transforming-growth-factor-alpha and Endometrial-Neoplasms

Endometrial carcinoma (EC) is one of the most lethal gynecologic cancers. Patients frequently have regional or distant metastasis at diagnosis. MicroRNAs are small non-coding RNAs that participate in numerous biological processes. Recent studies have demonstrated that miR-505 is associated with several types of cancer; however, the expression and function of miR-505 have not been investigated in EC.. miR-505 expression in normal endometrial tissue, endometrial carcinomas were quantified by Quantitative reverse transcription PCR. The endometrial carcinoma cell lines HEC-1B and Ishikawa were each transfected with miR-505 or scrambled mimics, after which cell phenotype and expression of relevant molecules were assayed. Dual-luciferase reporter assay and a xenograft mouse model were used to examine miR-505 and its target gene TGF-α.. RT-PCR results demonstrated that miR-505 was significantly downregulated in human EC tissues compared to normal endometrial tissues. Besides, miR-505 expression was negatively associated with FIGO stage (stage I-II vs. III-IV), and lymph node metastasis (negative vs. positive). In vitro, overexpression of miR-505 significantly suppressed EC cell proliferation, increased apoptosis and reduced migratory and invasive activity. A miR-505 binding site was identified in the 3' untranslated region of TGF-α mRNA (TGFA) using miRNA target-detecting software; a dual luciferase reporter assay confirmed that miR-505 directly targets and regulates TGFA. RT-PCR and Western-blotting results indicated that overexpressing miR-505 reduced the expression of TGF-α and the TGF-α-regulated proteins MMP2, MMP9, CDK2, while induced Bax and cleaved-PARP expression in EC cells. In vivo, overexpression of miR-505 reduced the tumorigenicity and inhibited the growth of xenograft tumors in a mouse model of EC.. Taken together, this study demonstrates that miR-505 acts as tumor suppressor in EC by regulating TGF-α.

Topics: Animals; Apoptosis; Base Sequence; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Endometrial Neoplasms; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Mice, Inbred BALB C; MicroRNAs; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Metastasis; Transfection; Transforming Growth Factor alpha; Xenograft Model Antitumor Assays

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate the translation of messenger RNAs by binding their 3'-untranslated region (3' UTR). MiR-490-3p has been reported to be a suppressor in various human cancers; however, little is known about the biological functions of miR-490-3p in endometrial cancer (EC). In our study, we found that MiR-490-3p mRNA expression was significantly lower in ECs than in normal endometrial tissues. MiR-490-3p mRNA expression was also negatively associated with depth of invasion (mucosa vs. muscular and serosa) and lymph node metastasis (negative vs. positive) in EC. MiR-490-3p overexpression reduced proliferation; promoted G1 arrest and apoptosis; suppressed migration and invasion; and reduced TGFα, NF-kB, cyclin D1, survivin, matrix metalloproteinase 2 (MMP2) mRNA and protein expression, and improved Bax mRNA and protein expression. The dual-luciferase reporter assay indicated that miR-490-3p directly targeted TGFα by binding its 3' untranslated region. MiR-490-3P transfection also suppressed tumor development and TGFα expression (as determined by immunohistochemistry and western blotting) in vivo in the xenograft mouse model. This is the first demonstration that miR-490-3P might act as a suppressor in EC tumorigenesis and progression by targeting TGFα. Our results provide a theoretical basis for the further study on the molecular target for endometrial cancer.

Topics: 3' Untranslated Regions; Aged; Animals; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Cyclin D1; Disease Progression; Endometrial Neoplasms; ErbB Receptors; Female; G1 Phase Cell Cycle Checkpoints; HEK293 Cells; Humans; Inhibitor of Apoptosis Proteins; Matrix Metalloproteinase 2; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Neoplasm Transplantation; NF-kappa B; RNA Interference; RNA, Messenger; RNA, Small Interfering; Survivin; Transforming Growth Factor alpha; Transplantation, Heterologous; Uterus

This study aimed to investigate serum levels of epidermal growth factor (EGF), transforming growth factor α (TGF-α), and c-erbB2 in patients with ovarian cancer.. In this retrospective cohort study, the study and control groups were composed of 43 women with a prediagnosis of ovarian cancer and 43 healthy women, respectively. Blood samples from all women were obtained and studied by enzyme-linked immunosorbent assay kits for EGF, TGF-α, and c-erbB2. After surgery of the study group, ovarian cancer was confirmed and compared with control group. Stage, grade, and histological types were defined after histopathologic examination, and subgroups were constructed and compared.. Serum EGF, TGF-α, and c-erbB2 levels were significantly increased in study group compared with those in the control group (P < 0.001). There were no differences in serum levels of EGF, TGF-α, and c-erbB2 among all stages, grades, and histological types of ovarian cancer. If 47.90 pg/mL was selected as the cutoff value, EGF has an 80% sensitivity and a 65% specificity for detecting ovarian cancer. The cutoff value of 41,095.00 pg/mL for TGF-α has a 90% sensitivity and a 72% specificity for detecting ovarian cancer. The c-erbB2 level of 4.63 pg/mL as the cutoff value has an 83% sensitivity and a 76% specificity for predicting ovarian cancer.. Serum levels of EGF, TGF-α, and c-erbB2 may be used for diagnosing ovarian cancer.

Topics: Adenocarcinoma, Clear Cell; Adenocarcinoma, Mucinous; Biomarkers, Tumor; Case-Control Studies; Cystadenocarcinoma, Serous; Endometrial Neoplasms; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Humans; Middle Aged; Neoplasm Grading; Neoplasm Staging; Ovarian Neoplasms; Prognosis; Receptor, ErbB-2; Retrospective Studies; ROC Curve; Transforming Growth Factor alpha

The over-expression of epidermal growth factor receptor (EGFR) and its ligands, epidermal growth factor (EGF) and transforming growth factor-alpha, is a common feature of epithelial carcinomas and correlates with neoplastic progression. Secretory leukocyte protease inhibitor (SLPI), a member of the Kazal superfamily of serine anti-proteases, induces proliferation and promotes malignancy of epithelial cells and is expressed at high levels in multiple tumor types. In the present study, we have demonstrated that EGF increases SLPI expression in the human endometrial epithelial cell line Ishikawa in a dose- and time-dependent manner. We have shown that this effect of EGF occurs, in part, at the level of the SLPI promoter and involves the MAP kinase signaling pathway. We have further shown that EGF promotion of cell proliferation, but not induction of cyclin D1 gene expression, involves SLPI. Our results suggest that the regulation of SLPI expression by EGFR ligand(s) may represent a 'feed-forward' mechanism by which the enhanced proliferative and migratory properties of EGFR over-expressing cancer cells are sustained. Increased SLPI expression is likely an important component of altered EGFR signaling in human tumors and may have significant therapeutic implications in cancer progression.

Topics: Carcinoma; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Endometrial Neoplasms; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Gene Expression Regulation; Humans; Proteinase Inhibitory Proteins, Secretory; Proteins; Secretory Leukocyte Peptidase Inhibitor; Time Factors; Transfection; Transforming Growth Factor alpha

The purpose of this study was to identify an endometrial cell line that maintained the E2 up-regulation of estrogen receptor (ER) mRNA by enhanced message stability and to assess its dependence on ER protein. Estradiol (E2) effects on gene expression were measured in three cell lines: one immortalized from sheep endometrial stroma (ST) and two from human endometrial adenocarcinomas (Ishikawa and ECC-1). E2 up-regulated ER mRNA levels in ST and Ishikawa cells, but down-regulated ER mRNA levels in ECC-1 cells. E2 up-regulated progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and transforming growth factor-alpha (TGF-alpha) in both Ishikawa and ECC-1 cells. The selective estrogen receptor modulator ICI 182,780 antagonized the E2-induced up-regulation of ER and/or PR mRNA levels in all three cells, while another, GW 5638, antagonized the up-regulation of PR mRNA in Ishikawa and ECC-1 cells. In mechanistic studies, E2 had no effect on ER mRNA stability in ST cells and it destabilized ER mRNA in ECC-1 cells. Thus, Ishikawa cells appear to be the most physiologically relevant cell line in which to study the up-regulation of ER mRNA levels by enhanced mRNA stability. Its antagonism by ICI 182,780 reveals that ER protein is involved in this E2 response.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents, Hormonal; Blotting, Northern; Cells, Cultured; Cloning, Molecular; Down-Regulation; Endometrial Neoplasms; Endometrium; Estradiol; Estrogen Antagonists; Female; Fulvestrant; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Receptors, Progesterone; RNA, Messenger; Selective Estrogen Receptor Modulators; Sheep; Stromal Cells; Time Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured; Up-Regulation

Tamoxifen is the endocrine treatment of choice for all stages of estrogen receptor (ER)-positive breast cancer, and it is the first drug approved to reduce the incidence of breast cancer in high-risk women. Unfortunately, tamoxifen also possesses some estrogen-like effects in the uterus that cause a modest increase in the risk of endometrial cancer. GW5638 is a tamoxifen derivative with a novel carboxylic acid side chain with no uterotropic activity in the rat (Willson et al., J Med Chem, 1994, 37:1550-1552). We have compared and contrasted the actions of 4-hydroxytamoxifen (4-OHT, the active metabolite of tamoxifen) with GW7604 [the presumed metabolite of GW5638 in breast (MCF-7) and endometrial (ECC-1) cell lines in vitro]. GW7604 did not cause the growth of ECC-1 cells at any concentration (10(-11)-10(-6) M), but 4-OHT was weakly estrogen-like at low concentrations (10(-11)-10(-10) M). Compounds (10(-7) M) blocked the growth promoting action of estradiol (10(-10) M) in both ECC-1 and MCF-7 cells. Western blotting was used to show that GW7604 and raloxifene did not affect ER levels significantly, compared with controls, in MCF-7 cells; whereas the pure antiestrogen ICI182,780 decreased ER levels (P < 0.05). An assay system was used that can classify compounds into tamoxifen-like, raloxifene-like, or pure antiestrogens. The assay depends on the activation of the transforming growth factor alpha (TGFalpha) gene in situ by wild-type or D351Y mutant ER stably transfected into MDA-MB-231 cells (MacGregor-Schafer et al., Cancer Res, 1999, 59:4308-4313). GW7604 inhibited both estradiol (10(-9) M) and 4-OHT (10(-8), 10(-7) M) induction of TGFalpha in a concentration related manner (10(-9)-10(-6) M). GW7604 and raloxifene stimulated TGFalpha with the D351Y ER. In contrast, ICI 182,780 (10(-6) M) did not initiate TGFalpha and blocked the induction of TGFalpha with GW7604, raloxifene, and 4-OHT in D351Y-transfected cells. Using computer-assisted molecular models of ER complexes, we found that the antiestrogenic side chain of 4-OHT weakly interacted with the surface amino acid 351 (aspartate), but the carboxylic acid of GW7604 caused a strong repulsion of aspartate 351. We propose that GW7604 is less estrogen-like than 4-OHT, because it disrupts the surface charge around aa351 required for coactivator docking in the 4-OHT:ER complex. This charge is restored in the D351Y ER, thus converting GW7604 from an antiestrogen to an estrogen-like molecule.

Topics: Carcinoma; Cell Division; Cinnamates; Endometrial Neoplasms; Estrogen Receptor alpha; Estrogen Receptor Modulators; Estrogens; Female; Gene Expression Regulation; Humans; Models, Molecular; Receptors, Estrogen; RNA, Messenger; Selective Estrogen Receptor Modulators; Stilbenes; Tamoxifen; Transforming Growth Factor alpha; Tumor Cells, Cultured

3,3'-Diindolylmethane (DIM), a major in vivo product of indole-3-carbinol (I3C), is a promising anticancer agent derived from vegetables of the Brassica genus including broccoli, Brussels sprouts and cabbage. We report here that DIM has a potent cytostatic effect in cultured human Ishikawa endometrial cancer cells. A combination of northern blot and quantitative PCR analyses revealed that DIM induced the level of TGF-alpha transcripts by approximately 4-fold within 24 h of indole treatment. DIM also induced a 4-fold increase in the activity of the estrogen response marker, alkaline phosphatase (AP). Co-treatment of cells with the estrogen receptor (ER) antagonist ICI, or with the inhibitor of PKA-mediated activation of the ER, H89, ablated the DIM induction of both TGF-alpha expression and AP activity. Furthermore, DIM increased the maximum stimulatory effect of estrogen on TGF-alpha expression. Co-treatment with the protein synthesis inhibitor, cycloheximide, abolished the inductive effects of DIM, indicating differences in the mechanistic requirements of DIM and estrogen. DIM treatment also stimulated levels of secreted TGF-alpha protein by >10-fold. The ectopic addition of TGF-alpha inhibited the growth of Ishikawa cells, whereas incubation with a TGF-alpha antibody partially reversed the growth inhibitory effects of DIM. Taken together, these results extend our previous findings of the ligand independent estrogen receptor agonist activity of DIM, and uncover an essential role for the stimulation in TGF-alpha expression and the TGF-alpha activated signal transduction pathway in the potent cytostatic effects of DIM in endometrial cancer cells.

Topics: Adenocarcinoma; Alkaline Phosphatase; Anticarcinogenic Agents; Blotting, Northern; Blotting, Western; Cell Division; Cycloheximide; DNA, Complementary; Endometrial Neoplasms; Enzyme-Linked Immunosorbent Assay; Estradiol; Estrogen Antagonists; Female; Humans; Indoles; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

We investigated tamoxifen's effects on the expression of growth regulatory genes in the endometrium to identify the mechanism by which tamoxifen induces proliferation.. Using immunohistochemical techniques, we analyzed 39 endometrial specimens for expression of Ki-67, lactoferrin, transforming growth factor-alpha, tumor necrosis factor receptor-II, adrenomedullin, estrogen receptors, and progesterone receptors. Twenty specimens were obtained from postmenopausal breast cancer patients treated with tamoxifen (20 mg/day) for at least 6 months to include two endometrial adenocarcinoma specimens. Five secretory phase, three proliferative phase, and seven atrophic endometrial specimens were used as controls. In addition, four endometrial adenocarcinoma specimens were reviewed from patients not treated with tamoxifen. Intensity of immunostaining was quantified using digitized imaging techniques.. Overexpression of both estrogen receptors and progesterone receptors, and an elevated proliferative index were the most consistent effects observed in benign endometrial specimens from tamoxifen-treated patients compared with atrophic controls (P <. 003). This staining pattern was also evident in adenocarcinomas from patients who received tamoxifen. Benign endometrium from tamoxifen-treated patients also expressed transforming growth factor-alpha, tumor necrosis factor receptor-II, lactoferrin, and adrenomedullin at levels comparable with those found in proliferative endometrial specimens.. These data provide further documentation that the uterotropic effects of tamoxifen may be due, at least in part, to the induction of estrogen receptors and progesterone receptors, as well as other genes associated with the proliferative phase. Furthermore, analysis of estrogen receptors, progesterone receptors, and Ki-67 may be useful in identifying postmenopausal individuals on tamoxifen, who are at increased risk for developing endometrial cancer.

Topics: Adrenomedullin; Antineoplastic Agents, Hormonal; Breast Neoplasms; Endometrial Neoplasms; Endometrium; Female; Gene Expression Regulation, Neoplastic; Genes, Regulator; Humans; Hyperplasia; Immunohistochemistry; Ki-67 Antigen; Lactoferrin; Peptides; Receptors, Estrogen; Receptors, Progesterone; Receptors, Tumor Necrosis Factor; Tamoxifen; Transforming Growth Factor alpha

We investigated the effects of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) on migration, invasion and proteinase expression of gynecological cultured cancer cells (SKG-IIIb cervical squamous cell carcinoma, OMC-4 cervical adenocarcinoma, SNG-M endometrial adenocarcinoma and OMC-3 ovarian adenocarcinoma), and whether these growth factors affect thymidine phosphorylase/platelet-derived endothelial cell growth factor expression of tumor cells. Tumor cell migration along a gradient of substratum-bound fibronectin and invasion into reconstituted basement membrane were stimulated by 0.1-10 nM EGF and TGF-alpha in a concentration-dependent manner. The zymography of tumor-conditioned medium showed that the treatment of tumor cells with EGF and TGF-alpha resulted in the increase of type IV collagenases, stromelysin and urokinase-type plasminogen activator which was partly confirmed by immunoblot analysis. The expression of thymidine phosphorylase/platelet-derived endothelial cell growth factor which has angiogenic activity, was also upregulated by these growth factors. These results suggest that EGF and TGF-alpha act as positive regulators on the invasion process of gynecological tumor cells which may be associated with their stimulatory action on the motility of tumor cells, the expression of proteinases secreted by tumor cells and the angiogenic phenotype.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Cell Movement; Endometrial Neoplasms; Epidermal Growth Factor; Female; Genital Neoplasms, Female; Humans; Metalloendopeptidases; Neoplasm Invasiveness; Ovarian Neoplasms; Thymidine Phosphorylase; Transforming Growth Factor alpha; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator; Uterine Cervical Neoplasms

Since the majority of endometrial carcinomas do not contain any detectable ras mutations, the precise contribution of aberrant Ras function, if any, to endometrial carcinoma development remains to be determined. Since there is considerable evidence that Ras transformation is associated with a decreased requirement for growth factors, we compared the growth response of endometrial carcinoma cells harbouring wild-type (Ishikawa cells) or mutated (HHUA cells) K-ras to epidermal growth factor (EGF). K-ras mutation did not significantly affect the level of the EGF receptor (EGFR) expressed in these carcinoma cells. EGF could stimulate the growth of Ishikawa, but not HHUA cells. Furthermore, EGF caused elevation of Ras-GTP levels in Ishikawa, but not HHUA cells. However, the introduction of mutated, but not normal, K-ras into Ishikawa cells rendered them non-responsive to EGF growth stimulation. Thus, the presence of mutated K-ras alone modulated the growth response of endometrial carcinoma cells to EGF. An inhibitor of the EGFR tyrosine kinase activity could prevent soft agar colony formation of Ishikawa cells, but not HHUA or mutant K-ras(12V)-transfected Ishikawa cells. Taken together, these results suggest that mutated K-ras causes a loss of responsiveness to EGF stimulation and that EGFR function is dispensable for the growth of mutant Ras-positive endometrial carcinoma cells.

Topics: Apoptosis; Cell Division; Endometrial Neoplasms; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; Genes, ras; Humans; Hydroquinones; Mutation; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured

Endometrial carcinoma cell lines were evaluated for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and insulin-like growth factor I (IGF-1) production and for autocrine stimulation.. Conditioned, serum-free media (CM) from cell lines RL95-2, KLE, HEC, and Ishikawa (ISH) were concentrated radioimmunoassayed (RIA). Samples for the IGF-1 assay were extracted with acid-ethanol to remove IGF-1 binding protein. Polymerase chain reaction (PCR) was used to validate the presence of mRNA for growth factors and receptors. Cells were incubated with Ab528, an antibody blocking EGF receptors, and alphaIR3, an antibody blocking IGF-1 receptors. Proliferation was quantified using [3H]thymidine incorporation.. TGF-alpha was detected in CM: RL95-2 (0.4 +/- 0.001 ng/ml), KLE (0.7 +/- 0.003 ng/ml), HEC (0.8 +/- 0.01 ng/ml), ISH (1.2 +/- 0.05 ng/ml). No EGF was detected in CM. In extracted samples, IGF-1 was detected in CM: RL95-2 (0.8 +/- 0. 03 ng/ml), KLE (1.25 +/- 0.02 ng/ml), HEC (1.6 +/- 0.01 ng/ml), ISH (1.6 +/- 0.08 ng/ml). Unconditioned media served as the control. EGF, TGF-alpha, and IGF-1 mRNA was identified in all cell lines, as was the mRNA for EGF and IGF-1 receptors. Incubation with Ab528 or alphaIR3 resulted in significant inhibition of DNA synthesis in HEC 1A, KLE, and ISH. No inhibition was detected in the RL95-2 cell line. A control antibody did not inhibit the cell lines.. Autocrine production and stimulation of endometrial carcinoma cell lines by TGF-alpha and IGF-1 are demonstrated in three of four endometrial cancer cell lines. No measurable EGF was produced by any of the cell lines.

Topics: Autocrine Communication; Cell Division; Culture Media, Conditioned; Culture Media, Serum-Free; DNA, Neoplasm; Endometrial Neoplasms; Epidermal Growth Factor; Female; Humans; Insulin-Like Growth Factor I; Neoplasm Proteins; Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

The role of estrogen and estrogen-related growth factors in the mechanism of hormone dependency of endometrial adenocarcinoma cells was investigated. The proliferation of hormone-responsive human endometrial adenocarcinoma cells (Ishikawa cells), which possess both estrogen and progesterone receptors, was optimally stimulated by 10 nM estradiol. Both transforming growth factor (TGF)-alpha and epidermal growth factor (EGF), added to the culture media, stimulated the proliferation of Ishikawa cells in a dose-dependent manner. Anti-TGF-alpha antibody completely eliminated the stimulatory effects of TGF-alpha. Anti-EGF receptor antibody inhibited the proliferation of these cells. The production of TGF-alpha into culture media was 5-40 pg/10 cells/24 h in 9 human endometrial adenocarcinoma cells. Ten nanomoles of estradiol increased the TGF-alpha production of Ishikawa cells by approximately 2.5-fold of the control level. In contrast, the production of TGF-alpha in hormone-unresponsive HEC-50 cels was not influenced by estradiol. C-erbB-2 oncoprotein expression of human endometrial adenocarcinoma cells, detected by both immunocytochemical staining and Western blot analysis, was associated with the tumor grade of the original tumor tissues. Ten nanomoles of estradiol clearly increased the c-erbB-2 oncoprotein levels at an optimal incubation period of 72 h, whereas estradiol did not affect the expression in HEC-50 cells.

Topics: Adenocarcinoma; Blotting, Western; Dose-Response Relationship, Drug; Endometrial Neoplasms; Epidermal Growth Factor; Estrogens; Female; Humans; Immunohistochemistry; Neoplasms, Hormone-Dependent; Receptor, ErbB-2; Time Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured

In the present study, we have investigated the effects of interferons-alpha (IFN-alpha) and -gamma (IFN-gamma), interleukin-10 (IL-10) and -13 (IL-13), transforming growth factor-beta1 (TGF-beta1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha) on cell proliferation and induction of transcription factors AP-1 and NF-kappaB in UM-EC-3 human endometrial adenocarcinoma cells and UT-OC-5 ovarian carcinoma cells in vitro. In addition, cellular DNA was extracted to study if any of these factors is able to induce apoptosis. In UM-EC-3 cell line DNA synthesis was inhibited by GM-CSF, IL-10, IL-13, TGF-beta1, IFN-alpha, and IFN-gamma after 48 and 72 h in culture, whereas TNF-alpha had no significant effect on cell proliferation in any of the experiments. The inhibition of DNA synthesis was similarly observed in UT-OC-5 ovarian carcinoma cells by IL-10, TNF-alpha, and IFN-gamma after 48 and 72 h, whereas IFN-alpha had no statistically significant effect. An inhibitory effect of GM-CSF was observed only after 48 h and TGF-beta after 72 h in culture, respectively. Transcription factors AP-1 and NF-kappaB were both constitutively active in UM-EC-3 and UT-OC-5 cells. The binding activity of AP-1 was found to be stimulated by all growth-inhibitory cytokines studied in both cell lines, whereas the specific binding activity of NF-kappaB was affected moderately only by TNF-alpha in UT-OC-5 ovarian carcinoma cells. No signs of DNA fragmentation typical of apoptosis were observed in any of these studies.

Topics: Adenocarcinoma; Aged; Cell Division; Cytokines; DNA, Neoplasm; Endometrial Neoplasms; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; NF-kappa B; Ovarian Neoplasms; Transcription Factor AP-1; Transcription Factors; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Six different ligands of the epidermal-growth-factor receptor (EGFR) have been identified in the past. In some cervical squamous-cell carcinomas, an increased amount of proteins binding to the EGFR has been reported. In order to identify the mRNA of EGFR ligands (EGFRL), which might be overexpressed in cervical and endometrial cancers, we performed semi-quantitative reverse-transcription/polymerase chain reactions (RT-PCR) for all 6 EGFRL in RNA extracts of normal and malignant tissue samples of the human uterus. PCR products from RNA extracts of 83 patients were quantitated relative to the housekeeping gene and internal standard pyruvate dehydrogenase by analyzing the PCR kinetics of product synthesis. In extracts of normal cervix, the level of mRNA expression of the EGFRL was significantly higher than in endometrium. No significant difference was detected between normal cervix and cervical carcinomas. However, both in cervical and in endometrial cancers, mRNA expression was non-parametrically distributed and in some cervical cancers overexpression of transforming growth factor alpha (TGF-alpha), amphiregulin or EGF was observed. In endometrial cancers, mRNA levels of all EGFRL were higher than in normal endometrium. This increase was significant (p < 0.005) for TGF-alpha and amphiregulin. Thus, TGF-alpha mRNA is overexpressed in approximately 10% of cervical cancers and in the majority of endometrial cancers. Since TGF-alpha anti-sense therapy might represent a future strategy in such cancers, we also determined the absolute level of TGF-alpha mRNA expression by quantitative PCR using a cloned standard.

Topics: Cloning, Molecular; DNA; DNA, Neoplasm; Endometrial Neoplasms; ErbB Receptors; Female; Humans; Ligands; Polymerase Chain Reaction; RNA; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Transforming Growth Factor alpha; Uterine Cervical Neoplasms; Uterus

The biological significance of epidermal growth factor (EGF)-related proteins in the development and progression of endometrioid endometrial carcinoma was studied. Expression of EGF-related proteins including EGF, transforming growth factor-alpha (TGF-alpha), cripto (CR), amphiregulin (AR), and EGF receptor (EGFR) were immunohistochemically examined. Results were then correlated with clinicopathologic findings and steroid receptor status in 12 specimens with normal endometrium, 13 with endometrial hyperplasia, and 40 with endometrioid endometrial carcinoma. EGFR, EGF, TGF-alpha, and CR immunoreactivities were observed in 58.3%, 66.7%, 91.6%, and 66.7% of normal endometrial specimens; 100%, 15.4%, 100%, and 30.8% of endometrial hyperplasia specimens; and 67.5%, 32.5%, 65.0%, and 65.0% of endometrial carcinoma specimens, respectively. AR immunoreactivity was not observed in any of the normal, hyperplastic, or neoplastic endometrium. The presence or absence of EGFR or TGF-alpha in endometrial carcinoma correlated with surgical stage, depth of myometrial invasion, and findings from peritoneal washing cytology. EGF expression significantly correlated with the age of the patients and that of CR with surgical stage and peritoneal washing cytological findings. There was a significant correlation between EGFR and TGF-alpha expression, and between EGF and TGF-alpha. Co-expression of EGFR and TGF-alpha, EGFR and CR, and TGF-alpha and CR in carcinoma specimens significantly correlated with advanced surgical stage, deeper myometrial invasion, and positive peritoneal washing cytology. In normal as well as hyperplastic endometrium, endometrial glands immunohistochemically positive for TGF-alpha were generally positive for ER, but in poorly differentiated endometrial carcinoma, cells positive for TGF-alpha tended to be negative for ER. The results of the present study show that among EGF-related proteins, expression of TGF-alpha and CR seem to be associated with the progression of human endometrioid endometrial carcinoma. Additionally, expression of TGF-alpha became increasingly estrogen independent with increasing histological carcinoma grades.

Topics: Adult; Amphiregulin; Carcinoma, Endometrioid; EGF Family of Proteins; Endometrial Hyperplasia; Endometrial Neoplasms; Endometrium; Epidermal Growth Factor; ErbB Receptors; Female; Glycoproteins; GPI-Linked Proteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Receptors, Estrogen; Receptors, Progesterone; Receptors, Steroid; Retrospective Studies; Transforming Growth Factor alpha

Biological effects of sex steroids (estradiol-17beta, E2; progesterone, P; medroxyprogesterone acetate, MPA; Danazol, DZ) and growth factors (epidermal growth factor, EGF; transforming growth factor, TGF-alpha,beta) on migration and invasion of endometrial adenocarcinoma SNG-M cells were investigated by haptotactic migration and haptoinvasion assay. The enzymatic degradation of the extracellular matrix by tumor cells was also examined. Tumor cell migration along a gradient of substratum-bound fibronectin was inhibited by 0.1-10 microM MPA and DZ, but promoted by 0.1-10 nM EGF and TGF-alpha in a concentration-dependent manner. E2, P and TGF-beta did not have any effect on the motility of tumor cells. These effects were also confirmed by wound assay. The invasive activity of SNG-M cells into reconstituted basement membrane (Matrigel) was inhibited by the presence of 0.1-10 microM MPA and DZ, but promoted by 0.1-10 nM EGF and TGF-alpha in a concentration-dependent manner. E2, P and TGF-beta did not have any effect on tumor cell invasion. The zymography of tumor-conditioned medium showed that the treatment of SNG-M cells with EGF and TGF-alpha resulted in the increase of the 68, 72 and 92 kDa type IV collagenases (matrix metalloproteinase, MMP-2 and 9). Sex steroids and TGF-beta did not have significant effects on MMP-2 and 9. Stromelysin (MMP-3), also secreted by SNG-M cells, was not affected by sex steroids and growth factors. These results suggest that EGF and TGF-alpha act as positive regulators on the invasion process of endometrial adenocarcinoma cells, which may partly be associated with the induction of type IV collagenase secretion by tumor cells. The inhibitory effects of MPA and DZ on tumor cell invasion may depend at least partly on their inhibitory action on the motility of tumor cells.

Topics: Adenocarcinoma; Caseins; Cell Division; Cell Movement; Culture Media, Conditioned; Danazol; Endometrial Neoplasms; Epidermal Growth Factor; Estrogen Antagonists; Female; Gelatin; Humans; Neoplasm Invasiveness; Progestins; Transforming Growth Factor alpha; Tumor Cells, Cultured

In a total of 113 cases of endometrial neoplasm, we studied the immunohistochemical expression of estradiol (E2), epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha), and epidermal growth factor receptor (EGFR). Positive immunoreactivity of E2 was found in 61% of the neoplasms. E2 immunoreactivity correlated well with high histologic grade and early clinical stage. Positive immunoreactivity for EGF or EGFR was found in 25.6% or 53.1% of the neoplasms, respectively. However, this was unrelated to histologic grade or clinical stage. On the other hand, TGFalpha immunoreactivity was found in 67% of endometrial neoplasias and was correlated with poor histologic grade and advanced stage. Contingency tables indicated a significant negative association between the status of E2 and that of TGFalpha. Simultaneous expression of E2, EGF and EGFR, or E2, TGFalpha and EGFR was found in 6.8% and 15.9% of endometrial carcinomas, respectively. These results suggest that a predominant number of endometrial carcinomas escape autocrine/paracrine growth regulation by EGF and E2 or TGFalpha and E2, and that TGFalpha may be involved in the progression of endometrial carcinoma.

Topics: Adult; Aged; Aged, 80 and over; Endometrial Neoplasms; Epidermal Growth Factor; ErbB Receptors; Estradiol; Female; Frozen Sections; Humans; Immunohistochemistry; Medroxyprogesterone Acetate; Middle Aged; Neoplasm Staging; Paraffin Embedding; Transforming Growth Factor alpha; Tumor Cells, Cultured

To study the regulation of c-erbB, EFG and TGF-alpha in endometrium, we examined the expression of their mRNA in 5 normal endometrial and 5 endometrial carcinoma tissues by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR). By Northern blot with 10 micrograms of total RNA, c-erbB mRNA was not detected in the normal endometrium, but it was detected in 3 samples of endometrial carcinoma tissues. On the other hand RT-PCR identified c-erbB mRNA in all the normal endometrium and endometrial carcinoma tissues by amplifying cDNA derived from c-erbB mRNA. EGF mRNA was detected by RT-PCR in normal endometrium except in the early follicular phase. It was detected in all cases of endometrial carcinoma tissues. TGF-alpha mRNA was also detected in all the normal and endometrial carcinoma tissues by RT-PCR. Our study suggests that an autocrine/paracrine mechanism of EGF may regulate the endometrial cycle. Because some endometrial carcinoma tissues express the c-erbB mRNA much more than normal endometrium, disruption of the autocrine/paracrine mechanism may trigger subsequent endometrial carcinogenesis.

Topics: Adenocarcinoma; Adult; Base Sequence; Blotting, Northern; Endometrial Neoplasms; Endometrium; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Middle Aged; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha

The expression of epidermal growth factor receptor (EGFr) and transforming growth factor alpha (TGF-alpha) was compared with the presence of "squamous differentiation" (SD) visualized in various histotypes of endometrial carcinoma by using a panel of monoclonal antibodies. The results of the current study demonstrate that EGFr and TGF-alpha are present in routinely processed endometrial carcinoma. The highest positive EGFr and TGF-alpha expression was seen in the group of adenocarcinomas with SD. The more intense EGFr and TGF-alpha immunoreactivity was observed in "squamous" foci both in adenoacanthomas (AA) and in adenosquamous carcinomas (AS). These EGFr- and TGF-alpha-positive squamous areas prevalently displayed a "stratification-related" cytokeratin (CK) immunoprofile characterized by the expression of CKs 1, 4, 5, 10, 13, 14, and 16. No correlation was found between EGFr- and TGF-alpha-positive status and depth of myometrial invasion or surgical stage. These results clearly demonstrate that EGFr and TGF-alpha expression is related remarkably to endometrial carcinoma with "squamous" areas both morphologically and immunophenotypically. This specific association leads us to suggest that EGFr and TGF-alpha expression in endometrial carcinoma may be prevalently involved in the equilibrium of cell differentiation of the "squamous" foci commonly observed in this group of neoplasias.

Topics: Adenocarcinoma; Aged; Biomarkers, Tumor; Carcinoma, Adenosquamous; Cell Differentiation; Endometrial Neoplasms; ErbB Receptors; Female; Humans; Immunohistochemistry; Keratins; Metaplasia; Middle Aged; Transforming Growth Factor alpha

Abnormal expression of p53, transforming growth factor alpha (TGF alpha), epidermal growth factor receptor (EGFR), and c-erbB-2 occurs in a variety of cancers and in some cases is associated with poor prognosis. Immunoperoxidase staining using these markers in formalin-fixed, paraffin-embedded endometrial carcinoma tissue was performed to determine whether immunoreactivity correlates with survival and known prognostic variables. Cases included 84 endometrioid adenocarcinomas, five adenoacanthomas, 12 adenosquamous carcinomas, 11 serous carcinomas, 15 clear cell carcinomas, and one carcinosarcoma for a total of 128 cases. Frequencies of immunoreactivity were as follows: p53, 37 of 128 (29%); TGF alpha, strong (2+) 23 of 128 (18%) and intermediate (1+) 26 of 128 (20%); EGFR, strong (3+) 21 of 128 (16%) and intermediate (2+ or 1+) 83 of 128 (65%); and c-erbB-2, strong (2+) four of 128 (2%) and intermediate (1+) three of 128 (1%). p53 and TGF alpha staining showed statistically significant correlations with decreased length of survival (P < .0017 and P < .0013, respectively, generalized Savage [Mantel Cox]). p53 immunoreactivity correlated with tumor types, grade, and stage. Transforming growth factor alpha staining correlated with increased depth of invasion and presence of vascular invasion. Epidermal growth factor receptor staining did not correlate with length of survival or known prognostic variables. c-erbB-2 staining correlated with tumor type. In the multivariate analysis p53 and TGF alpha staining were not independent predictors of survival when other variables were taken into account, including grade, stage, tumor type, presence of vascular invasion, and depth of invasion. Grade and stage were the only independent predictors of survival when used in combination in a Cox proportional hazards model.

Topics: Biomarkers, Tumor; Carcinoma; Endometrial Neoplasms; ErbB Receptors; Female; Humans; Neoplasm Staging; Prognosis; Receptor, ErbB-2; Survival Rate; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

We have previously shown that estrogen and progestins regulate both cellular proliferation and transforming growth factor (TGF) expression in human endometrial adenocarcinoma cells in vitro. In the current study we examined the regulation of TGF-alpha and -beta 1 expression in endometrial adenocarcinoma xenografts. Four human endometrial adenocarcinoma cell lines were inoculated into female BALB/c nude mice. Administration of 17 beta-estradiol (E2) increased tumor size in intact mice inoculated with Ishikawa, HEC-50 and HEC-1B cells but inhibited growth of HEC-1A xenografts. 4-Hydroxy tamoxifen (OH-Tam) had similar effects to E2 in animals carrying Ishikawa and HEC-1A cell xenografts but had no significant effect on growth of HEC-50 or HEC-1B xenografts. In intact mice inoculated with OH-Tam pellets and Ishikawa cells, the tumors were larger and had lower levels of TGF-alpha mRNA than in untreated or E2 treated mice. In mice carrying Ishikawa, HEC-50 and HEC-1B cell xenografts none of the hormones or agents tested altered TGF-beta 1 mRNA levels. In contrast, both E2 and OH-Tam significantly increased xenografts TGF-beta 1 mRNA levels in HEC-1A xenografts as well as significantly reduced tumor size. Medroxyprogesterone acetate (MPA) had no effect on tumor size of Ishikawa, HEC-1A and HEC-1B cell cell xenografts but significantly increased the size of HEC-50 xenografts. MPA significantly reduced TGF-alpha expression in Ishikawa cell xenografts but had no effect in the other cell xenografts. MPA had no effect on TGF-beta 1 expression in any of the xenografts. These observations demonstrate a discordance between the hormonal effects on TGF expression and cellular proliferation and argue against a major role for the TGFs in regulation of human endometrial adenocarcinoma cell proliferation in vivo.

Topics: Adenocarcinoma; Animals; Blotting, Northern; Cell Division; Dexamethasone; Dihydrotestosterone; Endometrial Neoplasms; Estradiol; Female; Gene Expression Regulation, Neoplastic; Hormones; Humans; Medroxyprogesterone Acetate; Mice; Mice, Inbred BALB C; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

The purpose of this study was to investigate the effect of medroxyprogesterone acetate on angiogenesis induced by endometrial cancer and to elucidate the possible mechanisms by which it inhibits the growth of the cancer.. Tumors were obtained from 29 patients with endometrial adenocarcinoma, and angiogenesis was assayed in corneas of white rabbits.. Transplantation of tumor tissues into rabbit corneas induced angiogenesis in 70.4% of the corneas, but their transplantation with a pellet of medroxyprogesterone acetate induced angiogenesis in only 21.5% of the corneas. This compound also inhibited angiogenesis induced by acidic fibroblast growth factor and transforming growth factor-alpha.. Inhibition of angiogenesis may be one mechanism by which medroxyprogesterone acetate inhibits the growth of endometrial adenocarcinoma, and its inhibition of neovascularization induced by adenocarcinoma may be through its direct action on endothelial cells.

Topics: Adenocarcinoma; Animals; Cornea; Endometrial Neoplasms; Female; Fibroblast Growth Factor 1; Humans; Medroxyprogesterone Acetate; Neoplasm Transplantation; Neovascularization, Pathologic; Rabbits; Transforming Growth Factor alpha

We have examined the effects of medroxyprogesterone acetate (MPA) and 4-hydroxytamoxifen (OH-TAM) on the cell proliferation and the expression of TGF-alpha and TGF-beta genes in Ishikawa cells and HEC-50 human endometrial adenocarcinoma cells. The effects of exogenous TGF-alpha, TGF-beta and anti-EGF receptor monoclonal antibody on cell proliferation were also determined. Antisense oligonucleotides were used to determine the effects of endogenous expression of TGF-alpha and TGF-beta. In both cell lines, MPA resulted in a time and dose-dependent inhibition of cell proliferation whereas OH-TAM had no effect on HEC-50 cell proliferation. The relative abundance of TGF-alpha mRNA was significantly reduced by MPA in Ishikawa cells but not in HEC-50 cells. In Ishikawa cells, a reduction in TGF-alpha mRNA abundance was observed with OH-TAM under conditions where both inhibition and stimulation of cell proliferation were demonstrated. Anti-EGF receptor monoclonal antibody inhibited Ishikawa cell growth but had little effect on HEC-50 cell proliferation. Exogenous TGF-alpha stimulated proliferation of both cell lines whereas exogenous TGF-beta inhibited proliferation of Ishikawa cells but stimulated proliferation of HEC-50 cells. Antisense oligonucleotides to TGF-beta inhibited proliferation of HEC-50 cells. From these data we conclude that the antiproliferative effects of progestins and OH-TAM on endometrial cancer cells appear to be mediated by different mechanisms.

Topics: Adenocarcinoma; Antineoplastic Agents; Cell Division; Cell Line; Endometrial Neoplasms; Estrogen Antagonists; Female; Gene Expression Regulation, Neoplastic; Humans; Medroxyprogesterone; Medroxyprogesterone Acetate; Oligonucleotides, Antisense; Steroids; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta

While antiestrogens are useful agents in the treatment of breast cancer, the usefulness of these agents in the treatment of endometrial cancer remains controversial. There is some concern that the currently available antiestrogens may have partial agonist activity in uterine tissue. To better understand the mechanisms by which estrogens and antiestrogens modulate growth of endometrial adenocarcinoma cells, we have compared the effects of 17-beta estradiol and three antiestrogens, 4-hydroxytamoxifen (OH-TAM), ICI 164384, and LY 117018 on proliferation and transforming growth factor (TGF) mRNA accumulation in two human endometrial adenocarcinoma cell lines. In HEC-50 cells, neither estradiol nor anti-estrogens had any effect on cell proliferation or TGF mRNA abundance under estrogen-depleted culture conditions [basal medium containing 1% twice charcoal-treated fetal bovine serum (ctFBS)] or in the presence of estrogen (basal medium containing 5% fetal bovine serum). At very high concentrations, both estradiol and OH-TAM caused a small decrease in HEC-50 cell proliferation in medium containing 5% serum. In contrast, the antiestrogens had different effects on Ishikawa cells, depending upon the culture conditions. In medium containing 5% fetal bovine serum, the antiestrogens inhibited cell proliferation and significantly decreased TGF-alpha mRNA abundance and TGF-alpha secretion. OH-TAM was more potent than the other antiestrogens. Under these culture conditions, estradiol had no effect on cell proliferation or TGF-alpha mRNA levels but increased TGF-alpha secretion. In medium supplemented with 1% ctFBS, estradiol increased cell proliferation and TGF-alpha mRNA (2.72-fold, P less than 0.005) and TGF-alpha secretion (700 +/- 156 versus 250 +/- 23 pg/10(6) cells/24 h, P less than 0.05), whereas OH-TAM, which also stimulated cell proliferation, reduced TGF-alpha mRNA abundance (P less than 0.05) but had no significant effect on TGF-alpha secretion. Under these conditions, ICI 164384 and LY 117018 had no effect on either cell proliferation or TGF-alpha expression. Estradiol treatment decreased, whereas OH-TAM increased, epidermal growth factor receptors in Ishikawa cells. Both estradiol and the antiestrogens decreased TGF-beta 1 mRNA abundance when cells were grown in media containing 1% ctFBS. In summary, the response of human endometrial adenocarcinoma cells to estrogen and antiestrogens varied between cell lines and was dependent upon the culture conditions use

Topics: Adenocarcinoma; Analysis of Variance; Cell Division; Cell Line; Dose-Response Relationship, Drug; Endometrial Neoplasms; ErbB Receptors; Estradiol; Estrogen Antagonists; Female; Gene Expression; Humans; Kinetics; Polyunsaturated Alkamides; Pyrrolidines; RNA, Messenger; Tamoxifen; Thiophenes; Transforming Growth Factor alpha; Tumor Cells, Cultured

The effects of the transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF) on the growth of cells from 2 endometrial cancer lines, Ishikawa and HEC-50 were evaluated by measuring rates of DNA synthesis and changes in cell numbers during culture. EGF at 17 and 1.7 nM concentrations consistently enhanced HEC-50 cell proliferation. TGF-beta 1 inhibited Ishikawa cell proliferation but, unexpectedly for epithelium-derived cells, stimulated HEC-50 cell growth. This effect is of interest as it indicates that endometrial cells can acquire an altered responsiveness to a growth inhibitor during the process of malignant transformation. Northern blot analyses showed expression of TGF-alpha, TGF-beta 1 and EGF receptors mRNA in both cell lines. Neither estradiol (E2) nor 4-hydroxytamoxifen (OHTam) affected mRNA levels for either TGF-alpha or TGF-beta in HEC-50 cells, a line unresponsive to E2 for proliferation. In Ishikawa cells, previously shown to respond to both E2 and OHTam by increasing proliferation rates, E2 increased TGF-alpha mRNA and reduced TGF-beta mRNA levels. OHTam lowered the levels of both mRNA species, although the effect was greater on TGF-beta than TGF-alpha mRNA. These data are consistent with, but do not prove, the existence of a possible autocrine regulation by TGF-alpha and TGF-beta of human cancer cell proliferation, which might be under E2 influence in Ishikawa cells.

Topics: Adenocarcinoma; Blotting, Northern; Cell Division; DNA; Endometrial Neoplasms; Epidermal Growth Factor; Estrogen Antagonists; Female; Humans; RNA, Messenger; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured