The Co-Expression of Estrogen Receptors ERα, ERß, and GPER in Endometrial Cancer.

Estrogens have important roles in endometrial cancer (EC) and exert biological effects through the classical estrogen receptors (ERs) ERα and ERß, and the G-protein-coupled ER, GPER. So far, the co-expression of these three types of ERs has not been studied in EC. We investigated ERα, ERß, GPER mRNA and protein levels, and their intracellular protein distributions in EC tissue and in adjacent control endometrial tissue. Compared to control endometrial tissue, immunoreactivity for ERα in EC tissue was weaker for nuclei with minor, but unchanged, cytoplasmic staining; mRNA and protein levels showed decreased patterns for ERα in EC tissue. For ERß, across both tissue types, the immunoreactivity was unchanged for nuclei and cytoplasm, although EC tissues again showed lower mRNA and protein levels compared to adjacent control endometrial tissue. The immunoreactivity of GPER as well as mRNA levels of GPER were unchanged across cancer and control endometrial tissues, while protein levels were lower in EC tissue. Statistically significant correlations of estrogen receptor α (ESR1) versus estrogen receptor ß (ESR2) and GPER variant 3,4 versus ESR1 and ESR2 was seen at the mRNA level. At the protein level studied with Western blotting, there was significant correlation of ERα versus GPER, and ERß versus GPER. While in clinical practice the expression of ERα is routinely tested in EC tissue, ERß and GPER need to be further studied to examine their potential as prognostic markers, provided that specific and validated antibodies are available.

The characterization of the human cell line Calu-3 under different culture conditions and its use as an optimized in vitro model to investigate bronchial epithelial function.

In this study we have investigated the effects of different cell culture conditions on the Calu-3 epithelial cell model. Calu-3 cells were cultured in media A-MEM at the air-liquid (A-L) or liquid-liquid (L-L) interface for one or three wks (weeks). Different cryomethods were tested and the cell line was characterized using histochemistry, immunofluorescence, transmission and scanning electron microscopy, transepithelial resistance (TEER) measurements, permeability studies, and gene profiling of 84 drug transporters. Cell culture was successful in A-MEM with only 2.5% FBS. Cell proliferation and viability depended on the cryopreservation method. All Calu-3 models expressed CK7, occludin, and E-cadherin. The A-L interface resulted in a more biomimetic native bronchial epithelium displaying pseudostratified columnar epithelium with more microvilli and secretory vesicles than at the L-L interface, where the epithelium was cuboidal, but exhibited higher TEER values and lower dextran permeabilities. Longer time in culture significantly decreased dextran permeability and increased the expression of specific drug transporters. Drug transporter expression was also notably influenced by the culture interface, where the A-L interface yielded a higher expression of drug transporter genes than the L-L interface. Since cell culture interface and time in culture affect Calu-3 cell differentiation, barrier integrity, permeability properties, and drug transporter expression, culture conditions need to be considered and standardized when using the Calu-3 cell line as an in vitro model for aerosol drug delivery and screening of bronchial drug candidates.

The characterization of the human nasal epithelial cell line RPMI 2650 under different culture conditions and their optimization for an appropriate in vitro nasal model.

PURPOSE: The further characterization of the cell line RPMI 2650 and the evaluation of different culture conditions for an in vitro model for nasal mucosa. METHODS: Cells were cultured in media MEM or A-MEM at air-liquid (A-L) or liquid-liquid (L-L) interfaces for 1 or 3 weeks. Different cryopreservation methods and cell culture techniques were evaluated with immunolabelling of junctional proteins, ultrastructural analysis using electron microscopy, transepithelial electrical resistance (TEER) measurements, permeation studies with dextran and jacalin, and gene expression profiling of 84 drug transporters. RESULTS: Cell proliferation and differentiation depended on the used medium. The established epithelia expressed occludin, claudin-1, and E-cadherin under all conditions. Cells grown at the A-L interface formed more layers and exhibited a higher TEER and lower dextran and jacalin permeability than at the L-L interface, where cells morphologically exhibited a more differentiated phenotype. The expression of ABC and SLC transporters depended on culture duration and interface. CONCLUSIONS: The RPMI 2650 cells form a polarized epithelium resembling nasal mucosa. However, different culture conditions have a significant effect on cell ultrastructure, barrier integrity, and gene expression, and should be considered when using this cell line as an in vitro model for drug permeability studies and screening of nasal drug candidates.

The endometrial cancer cell lines Ishikawa and HEC-1A, and the control cell line HIEEC, differ in expression of estrogen biosynthetic and metabolic genes, and in androstenedione and estrone-sulfate metabolism.

Estrogens have important roles in the pathogenesis of endometrial cancer. They can have carcinogenic effects through stimulation of cell proliferation or formation of DNA-damaging species. To characterize model cell lines of endometrial cancer, we determined the expression profiles of the estrogen receptors (ERs) ESR1, ESR2 and GPER, and 23 estrogen biosynthetic and metabolic genes, and investigated estrogen biosynthesis in the control HIEEC cell line and the Ishikawa and HEC-1A EC cell lines. HIEEC and Ishikawa expressed all ERs to different extents, while HEC-1A cells lacked expression of ESR1. Considering the estrogen biosynthetic and metabolic enzymes, these cells showed statistically significant different gene expression profiles for SULT2B1, HSD3B2, CYP19A1, AKR1C3, HSD17B1, HSD17B7, HSD17B12, CYP1B1, CYP3A5, COMT, SULT1A1, GSTP1 and NQO2. In these cells, E2 was formed from E1S and E1, while androstenedione was not converted to estrogens. HIEEC and Ishikawa had similar profiles of androstenedione and E1 metabolism, but hydrolysis of E1S to E1 was weaker in Ishikawa cells. HEC-1A cells were less efficient for activation of E1 into the potent E2, but metabolized androstenedione to other androgenic metabolites better than HIEEC and Ishikawa cells. This study reveals that HIEEC, Ishikawa, and HEC-1A cells can all form estrogens only via the sulfatase pathway. HIEEC, Ishikawa, and HEC-1A cells expressed all the major genes in the production of hydroxyestrogens and estrogen quinones, and in their conjugation. Significantly higher CYP1B1 mRNA levels in Ishikawa cells compared to HEC-1A cells, together with lack of UGT2B7 expression, indicate that Ishikawa cells can accumulate more toxic estrogen-3,4-quinones than HEC-1A cells, as also for HIEEC cells. This study provides further characterization of HIEEC, Ishikawa, and HEC-1A cells, and shows that they differ greatly in expression of the genes investigated and in their capacity for E2 formation, and thus they represent different in vitro models.

Increased levels of biglycan in endometriomas and peritoneal fluid samples from ovarian endometriosis patients.

In our previous low-density-array gene-expression analysis we found an increased expression of biglycan gene in ovarian endometriosis patients. In the present study we evaluated biglycan expression at the protein level in tissue, serum and peritoneal fluid (PF) from ovarian endometriosis patients, patients with benign ovarian cysts and healthy women. Twenty samples of endometriomas and 27 of control tissues (benign ovarian cysts and eutopic endometrium of healthy women) were obtained laparoscopically or by curettage. Serum and PF samples were collected from 56 ovarian endometriosis patients and 40 controls (patients with benign cysts and healthy women). Tissue biglycan levels and serum and PF biglycan concentrations were determined by Western blotting and ELISA, respectively. Biglycan was detected in endometriomas and in benign cysts tissues but differed in glycosylation levels. The PF biglycan concentrations were significantly increased in ovarian endometriosis patients (mean ± SD=220.3 ± 190.5 pg/mg protein) compared to the whole control group (101.9 ± 94.7 pg/mg protein, p<0.001), while serum concentrations did not differ significantly. Biglycan appears to be involved in ovarian pathologies and probably has different roles in benign cysts as compared to ovarian endometriomas.