldehyde (FOR), isoprene (ISO), nicotine-derived nitrosamine ketone (NNK), which are the main components of CS, on the proliferation, invasion, and the epithelial-mesenchymal transition (EMT) process of human Ishikawa endometrial adenocarcinoma cells were investigated. Treating Ishikawa cells with CS components resulted in increased cell growth and altered expression of cell cycle-related genes: the protein expression of cyclin D & E increased, while the levels of p21 & p27 were reduced following treatment of these five CS components. In addition, CS components increased the invasion capacity of Ishikawa cells. The expression of the epithelial markers, E-cadherin and occludin, were significantly decreased, while the expression of the mesenchymal marker, N-cadherin, was significantly increased by CS components. In dichloro-dihydro-fluorescein diacetate (H2DCF-DA) assay, ROS production increased by treatment of CS components. The CS components activated the ROS-p38 MAPK-EMT pathway by increasing the level of phosphorylated p38 MAPK and p44/42 (ERK1/2), and by up-regulating Snail and Slug, the transcription factors for EMT. Taken together, these results indicate that CS components can promote progression of endometrial adenocarcinoma via increasing cell proliferation and the ROS-mediated EMT process.

In vitro evidence that platelet-rich plasma stimulates cellular processes involved in endometrial regeneration.

The study aims to test the hypothesis that platelet-rich plasma (PRP) stimulates cellular processes involved in endometrial regeneration relevant to clinical management of poor endometrial growth or intrauterine scarring. Human endometrial stromal fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone marrow-derived mesenchymal stem cells (BM-MSC), and Ishikawa endometrial adenocarcinoma cells (IC) were cultured with/without 5% activated (a) PRP, non-activated (na) PRP, aPPP (platelet-poor-plasma), and naPPP. Treatment effects were evaluated with cell proliferation (WST-1), wound healing, and chemotaxis Transwell migration assays. Mesenchymal-to-epithelial transition (MET) was evaluated by cytokeratin and vimentin expression. Differential gene expression of various markers was analyzed by multiplex Q-PCR. Activated PRP enhanced migration of all cell types, compared to naPRP, aPPP, naPPP, and vehicle controls, in a time-dependent manner (p < 0.05). The WST-1 assay showed increased stromal and mesenchymal cell proliferation by aPRP vs. naPRP, aPPP, and naPPP (p < 0.05), while IC proliferation was enhanced by aPRP and aPPP (p < 0.05). There was no evidence of MET. Expressions of MMP1, MMP3, MMP7, and MMP26 were increased by aPRP (p < 0.05) in eMSC and eSF. Transcripts for inflammation markers/chemokines were upregulated by aPRP vs. aPPP (p < 0.05) in eMSC and eSF. No difference in estrogen or progesterone receptor mRNAs was observed. This is the first study evaluating the effect of PRP on different human endometrial cells involved in tissue regeneration. These data provide an initial ex vivo proof of principle for autologous PRP to promote endometrial regeneration in clinical situations with compromised endometrial growth and scarring.

LRIG2 is a growth suppressor of Hec-1A and Ishikawa endometrial adenocarcinoma cells by regulating PI3K/AKT- and EGFR-mediated apoptosis and cell-cycle

Although endometrial cancer is the most common type of gynecological malignancy in developed countries, its molecular etiology is not well understood. Leucine-rich repeat and immunoglobulin-like domain 2 (LRIG2) is an evolutionarily conserved gene, but its functions in the endometrium are unknown. In this study, we found that LRIG2 is highly downregulated in endometrial adenocarcinoma patients and that it functions as a tumor suppressor. LRIG2 induced the mitochondrion-mediated apoptotic pathways by regulating stoichiometric balance among BCL-2 family proteins, whereby pro-survival members, MCL-1 and BCL-xL, were downregulated and pro-apoptotic BAK and BAX were upregulated. LRIG2 also inhibited proliferation of the Hec-1A and Ishikawa endometrial adenocarcinoma cells by upregulating p21. LRIG2 induced BAX- and BAK-dependent cell death that was efficiently prevented by MCL-1 overexpression. Furthermore, we found that LRIG2 unexpectedly phosphor-activates phosphoinositide 3-kinase (PI3K)/AKT and epidermal growth factor receptor (EGFR), which are conventionally accepted as survival signaling cues in diverse types of cancer. We observed that PI3K/AKT and EGFR serve as key kinases that have roles as growth suppressors of Hec-1A endometrial cancer cells by mediating the LRIG2-induced modulation of the BCL-2 family of proteins and p21. In vivo delivery of antisense DNAs against LRIG2 promoted the Hec-1A endometrial tumor growth in a xenograft mouse model, and immunoblotting of these tumor extracts showed consistent modulation of AKT, EGFR, the BCL-2 family members, and p21. Thus, our results demonstrated that LRIG2 is a growth suppressor of endometrial adenocarcinoma cells.

Proliferation and metastatic potential of endometrial cancer cells in response to metformin treatment in a high versus normal glucose environment.

In order to improve our understanding of the potential preventive and therapeutic role of metformin, the present study aimed to investigate the capability of low-dose metformin in the efficient inhibition of cancer development and the reduction of the metastasis of endometrial adenocarcinoma type I and primary endometrial epithelial cells (eEPs), with the drug acting as a treatment in a hyperinsulinemic environment exposed to high and normal glucose conditions. The Ishikawa endometrial adenocarcinoma cell line and primary eEPs were exposed to an environment with high (17 mM) or normal glucose (5 mM) and treated with insulin, low-dose metformin (0.1 mM) or a combined treatment. Metastatic potential was assessed by migration and invasion assays, and relative cell proliferation was determined. Metformin at a low dose potently inhibited the insulin action, decreasing the ability of the endometrial cancer (EC) cell line to migrate and invade in a high and normal glucose environment, and decreasing the migration ability of the primary eEPs. In the EC cell line, the insulin treatment increased the proliferation, without any subsequent reduction of proliferation by the addition of 0.1 mM metformin; however, relative cell proliferation sensitivity to metformin was observed in the range between 1 and 5 mM regardless of the glucose concentration present. Overall, metformin at 0.1 mM is not efficient enough to decrease the proliferation in an EC cell line. However, at this concentration, metformin can inhibit the insulin action in endometrial epithelial cancer cells, demonstrating an anti-metastatic effect in high and normal glucose environments.

The anti-proliferative effect of 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b) pyran is potentiated via induction of estrogen receptor beta and p21 in human endometrial adenocarcinoma cells

In an effort to develop novel therapeutic agents for endometrial cancer, benzopyran derivatives synthesized at our institute display significant inhibitory activity on cellular growth in uterine cancer cells. The current study was undertaken to demonstrate and explore the estrogen receptor (ER) subtype mediated mechanism of action of benzopyran derivative 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b) pyran (K-1) in human endometrial cancer cells. K-1 competitively inhibited the estradiol binding to human ERα and ERβ and showed growth inhibitory activity in human endometrial Ishikawa, HEC1B and primary endometrial adenocarcinoma cells. Transient transactivation assays carried out in COS-1 cells have demonstrated the diminished ERα-ERE mediated- and induced the ERβ-ERE mediated-transactivation triggered by compound. It also induced ER-mediated transactivation of the cyclin-dependent kinase inhibitor (CDKI) p21WAF-1 in both COS-1 cells and in Ishikawa cells. ERβ inducing effects of compound were blocked by ICI182,780. In endometrial adenocarcinoma cells, it induced ERβ and p21 expression significantly whereas the expression of fos, jun and ERα were significantly reduced. In addition, compound promoted ERα–β heterodimerization as observed in Ishikawa cells. These results demonstrate that the benzopyran compound suppressed the cellular growth via ERβ agonism, induction of p21 and via promoting the ERα–β heterodimerization, in addition to its antagonistic effects exerted on ERα, in human endometrial cancer cells. The study suggests that the dual action of benzopyran molecule may be of significant therapeutic value in ERα/β-positive cases of endometrial cancer.

Altered Claudin-4 Expression in Progesterone-Treated Endometrial Adenocarcinoma Cell Line Ishikawa

To detect the expression change of claudin-4 in Ishikawa endometrial adenocarcinoma cell line in response to progesterone. To determine whether claudin-4 is involved in the anticancer effect of progesterone. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the 50% inhibitory concentration (IC50) of megestrol acetate (MA) in treating Ishikawa cells. After the Ishikawa cells were treated with MA at IC50, cell apoptosis was examined by flow cytometry and transmission electron microscopy. The messenger RNA and protein expression levels of claudin-4 were further quantified by real-time polymerase chain reaction and Western blot. The localization of claudin-4 was examined by immunofluorescent staining. The IC50 of MA on Ishikawa cells was 15 mg/L incubated for 72 hours. Apoptosis percentage was elevated from 0.07% ± 0.02% to 3.93% ± 0.81% after MA treatment. The expression of claudin-4 at both protein and messenger RNA levels was significantly decreased after the treatment of MA (P < 0.05). The localization of claudin-4 transferred from cytomembrane to cytoplasm and nucleus. Megestrol acetate can inhibit the growth of Ishikawa cells. It may work through decreasing claudin-4 expression and cell apoptosis. The localization change of claudin-4 may also be involved in the anticancer effect of progesterone.

2,3-Diaryl-2H-1-benzopyran derivatives interfere with classical and non-classical estrogen receptor signaling pathways, inhibit Akt activation and induce apoptosis in human endometrial cancer cells

Objectives The present study was undertaken to explore the mechanism of anti-proliferative action of benzopyran compound D1 (2-[piperidinoethoxyphenyl]-3-phenyl-2H-benzopyran) and its hydroxy-(D2) and methoxy-(D3) derivatives in Ishikawa and human primary endometrial adenocarcinoma cells. Methods Transcriptional activation assays were performed using luciferase reporter system and cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The stage of cell cycle was determined by flow-cytometry and real time analysis of cyclinE1 and cdc2 genes. The apoptotic effects were measured by AnnexinV/PI staining and TUNEL. The expression of PCNA, cyclinD1, pAkt, XIAP, cleaved caspase-9, -3, PARP, Bax and Bcl2 were determined by immunoblotting. The caspase-3 activity and mitochondrial membrane potential were measured by colorimetric assay. Results All three compounds inhibited E 2-induced ERE- and AP-1-mediated transactivation and proliferation in endometrial adenocarcinoma cells dose-dependently. Compound D1 caused the arrest of cells in the G 2 phase while D2 and D3 caused arrest in G 1 phase of the cell cycle. All compounds interfered with Akt activation, decreased XIAP expression leading to an increased cleavage of caspase-9, -3, PARP, increased Bax/Bcl2 ratio and caspase-3 activity. Conclusion Findings suggest that benzopyran derivatives inhibit cellular proliferation via modulating ER-dependent classical and non-classical signaling mechanisms, interfere with Akt activation and induce apoptosis via intrinsic pathway in endometrial adenocarcinoma cells.

Hypoxia and prostaglandin E receptor 4 signalling pathways synergise to promote endometrial adenocarcinoma cell proliferation and tumour growth.

The prostaglandin endoperoxide synthase (PTGS) pathway is a potent driver of tumour development in humans by enhancing the biosynthesis and signalling of prostaglandin (PG) E2. PTGS2 expression and PGE2 biosynthesis is elevated in endometrial adenocarcinoma, however the mechanism whereby PTGS and PGE2 regulate endometrial tumour growth is unknown. Here we investigated (a) the expression profile of the PGE synthase enzymes (PTGES, PTGES-2, PTGES-3) and PGE receptors (PTGER1–4) in endometrial adenocarcinomas compared with normal endometrium and (b) the role of PTGER4 in endometrial tumorigenesis in vivo. We found elevated expression of PTGES2 and PTGER4 and suppression of PTGER1 and PTGER3 in endometrial adenocarcinomas compared with normal endometrium. Using WT Ishikawa endometrial adenocarcinoma cells and Ishikawa cells stably transfected with the full length PTGER4 cDNA (PTGER4 cells) xenografted in the dorsal flanks of nude mice, we show that PTGER4 rapidly and significantly enhances tumour growth rate. Coincident with enhanced PTGER4-mediated tumour growth we found elevated expression of PTGS2 in PTGER4 xenografts compared with WT xenografts. Furthermore we found that the augmented growth rate of the PTGER4 xenografts was not due to enhanced angiogenesis, but regulated by an increased proliferation index and hypoxia. In vitro, we found that PGE2 and hypoxia independently induce expression of PTGER4 indicating two independent pathways regulating prostanoid receptor expression. Finally we have shown that PGE2 and hypoxia synergise to promote cellular proliferation of endometrial adenocarcinoma cells.

Genistein effects on stromal cells determines epithelial proliferation in endometrial co-cultures

Background Estrogen is the leading etiologic factor for endometrial cancer. Estrogen-induced proliferation of endometrial epithelial cells normally requires paracrine growth factors produced by stromal cells. Epidemiologic evidence indicates that dietary soy prevents endometrial cancer, and implicates the phytoestrogen genistein in this effect. However, results from previous studies are conflicting regarding the effects of genistein on hormone responsive cancers. Methods The effects of estrogen and genistein on proliferation of Ishikawa (IK) endometrial adenocarcinoma cells were examined in co-cultures of IK cells with endometrial stromal cells, recapitulating the heterotypic cell-to-cell interactions observed in vivo. The roles of estrogen receptor (ER)α and ERβ were evaluated using ERα and ERβ specific agonists. ER activation and cell proliferation in the IK epithelial cells were determined by alkaline phosphatase assay and Coulter counter enumeration, respectively. Results Both estrogen and genistein increased estrogen receptor-induced gene activity in IK cells over a range of concentrations. Estrogen alone but not genistein increased IK proliferation in co-cultures. When primed by estrogen treatment, increasing concentrations of genistein produced a biphasic effect on IK proliferation: nM concentrations inhibited estrogen-induced proliferation while μM concentrations increased proliferation. Studies with an ERβ-specific agonist produced similar results. Genistein did not influence the effects of estrogen on IK proliferation in monoculture. Conclusions Our study indicates that nutritionally relevant concentrations (nM) of genistein inhibit the proliferative effects of estrogen on endometrial adenocarcinoma cells presumably through activation of stromal cell ERβ. We believe that sub-micromolar concentrations of genistein may represent a novel adjuvant for endometrial cancer treatment and prevention.

Apicidin suppresses transcription of 17β-hydroxysteroid dehydrogenase type 1 in endometrial adenocarcinoma cells

It has recently been reported that endometrial cancer cells are able to convert estron (E1) to 17β estradiol (E2). We observed the presence of 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1) transcript and protein in receptor positive ER(+) and negative ER(-) Ishikawa endometrial adenocarcinoma (ISH) cells. ER(+) ISH, but not ER(-)02 ISH, cells were significantly susceptible to apicidin induced death, and we further used ER(-)ISH cells to study the effect of apicidin on cellular levels of HSD17B1 transcript and protein. We showed that apicidin significantly lowered HSD17B1 transcript and protein levels in ISH cells. There was no significant effect on HSD17B1 transcript stability. However, chromatin immunoprecipitation analysis revealed that apicidin significantly decreased occupation of the first exon of the HSD17B1 gene by Polymerase II. Since intratumoral E1 to E2 conversion is a significant contributor to the progression of estrogen dependent cancers, and HDAC inhibitors are being tested in anticancer clinical trials, our observations may have clinical value.

Interleukin-11 in Endometrial Adenocarcinoma Is Regulated by Prostaglandin F 2α-F-Prostanoid Receptor Interaction via the Calcium-Calcineurin-Nuclear Factor of Activated T Cells Pathway and Negatively Regulated by the Regulator of Calcineurin-1

Interleukin-11 (IL-11) up-regulates the proliferative and invasive capacity of many cancers. Coexpression of glycoprotein 130 (GP130) and IL-11 receptor alpha (IL-11Ralpha) is necessary for high-affinity binding of IL-11 to IL-11Ralpha. This study investigated the expression of IL-11 and role of prostaglandin F(2alpha)-F-prostanoid receptor (FP receptor) signaling in the modulation of IL-11 expression in endometrial adenocarcinoma cells. Localization of IL-11, IL-11Ralpha, and GP130 expression was performed by immunohistochemistry. IL-11 and regulator of calcineurin 1 isoform 4 (RCAN1-4) mRNA and protein expression were determined by real-time RT-PCR and/or enzyme-linked immunosorbent assay/Western blot analysis using Ishikawa endometrial adenocarcinoma cells stably expressing the FP receptor (FPS cells) and endometrial adenocarcinoma explants. IL-11 mRNA expression was significantly elevated in endometrial adenocarcinoma samples compared with normal endometrium and increased with tumor grade. IL-11 protein expression localized with FP receptor, IL-11Ralpha, and GP130 in the neoplastic glandular epithelium of endometrial adenocarcinomas. Prostaglandin F(2alpha)-FP receptor signaling significantly elevated the expression of IL-11 mRNA and protein in a Gq-protein kinase C-calcium-calcineurin-nuclear factor of activated T cells-dependent manner in FPS cells. The calcineurin signaling pathway is known to be controlled by the RCAN (RCAN1-4). Indeed, RCAN1-4 expression was significantly elevated in well-differentiated endometrial adenocarcinoma compared with normal endometrium and was found to decrease with tumor grade and negatively regulate IL-11 expression in vitro. This study has highlighted a new mechanism regulating IL-11 expression in endometrial adenocarcinoma cells by the FP receptor via the calcium-calcineurin-nuclear factor of activated T cells pathway.

Inhibin/activin-betaC and -betaE subunits in the Ishikawa human endometrial adenocarcinoma cell line

Inhibins and activins are important regulators of the female reproductive system. Recently, two novel inhibin subunits, named betaC and betaE, have been identified and shown to be expressed in several human tissues. However, only limited data on the expression of these novel inhibin subunits in normal human endometrial tissue and endometrial adenocarcinoma cell lines exist. Samples of proliferative and secretory human endometrium were obtained from five premenopausal, non-pregnant patients undergoing gynecological surgery for benign diseases. Normal endometrial tissue and Ishikawa endometrial adenocarcinoma cell lines were analyzed by immunohistochemistry, immunofluorescence and RT-PCR. Expression of the inhibin betaC and betaE subunits could be demonstrated at the protein level by means of immunohistochemical evaluation and at the transcriptional level by establishing a betaC- and betaE-specific RT-PCR analysis in normal human endometrial tissue and the parental Ishikawa cell line. Interestingly, in a highly de-differentiated subclone of the Ishikawa cell line lacking estrogen receptor expression, the expression of the inhibin-betaC subunit appeared strongly reduced. Here, we show for the first time that the novel inhibin/activin-betaC and -betaE subunits are expressed in normal human endometrium and the estrogen receptor positive human endometrial carcinoma cell line Ishikawa using RT-PCR and immunohistochemical detection methods. Interestingly, the Ishikawa minus cell line (lacking estrogen receptor expression) demonstrated no to minimal expression of the betaC subunit as observed with immunofluorescence and RT-PCR, suggesting a possible hormone- dependency of this subunit in human endometrial cancer cells. Moreover, because the Ishikawa cell line minus is thought to be a more malignant endometrial cell line than its estrogen receptor positive counterpart, inhibin-betaC subunit might be substantially involved in the pathogenesis and malignant transformation in human endometrium.

Estrogen-dependent Regulation of PPAR-γ Signaling on Collagen Biosynthesis in Adenocarcinoma Endometrial Cells

The link between estrogen and metabolic developmental factors of endometrial carcinoma is well established. PPAR- gamma, (an important modulator of metabolism) and estrogen receptor belong to a family of nuclear hormone receptors that were shown to interact with each other. The interaction may affect transcriptional activity of these transcription factors. The anti-diabetic troglitazone (TGZ) is well known PPAR- gamma ligand. The effect of troglitazone-induced PPAR- gamma activation on estrogen-dependent stimulation of collagen biosynthesis was studied in the Ishikawa endometrial adenocarcinoma cell line. We have found that the presence of estrogen activity in growth medium (1nM) augmented collagen biosynthesis in the cells. An addition of PPAR- gamma agonists, as troglitazone or clofibrat to the growth medium induced inhibition of collagen biosynthesis. The inhibition was effective only when estrogen receptor was stimulated, since removal of estrogen receptor by ICI 182- 780-dependent degradation did not affect collagen biosynthesis. The mechanism of the inhibition was found at the level of NF-kB (known inhibitor of collagen gene expression) and MAPK signaling. PPAR- gamma ligands stimulated expression of NF-kB, while they inhibited expression of p-38 but not ERK1/ERK2. The data document for the first time that inhibitory effect of PPAR- gamma ligands on collagen biosynthesis in endometrial adenocarcinoma cells requires functional estrogen receptor.

Estrogen regulates DNA methyltransferase 3B expression in Ishikawa endometrial adenocarcinoma cells

It is well-known that exposure to unopposed estrogen is considered as an important risk factor for endometrial cancer. Recent studies have shown that over-expression of DNA methyltransferases (DNMTs) are involved in the development of endometrial cancer. Therefore, the present study was undertaken to elucidate the impact of estrogen on the expression of DNMTs in endometrial cancer. Ishikawa cell line was used. Flow cytometry analysis demonstrated that 17 beta-estradiol (E(2)) enhanced the cell proliferation with a peak at 10(-8) M. Over-expression of DNMT3B treated with E(2) was confirmed by real-time PCR and western blotting analysis. Furthermore, the up-regulation of DNMT3B expression induced by E(2) was suppressed by the addition of ICI182780. However, we did not observe changes in the expression of DNMT1. Our study suggests that estrogen up-regulating the expression of DNMT3B in an ER-dependent pathway may be a possible mechanism for estrogen facilitates the malignant transformation of endometrial cancer cells.

Antiproliferative and apoptotic activities of tosylcyclonovobiocic acids as potent heat shock protein 90 inhibitors in human cancer cells

We evaluated whether inhibition of heat shock protein 90 (hsp90) function by novobiocin derivatives could induce the degradation of signal transducers that drive cancer cell growth and thereby promote apoptosis. Removal of the noviose moiety in novobiocin and introduction of a tosyl substituent at C-4 or C-7 coumarin nucleus provided derivatives 4TCNA and 7TCNA which compared favourably with novobiocin in MCF-7 breast cancer cells. Here we extend the antiproliferative and apoptotic properties of these analogues to a panel of cancer cell lines. Destabilization of hsp90 client proteins Raf-1, HER2, and cdk4 suggests inhibition of hsp90 chaperoning function. In HT29 colon and IGROV1 ovarian cancer cells, the growth inhibiting effect of 4TCNA and 7TCNA was consistent with the stimulation of cell death as assessed by the processing and activation of caspase 9, 8, 7 and 3 and the subsequent cleavage of poly(ADP-ribose) polymerase (PARP). In Ishikawa endometrial adenocarcinoma cells, 4TCNA also promoted apoptosis and the processing of PARP. These derivatives impacting multiple pathways involved in the neoplastic process may represent promising drugs for cancer therapy.