Different Culture Conditions Affect Drug Transporter Gene Expression, Ultrastructure, and Permeability of Primary Human Nasal Epithelial Cells.

PURPOSE: This study aimed to characterize a commercially available primary human nasal epithelial cell culture and its gene expression of a wide range of drug transporters under different culture conditions. METHODS: Human nasal cells were cultured in three different types of culture media at the air-liquid (A-L) or liquid-liquid (L-L) interfaces for 1 or 3 wks. The effects of the different cell culture conditions were evaluated using light and electron microscopy, transepithelial electrical resistance (TEER) measurements, permeation studies with dextran, and gene expression profiling of 84 drug transporters. RESULTS: The type of culture medium affected cell ultrastructure, TEER, and dextran permeation across epithelia. The expression of 20 drug transporter genes depended on the culture interface and/or time in culture; the A-L interface and longer time in culture favored higher expression levels of five ABC and seven SLC transporters. CONCLUSIONS: Culture conditions influence the morphology, barrier formation, permeation properties, and drug transporter expression of human nasal epithelial cells, and this must be taken into consideration during the establishment and validation of in vitro models. A thorough characterization of a nasal epithelial model and its permeability properties is necessary to obtain an appropriate standardized model for the design of aerosol therapeutics and drug transport studies.

Demonstrating suitability of the Caco-2 cell model for BCS-based biowaiver according to the recent FDA and ICH harmonised guidelines.

OBJECTIVE: According to the regulatory guidelines, one of the critical steps in using in-vitro permeability methods for permeability classification is to demonstrate the suitability of the method. Here, suitability of the permeability method by using a monolayer of cultured epithelial cells was verified with different criteria. METHODS: Imaging with a transmission electron microscope was used for characterisation of the cells. Monolayer integrity was confirmed by transepithelial electrical resistance measurements and permeability of zero permeability marker compounds. Real-time polymerase chain reaction was employed to evaluate expression levels of 84 known transporters. Samples for bidirectional permeability determination were quantified by ultra-performance liquid chromatography. KEY FINDINGS: The Caco-2 cells grow in an intact monolayer and morphologically resemble enterocytes. Genes of 84 known transporters were expressed at different levels; furthermore, expression was time depended. Functional expression of efflux transporter P-glycoprotein was confirmed. We established a correlation between permeability coefficients of 21 tested drug substances ranging from low, moderate and high absorption with human fraction absorbed literature data (R2  = 0.84). CONCLUSIONS: Assay standardisation assures the consistency of experimental data. Only such fully characterised model has the ability to accurately predict drug's intestinal permeability at the early stage of research or for the BCS-based biowaiver application.

The Significance of the Sulfatase Pathway for Local Estrogen Formation in Endometrial Cancer.

Endometrial cancer (EC) is the most common estrogen-dependent gynecological malignancy in the developed World. To investigate the local formation of estradiol (E2), we first measured the concentrations of the steroid precursor androstenedione (A-dione) and the most potent estrogen, E2, and we evaluated the metabolism of A-dione, estrone-sulfate (E1-S), and estrone (E1) in cancerous and adjacent control endometrium. Furthermore, we studied expression of the key genes for estradiol formation via the aromatase and sulfatase pathways. A-dione and E2 were detected in cancerous and adjacent control endometrium. In cancerous endometrium, A-dione was metabolized to testosterone, and no E2 was formed. Both, E1-S and E1 were metabolized to E2, with increased levels of E2 seen in cancerous tissue. There was no significant difference in expression of the key genes of the aromatase (CYP19A1) and the sulfatase (STS, HSD17B1, HSD17B2) pathways in cancerous endometrium compared to adjacent control tissue. The mRNA levels of CYP19A1 and HSD17B1 were low, and HSD17B14, which promotes inactivation of E2, was significantly down-regulated in cancerous endometrium, especially in patients with lymphovascular invasion. At the protein level, there were no differences in the levels of STS and HSD17B2 between cancerous and adjacent control tissue by Western blotting, and immunohistochemistry revealed intense staining for STS and HSD17B2, and weak staining for SULT1E1 and HSD17B1 in cancerous tissue. Our data demonstrate that in cancerous endometrium, E2 is formed from E1-S via the sulfatase pathway, and not from A-dione via the aromatase pathway.

Correlation between erythropoietin receptor(s) and estrogen and progesterone receptor expression in different breast cancer cell lines.

Erythropoietin (EPO) receptor (EPOR) expression in breast cancer has been shown to correlate with the expression of estrogen receptor (ESR) and progesterone receptor (PGR) and to be associated with the response to tamoxifen in ESR+/PGR+ tumors but not in ESR- tumors. In addition, the correlation between EPOR and G protein-coupled estrogen receptor 1 [GPER; also known as G protein-coupled receptor 30 (GPR30)] has been reported, suggesting the prognostic potential of EPOR expression. Moreover, the involvement of colony stimulating factor 2 receptor, ß, low­affinity (CSF2RB) and ephrin type-B receptor 4 (EPHB4) as EPOR potential receptor partners in cancer has been indicated. This study analyzed the correlation between the expression of genes for EPO, EPOR, CSF2RB, EPHB4, ESR, PGR and GPER in the MCF-7, MDA-MB-361, T-47D, MDA-MB-231, Hs578Bst, SKBR3, MCF-10A and Hs578T cell lines. The cell lines were also treated with recombinant human EPO (rHuEPO) in order to determine its ability to activate the Jak/STAT5, MAPK and PI3K signaling pathways and modify cell growth characteristics. Expression analysis stratified the cell lines in 2 main clusters, hormone-dependent cell lines expressing ESR and PGR and a hormone-independent cluster. A significant correlation was observed between the expression levels of ESR and PGR and their expression was also associated with that of GPER. Furthermore, the expression of GPER was associated with that of EPOR, suggesting the connection between this orphan G protein and EPO signaling. A negative correlation between EPOR and CSF2RB expression was observed, questioning the involvement of these two receptors in the hetero-receptor formation. rHuEPO treatment only influenced the hormone-independent cell lines, since only the MDA-MB-231, SKBR3 and Hs578T cells responded to the treatment. The correlation between the expression of the analyzed receptors suggests that the receptors may interact in order to activate signaling pathways or to evade their inhibition. Therefore, breast cancer classification upon ESR, PGR and human epidermal growth factor receptor 2 (HER2) may not be sufficient for the selection of suitable treatment protocol. The expression of EPOR, GPER and EPHB4 may be considered as additional classification factors.

Disturbed balance between phase I and II metabolizing enzymes in ovarian endometriosis: a source of excessive hydroxy-estrogens and ROS?

Oxidative metabolism of estrogens was studied in 31 ovarian endometriosis and 29 normal endometrium samples, by qPCR. Expression was monitored for genes encoding five estrogen hydroxylating, five hydroxy (OH)-estrogen conjugating, and three estrogen quinone detoxifying enzymes. CYP1B1, COMT, NQO1, and GSTP1 protein levels were determined using Western blotting and immunohistochemistry staining. Increased expression of CYP1A1, CYP3A7 and COMT, and higher levels of MB-COMT were seen in endometriosis, as compared to normal endometrium. Expression of CYP1B1, CYP3A5, SULT1A1 and NQO2 was unchanged, with comparable CYP1B1 protein levels. Expression of SULT1E1, SULT2B1, UGT2B7, NQO1, and GSTP1 was decreased. Three NQO1 isoforms were detected; NQO1c appears to be endometriosis-specific. Our data indicate a disturbed balance between phase I and II metabolizing enzymes in endometriosis, potentially leading to excessive OH-estrogen and altered ROS formation, and stimulation of proliferation of ectopic endometrium. This is the first report on disturbed expression of estrogen oxidative metabolism genes in ovarian endometriosis.

Altered expression of genes involved in progesterone biosynthesis, metabolism and action in endometrial cancer.

Endometrial cancer (EC) is one of the most common gynecological malignancies worldwide. It is associated with prolonged exposure to estrogens that is unopposed by the protective effects of progesterone, which suggests that altered progesterone biosynthesis, metabolism and actions might be implicated in the development of EC. Our aim was to evaluate these processes through quantitative real-time PCR expression analysis in up to 47 pairs of EC tissue and adjacent control endometrium. First, we examined the expression of genes encoding proteins associated with progesterone biosynthesis: steroidogenic acute regulatory protein (STAR); a side chain cleavage enzyme (CYP11A1); and 3ß-hydroxysteroid dehydrogenase/ketosteroid isomerase (HSD3B). There were 1.9- and 10.0-fold decreased expression of STAR and CYP11A1, respectively, in EC versus adjacent control endometrium, with no significant differences in the expression of HSD3B1 and HSD3B2. Next, we examined expression of genes encoding five progesterone metabolizing enzymes: the 3-keto and 20-ketosteroid reductases (AKR1C1-AKR1C3) and 5α-reductases (SRD5A1 and SRD5A2); and the opposing 20α-hydroxysteroid dehydrogenase (HSD17B2). These genes are expressed in EC and adjacent control endometrium. No statistically significant differences were seen in mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1. Expression of HSD17B2 was 3.0-fold increased, and expression of SRD5A2 was 3.7-fold decreased, in EC versus adjacent control endometrium. We also examined mRNA levels of progesterone receptors A and B (PGR), and separately the expression of progesterone receptor B (PR-B). Here we saw 1.8- and 2.0-fold lower mRNA levels of PGR and PR-B, respectively, in EC versus adjacent control endometrium. This down-regulation of STAR, CYP11A1 and PGR in endometrial cancer may lead to decreased progesterone biosynthesis and actions although the effects on progesterone levels should be further studied.

Decreased levels of AKR1B1 and AKR1B10 in cancerous endometrium compared to adjacent non-cancerous tissue.

Endometrial cancer is associated with enhanced cell proliferation due to high concentrations of estrogens, and decreased cell differentiation due to low levels of progesterone and retinoic acid. It is also associated with aberrant inflammatory responses and concomitant increased production of prostaglandins. The human members of the aldo-keto reductase 1B (AKR1B) subfamily, AKR1B1 and AKR1B10, have roles in these processes and can thus be implicated in endometrial cancer. To date, there have been no reports on the expression of AKR1B1 in endometrial cancer, while AKR1B10 has only been studied at the cellular level. To evaluate the roles of these AKR1B enzymes, we investigated expression of AKR1B1 and AKR1B10 in 47 paired samples of cancerous and adjacent control endometrium at the mRNA and protein levels, by quantitative PCR, Western blotting and immunohistochemistry staining. There were significantly lower mRNA and protein levels of AKR1B1 in cancerous tissues compared to adjacent endometrium. The gene expression of AKR1B10 at the mRNA level was significantly increased, while there were significantly decreased protein levels. Immunohistochemistry revealed that both of these enzymes were present in all of the samples, and are located in epithelial cells of cancerous and control endometrial glands. Elevated levels in adjacent non-cancerous tissues imply that these enzymes are more important in the initiation of endometrial cancer than in its progression. To the best of our knowledge, this is the first report on the expression of AKR1B1 and AKR1B10 in endometrial cancer. Further studies are needed to define the precise roles of these enzymes in the pathogenesis of endometrial cancer.

Disturbed expression of phase I and phase II estrogen-metabolizing enzymes in endometrial cancer: lower levels of CYP1B1 and increased expression of S-COMT.

Expression levels of genes encoding phase I and phase II estrogen-metabolizing enzymes: CYP1A1, CYP1A2, CYP1B1, CYP3A5, CYP3A7, SULT1A1, SULT1E1, SULT2B1, COMT, UGT2B7, and GSTP1 were studied by real-time PCR in 38 samples of cancerous and adjacent control endometrium. We found significantly lower levels of CYP1B1 and CYP3A7, higher levels of SULT2B1, UGT2B7 and GSTP1, and no differences in expression of COMT, CYP1A1, CYP3A5, SULT1E1 and SULT2A1 in the endometrial cancers. The CYP1B1 and COMT proteins were also examined by Western blotting and immunohistochemical staining, supporting the real-time PCR analysis. Lower levels of CYP1B1 detected in cancerous endometrium suggest its important role in control, precancerous tissue. Additionally, we showed for the first time higher protein levels of soluble COMT in cancerous endometrium, and higher levels of membrane-bound COMT in control, precancerous endometrium. The importance of the changed ratio between soluble and membrane-bound COMT still needs to be evaluated in further studies.

Disturbed estrogen and progesterone action in ovarian endometriosis.

Endometriosis is a very common disease in pre-menopausal women, where defective metabolism of steroid hormones plays an important role in its development and promotion. In the present study, we have examined the expression of 11 estrogen and progesterone metabolizing enzymes and their corresponding receptors in samples of ovarian endometriomas and control endometrium. Expression analysis revealed significant up-regulation of enzymes involved in estradiol formation (aromatase, sulfatase and all reductive 17beta-hydroxysteroid dehydrogenases) and in progesterone inactivation (AKR1C1 and AKR1C3). Among the estrogen and progesterone receptors, ERalpha was down-regulated, ERbeta was up-regulated, and there was no significant difference in expression of progesterone receptors A and B (PRAB). Our data indicate that several enzymes of estrogen and progesterone metabolism are aberrantly expressed in endometriosis, which can lead to increased local levels of mitogenic estradiol and decreased levels of protective progesterone. Changes in estrogen receptor expression suggest that estradiol may also act via non-estrogen receptor-mediated pathways, while expression of progesterone receptors still needs further investigation.