Antonin Libra

Expression and Transport Activity of Breast Cancer Resistance Protein (Bcrp/Abcg2) in Dually Perfused Rat Placenta and HRP-1 Cell Line

Breast cancer resistance protein (BCRP/ABCG2) is a member of the ATP-binding cassette transporter family that recognizes a variety of chemically unrelated compounds. Its expression has been revealed in many mammal tissues, including placenta. The purpose of this study was to describe its role in transplacental pharmacokinetics using rat placental HRP-1 cell line and dually perfused rat placenta. In HRP-1 cells, expression of Bcrp, but not P-glycoprotein, was revealed at mRNA and protein levels. Cell accumulation studies confirmed Bcrp-dependent uptake of BODIPY FL prazosin. In the placental perfusion studies, a pharmacokinetic model was applied to distinguish between passive and Bcrp-mediated transplacental passage of cimetidine as a model substrate. Bcrp was shown to act in a concentration-dependent manner and to hinder maternal-to-fetal transport of the drug. Fetal-to-maternal clearance of cimetidine was found to be 25 times higher than that in the opposite direction; this asymmetry was partly eliminated by BCRP inhibitors fumitremorgin C (2 μM) or N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918; 2 μM) and abolished at high cimetidine concentrations (1000 μM). When fetal perfusate was recirculated, Bcrp was found to actively remove cimetidine from the fetal compartment to the maternal compartment even against a concentration gradient and to establish a 2-fold maternal-to-fetal concentration ratio. Based on our results, we propose a two-level defensive role of Bcrp in the rat placenta in which the transporter 1) reduces passage of its substrates from mother to fetus but also 2) removes the drug already present in the fetal circulation.

EXPRESSION AND FUNCTIONAL ACTIVITY OF BREAST CANCER RESISTANCE PROTEIN (BCRP, ABCG2) TRANSPORTER IN THE HUMAN CHORIOCARCINOMA CELL LINE BEWO

1 Breast cancer resistance protein (BCRP, ABCG2) is a drug efflux transporter that is believed to affect the drug disposition of several drugs and xenobiotics. In the present study, we evaluated the localization and functional expression of BCRP in the human choriocarcinoma cell line BeWo, an in vitro model of the human trophoblast, and compared it with the expression of P‐glycoprotein (MDR1, ABCB1) as the most widely studied placental transporter. In addition, the expression of BCRP at the mRNA level was compared with that of MDR1 in the human term placenta. 2 Western blotting analysis revealed high endogenous expression of BCRP protein in BeWo cells. Using indirect immunofluorescence microscopy, we found that the BCRP transporter appears to be localized predominantly at the apical plasma membrane. Functional studies showed a significant effect of the BCRP inhibitors GF120918 (5 mmol/L) and Ko143 (1 mmol/L) on mitoxantrone accumulation and, thus, confirmed efflux activity of BCRP in BeWo cells. 3 Using absolute mRNA quantification with real‐time reverse transcription–polymerase chain reaction, we found high expression of BCRP in BeWo cells, whereas no transcript of MDR1 (P‐glycoprotein), the most extensively studied drug transporter, was detected. 4 In the human placenta, BCRP was localized predominantly in the syncytiotrophoblast layer; however, immunopositivity for the BXP‐21 antibody was also observed in fetal vessels of the chorionic villi. The number of BCRP transcripts in the human term placenta was found to be more than 10‐fold higher compared with the expression of MDR1. 5 In conclusion, we suggest that BeWo cells could serve as a suitable in vitro model to study trans‐trophoblast transport of BCRP substrates and that placental BCRP can play an important role in preventing the accumulation of potentially toxic xenobiotics in the trophoblast cells.

Molecular determinants in the transport of a bile acid-derived diagnostic agent in tumoral and nontumoral cell lines of human liver.

Contrast-enhanced magnetic resonance imaging (CE-MRI) is a valuable technique for the diagnosis of liver diseases. As gadocoletic acid trisodium salt (B22956/1), a new contrast agent showing high biliary excretion, may be potentially advantageous in hepatobiliary imaging, the aim of the study was to investigate the molecular mechanisms of hepatic transport of the B22956 ion in a cellular model of hepatic tumor. B22956 ion uptake was measured in tumoral (HepG2) and nontumoral (Chang liver) hepatic cell lines. Absolute quantitative real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) analyses, using cloned PCR products as standards, were performed on total RNA of both cell lines and normal liver to evaluate the transcription of 12 transport genes: SLCO1A2, SLCO2B1, SLCO1B1, SLCO3A1, SLCO4A1, SLCO1B3, SLC22A7, SLC22A8, SLC22A1, SLC10A1, SLC15A1, and SLC15A2. B22956 transport was more efficient in Chang liver than in HepG2 cells and was inhibited by cholecystokinin-8, a specific substrate of OATP1B3. Real-time RT-PCR analyses revealed different transcription profiles in the tumoral and nontumoral cell lines. Compared with normal liver, the expression of SLCO1B1, SLCO3A1, and SLCO1B3 was greatly repressed in HepG2 cells, whereas SLCO2B1, SLC22A7, and SLC22A8 expression was either maintained or increased. On the contrary, in Chang liver cells, SLC22A7 and SLC22A8 genes were undetectable, whereas the expression of SLCO3A1, SLCO4A1, and SLCO1B3 was similar to normal liver. Transport studies and gene expression analyses indicated that B22956 ion is a good substrate to the liver-specific OATP1B3, reported to be poorly expressed or absent in human liver tumors. Therefore, B22956 may be helpful in detecting hepatic neoplastic lesions by CE-MRI.

Effect of ABCG2 on cytotoxicity of platinum drugs: Interference of EGFP

ATP-binding drug efflux transporters decrease intracellular concentrations of cytotoxic drugs, causing multidrug resistance in cancer. In this study, we examined possible interactions of ABCG2 transporter with platinum cytotoxic drugs. We demonstrate here an interference of platinum drugs with enhanced green fluorescence protein (EGFP) in the cellular models, where EGFP was employed as a reporter gene. Cytotoxicity of cisplatin (CIP), carboplatin (CAP) and oxaliplatin (OXP) was significantly lowered in MDCKII cells transfected with ABCG2 transporter and EGFP reporter. The IC(50) values in MDCKII-ABCG2 were 25.7, 164 and 165 microM for CIP, CAP and OXP, respectively, whereas IC(50) for the same cytostatics in MDCKII cells were as follows: 15.4, 133 and 50.3 microM. Addition of fumitremorgin C (FTC), a potent ABCG2 inhibitor, significantly suppressed the resistance of MDCKII-ABCG2 to OXP, suggesting that OXP interacts with ABCG2. However, FTC did not change the sensitivity of the cells to CIP and CAP. We assume that EGFP rather than ABCG2 causes the diminished toxicity of the platinum cytostatics in the transfected cells. This hypothesis was confirmed in human Hep2 cells expressing EGFP: using MTT test, IC(50) of 30.0, 247 and 27.9 microM were obtained for CIP, CAP and OXP, respectively, while 12.3, 106 and 20.5 microM were observed in the parent Hep2 cells. Employing neutral red cytotoxicity assay, similar data were obtained (IC(50) 7.73, 685 and 112 microM for CIP, CAP, and OXP, respectively, in the Hep2-EGFP cells and 1.65, 79.4 and 24.5 microM in the parent Hep2 cells). Caspase-3/7 assay revealed lower susceptibility of EGFP expressing Hep2 cells to apoptosis induced by CIP when compared to the parent cell line. We therefore conclude that EGFP in transfected cells interferes with cytotoxicity of platinum drugs by hindering the drug induced apoptosis and could cause misinterpretation of results obtained in cytotoxicity studies.

Dexamethasone and betamethasone administration during pregnancy affects expression and function of 11β-hydroxysteroid dehydrogenase type 2 in the rat placenta

Placental 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) is the key enzyme which protects the fetus from overexposure to glucocorticoids (GCs) by their oxidation into inactive derivates. Several recent studies suggest that 11 beta-HSD2 expression is subjected to regulation by antenatal steroid therapy. In our study we investigated the effect of two commonly used synthetic steroids, dexamethasone (DXM) and betamethasone (BTM), on the expression and function of 11 beta-HSD2 in the rat placenta. Pregnant rats were pretreated with low (0.2mg/kg) or high (5mg/kg and 11.5mg/kg for DXM and BTM, respectively) i.m. doses of GCs. 11 beta-HSD2 expression was investigated using real-time RT-PCR and Western blotting; conversion capacity of 11 beta-HSD2 was assessed by dual perfusion of the rat placenta. Significant increase in placental 11 beta-HSD2 mRNA expression was found in rats treated with DXM, however, this alteration was not observed on protein level. BTM had no effect on either mRNA or protein levels of 11 beta-HSD2. Functional studies revealed that both GCs significantly reduced the metabolism of corticosterone by the placenta. Our data indicate that placental barrier function mediated by 11 beta-HSD2 might be considerably impaired by the antenatal therapy with DXM and BTM. In addition, the discrepancy between expressional and functional studies suggests that sole analysis of expressional changes of 11 beta-HSD2 at mRNA and/or protein levels cannot convincingly predict the role of GC treatment on 11 beta-HSD2 function in the placental barrier.

Modulation of keratin 1, 10 and involucrin expression as part of the complex response of the human keratinocyte cell line HaCaT to ultraviolet radiation

ABSTRACT Skin exposure to ultraviolet (UV) light evokes a complex stress response in keratinocytes. Keratin filament organization provides structural stability and mechanical integrity of keratinocytes. Involucrin is a transglutaminase substrate protein contributing to the formation of insoluble cornified envelopes. However, a more complex role for keratins and involucrin has been proposed, including the regulation of cell stress response. The aim was to evaluate modulations of keratin 1, 10 and involucrin expression in HaCaT in the light of the complex response of these cells to UV-B radiation, including effects on c-Jun and matrix metalloproteinase 1 (MMP-1) gene expression and production of interleukin (IL) 6 and 8. A UV-B (300±5 nm) dose of 10 mJ/cm2 was selected since this dose resulted in a partial decrease in cell viability in contrast to higher UV-B doses, which induced complete cell death 48 h after treatment. The UV-B radiation induced significant expression of keratin 1 and 10 and decreased expression of involucrin. This was accompanied by increased expression of c-Jun and MMP-1 and IL-6 and IL-8 production. The data suggest that the expression of keratin 1, 10 and involucrin is modulated in HaCaT keratinocytes as a part of the complex stress response to UV radiation

Mechanisms of ellipticine-mediated resistance in UKF-NB-4 neuroblastoma cells.

Most high‐risk neuroblastomas develop resistance to cytostatics and therefore there is a need to develop new drugs. In previous studies, we found that ellipticine induces apoptosis in human neuroblastoma cells. We also investigated whether ellipticine was able to induce resistance in the UKF‐NB‐4 neuroblastoma line and concluded that it may be possible after long‐term treatment with increasing concentrations of ellipticine. The aim of the present study was to investigate the mechanisms responsible for ellipticine resistance. To elucidate the mechanisms involved, we used the ellipticine‐resistant subline UKF‐NB‐4ELLI and performed comparative genomic hybridization, multicolor and interphase FISH, expression microarray, real‐time RT‐PCR, flow cytometry and western blotting analysis of proteins. On the basis of our results, it appears that ellipticine resistance in neuroblastoma is caused by a combination of overexpression of Bcl‐2, efflux or degradation of the drug and downregulation of topoisomerases. Other mechanisms, such as upregulation of enzymes involved in oxidative phosphorylation, cellular respiration, V‐ATPases, aerobic respiration or spermine synthetase, as well as reduced growth rate, may also be involved. Some changes are expressed at the DNA level, including gains, amplifications or deletions. The present study demonstrates that resistance to ellipticine is caused by a combination of mechanisms. (Cancer Sci 2012; 103: 334–341)

STUDYING LIVER REGENERATION BY MEANS OF MOLECULAR BIOLOGY: HOW FAR WE ARE IN INTERPRETING THE FINDINGS?

Liver regeneration in mammals is a unique phenomenon attracting scientific interest for decades. It is a valuable model for basic biology research of cell cycle control as well as for clinically oriented studies of wide and heterogeneous group of liver diseases. This article provides a concise review of current knowledge about the liver regeneration, focusing mainly on rat partial hepatectomy model. The three main recognized phases of the regenerative response are described. The article also summarizes history of molecular biology approaches to the topic and finally comments on obstacles in interpreting the data obtained from large scale microarray-based gene expression analyses.