Breast Cancer Resistance Protein Expression Research Articles

Reduction in ABCG2 mRNA Expression in Human Immortalised Brain Microvascular Endothelial Cells by Ferric Ammonium Citrate is Mediated by Reactive Oxygen Species and Activation of ERK1/2 Signalling.

The ATP-binding cassette (ABC) transport protein ABCG2 (also known as breast cancer resistance protein (BCRP)) is expressed at the luminal face of the blood-brain barrier (BBB), where it limits the brain uptake of a number of therapeutic drugs. We recently reported that the ABC efflux transporter P-glycoprotein (P-gp) was downregulated in human immortalised brain endothelial (hCMEC/D3) cells treated with ferric ammonium citrate (FAC). The aim of the present study, therefore, was to assess whether BCRP expression is also affected by FAC and identify any signalling mechanisms involved. ABCG2 mRNA was assessed by RT-qPCR. Protein levels of BCRP, phosphorylated extracellular-regulated kinases 1 and 2 (p-ERK1/2) and total ERK 1/2 were assessed by Western blot. Reactive oxygen species (ROS) levels were determined using 2',7'-dichlorofluorescin diacetate. Treatment of hCMEC/D3 cells with FAC (250 µM, 72 h) significantly reduced ABCG2 mRNA levels (32.2 ± 3.7%) without a concomitant reduction in BCRP protein expression. ABCG2 mRNA levels were restored to control levels when co-treated with the antioxidant N-acetylcysteine (NAC), suggesting the effect of FAC was mediated by a ROS-sensitive pathway. We also found that FAC-treatment was associated with increased levels of p-ERK1/2, suggesting involvement of the ERK1/2 signalling pathway in the observed ABCG2 mRNA downregulation. The ERK1/2 signalling pathway inhibitor U0126 restored p-ERK1/2 levels and partially attenuated the FAC-induced reduction in ABCG2 mRNA. This study suggests that FAC-induced downregulation of ABCG2 mRNA isdriven byROS and ERK1/2 signalling, mechanisms which may be exploited to modulate BCRP expression at the BBB.

Regulation of P-glycoprotein and Breast Cancer Resistance Protein Expression Induced by Focused Ultrasound-Mediated Blood-Brain Barrier Disruption: A Pilot Study.

The blood-brain barrier (BBB) controls brain homeostasis; it is formed by vascular endothelial cells that are physically connected by tight junctions (TJs). The BBB expresses efflux transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), which limit the passage of substrate molecules from blood circulation to the brain. Focused ultrasound (FUS) with microbubbles can create a local and reversible detachment of the TJs. However, very little is known about the effect of FUS on the expression of efflux transporters. We investigated the in vivo effects of moderate acoustic pressures on both P-gp and BCRP expression for up to two weeks after sonication. Magnetic resonance-guided FUS was applied in the striatum of 12 rats. P-gp and BCRP expression were determined by immunohistochemistry at 1, 3, 7, and 14 days postFUS. Our results indicate that FUS-induced BBB opening is capable of (i) decreasing P-gp expression up to 3 days after sonication in both the treated and in the contralateral brain regions and is capable of (ii) overexpressing BCRP up to 7 days after FUS in the sonicated regions only. Our findings may help improve FUS-aided drug delivery strategies by considering both the mechanical effect on the TJs and the regulation of P-gp and BCRP.

Modulation of monepantel secretion into milk by soy isoflavones.

Monepantel (MNP), a novel anthelmintic drug from amino-acetonitrile derivatives, is a substrate for breast cancer resistance protein (BCRP). BCRP-mediated milk secretion of drugs can be altered by isoflavones. In this study, we aimed to show how soy isoflavones and BCRP inhibitors genistein (GEN) and daidzein (DAI) can modulate the secretion of MNP into milk. Moreover, we observed that the expression of BCRP in the lactating mammary gland of sheep was significantly higher than in non-lactating sheep using Western blot analysis. These properties of MNP and MNPSO2 (monepantel sulfone, the major active metabolite of MNP), identified as a BCRP substrate in determining the interaction with BCRP, were examined by vesicular transport (VT) inhibition assays. In pharmacokinetic studies, we demonstrated the transport of MNP into milk in three experimental groups: G1 fed standard forage; G2 fed soy-enriched forage; G3 fed standard forage paired with orally administered exogenous GEN and DAI. The concentrations of MNP and MNPSO2 were analyzed by high-performance liquid chromatography. Compared to the control group (3.27 ± 1.13 vs. 5.46 ± 2.23), the AUC (0-840 h) milk/plasma ratio decreased by 40% in the soy-enriched diet group. The concentrations of GEN and DAI were determined using liquid chromatography coupled with tandem mass spectrometry in soy. A VT inhibition assay was conducted to determine the IC50 values for MNP and MNPSO2 as BCRP inhibitors. This study showed that milk excretion of a BCRP substrate, such as monepantel, can be diminished by the presence of isoflavones in the diet.

Mechanisms Underlying the Differences in the Pharmacokinetics of Six Active Constituents of Huangqi Liuyi Decoction between Normal and Diabetic Nephropathy Mouse Models.

The aim of this study was to explore the mechanisms underlying the differences in the pharmacokinetics of Huangqi Liuyi decoction extract (HQD) under physiological and pathological conditions. The roles of liver cytochrome P450 metabolic enzymes (Cyp450) and small intestinal transporters were also investigated. The cocktail probe drug method was used to investigate the effects of diabetic nephropathy (DN) and HQD on metabolic enzyme activity. The expression levels of liver Cyp450 metabolic enzymes (Cyp1A2, Cyp2C37, Cyp3A11, Cyp2E1, and Cyp2C11) and small intestinal transporters (breast cancer resistance protein (BCRP), P-glycoprotein (P-gp), organic cation transporters (OCTs), and multidrug resistance-associated protein (MRPs) were determined using western blot. Compared to normal mice, the expression of OCT1, OCT2, MRP1, and MRP2 was increased in DN mice, while that of P-gp and BCRP (P < 0.05 and P < 0.001) was inhibited. HQD inhibited expression of Cyp1A2 and Cyp3A11 and increased the expression of P-gp and BCRP in normal mice. In DN mice, HQD induced expression of BCRP and inhibited expression of Cyp2C37, Cyp3A11, OCT2, MRP1, and MRP2. The activity of each Cyp450 enzyme was consistent with changes in expression. The changes in pharmacokinetic parameters of HQD in DN might, in part, be secondary to decreased expression of P-gp and BCRP. HQD varied in regulating transporter activities between health and disease. These findings support careful application of HQD-based treatment in DN, especially in combination with other drugs.

Quantitative Comparison of Breast Cancer Resistance Protein (BCRP/ABCG2) Expression and Function Between Maternal Blood-Brain Barrier and Placental Barrier in Mice at Different Gestational Ages

Breast cancer resistance protein (BCRP) is expressed by brain capillary endothelial cells and at the interface between two placental syncytiotrophoblast layers in rodents and serves to suppress drug distribution to the brain and the fetus. The purpose of the present study is to determine and compare the apparent impact of a single BCRP molecule on drug transfer between the maternal blood-brain barrier and placental barrier in pregnant mice at different gestation ages. BCRP protein was quantified by liquid chromatography-tandem mass spectrometry. Genistein or dantrolene was continuously administered to pregnant Bcrp−/− or wild-type (WT) mice, and the brain-to-plasma concentration ratio in the mother (Kp,brain) and the fetal-to-maternal ratio of plasma concentrations (Kp,fp) were determined. At gestational day 15.5 (GD15.5), the protein amount of BCRP at the murine placental barrier was estimated to be approximately three times higher than at the maternal blood-brain barrier, but the levels were approximately the same at GD17.5 due to the decline of placental BCRP expression during gestation. On the other hand, the values of Bcrp−/−/WT ratio of Kp,brain for genistein and dantrolene were 6.1 and 3.8, respectively, while the Kp,fp ratios were all less than 2.0. These results indicate that the apparent impact of a single placental BCRP molecule on the restriction of drug distribution is much less than that of a single brain BCRP molecule, probably because the function of placental BCRP is attenuated by bypass transfer through the connexin26 gap junctions between adjacent syncytiotrophoblast layers. The present study also found that the expression amount of BCRP protein at the human placental barrier formed by the monolayer of syncytiotrophoblasts was lower than that in mice, but this species difference appears to be functionally compensated by the murine-specific bypass route through gap junctions, at least in part.

The Intake of Coffee Increases the Absorption of Aspirin in Mice by Modifying Gut Microbiome

The absorption of orally administered aspirin into the blood was affected by gastrointestinal environmental factors such as gut pH, digestive enzymes, and microbiota. The intake of coffee affects the pharmacological effects of aspirin. Therefore, we examined the gut microbiota-mediated effect of coffee bean extract (CBE) intake on the pharmacokinetics of aspirin in mice. The intake of CBE modified the gut microbiota composition and their α- and β-diversities: It decreased the Proteobacteria, Helicobacteriaceae, and Bacteroidaceae populations in the fecal microbiota composition, while the S24-7_f (Muribaculaceae) and Lactobacillaceae populations increased. The fecal aspirin-hydrolyzing activities of humans and mice to salicylic acid were 0.045 ± 0.036 μmole/h/g and 0.032 ± 0.003 μmole/h/g, respectively. However, CBE treatment significantly suppressed the aspirin-hydrolyzing activity in mice. Furthermore, the area under the serum concentration–time curves (AUCs) of aspirin and salicylic acid were 0.265 ± 0.050 µg·h/mL and 16.224 ± 5.578 µg·h/mL in CBE-treated mice, respectively, and 0.248 ± 0.042 µg·h/mL and 10.756 ± 2.071 µg·h/mL in control mice, respectively. Moreover, CBE treatment suppressed the multidrug resistance protein 4 (Mrp4) expression in the intestines of mice, while the P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) expression was not affected. Furthermore, the CBE-treated mouse fecal lysate suppressed Mrp4 expression in Caco-2 cells compared to that of vehicle-treated mice, while CBE treatment did not affect Mrp4 expression. Oral gavage of caffeine also suppressed the Mrp4 expression in the intestines of mice. These findings suggest that intake of coffee can increase the absorption of aspirin by modifying the gut microbiome.

Ko143 Reverses MDR in Glioblastoma via Deactivating P-Glycoprotein, Sensitizing a Resistant Phenotype to TMZ Treatment.

Over-expression of both P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) has been associated with multidrug-resistance in glioblastoma (GBM). Though previously studied broad-spectrum inhibitors of drug efflux pumps have failed to progress in clinical studies due to in vivo toxicity, research into clinically viable targeted inhibitors is needed. This study evaluated the effects of Ko143, a non-toxic analog of fumitremorgin C, on temozolomide (TMZ) efficacy in resistant glioblastoma stem cells. We used ATP-Glo assay to determine cell viabilities and flow cytometry to perform cell cycle analysis. Comparative gene expression was analysed through RT-qPCR. TMZ IC50 decreased 41.07% (p<0.01) in the resistant phenotype when delivered in combination with Ko143. Additionally, the TMZ-resistant phenotype (GBM146) displayed 44-fold greater P-gp expression than the TMZ-sensitive phenotype (GBM9) (p<0.01), yet a 0.6-fold lower BCRP expression. Ko143 potentiates TMZ efficacy and likely inhibits P-glycoprotein more potently than previously indicated. Further development of non-toxic, targeted inhibitors of drug efflux pumps for use in combinatorial chemotherapy may improve glioblastoma patient prognosis.

Fetal Membranes Contribute to Drug Transport across the Feto-Maternal Interface Utilizing the Breast Cancer Resistance Protein (BCRP).

During pregnancy, the placenta is established as a primary organ for drug transport at the maternal-fetal interface. The fetal membranes (FM) also form an interface with maternal tissues; however, their role in drug transport has not been previously investigated. Knowledge of drug transport across this feto-maternal interface along with the placenta can improve new drug development and testing for use during pregnancy. We also hypothesize that extracellular vesicles (exosomes 30–160 nm) released from the FM and placental cells may also contain drug transport proteins and might impact drug trafficking across the feto-maternal interfaces. The objectives were to (1) localize the breast cancer resistance protein (BCRP) in human FM; (2) determine the drug transport function of BCRP in chorion trophoblast cells (CTCs) of the FM; and (3) investigate the presence of BCRP in FM cell-derived exosomes, as a paracrine modifier of the tissue environment for transport functions. The gene and protein expressions of ABCG2/BCRP in FMs were determined by quantitative real-time PCR (qRT-PCR) and western blotting (WB) and were localized by immunohistochemistry (IHC). The surface expression of BCRP in FM cells was determined by flow cytometry. The functional role of BCRP was assessed by an EFFLUX dye multidrug resistance assay. The presence of BCRP in exosomes derived from CTCs and BeWo cells was examined using ExoView®. Data derived from CTCs are compared with placental trophoblast cells (BeWo). BCRP is expressed and localized in the fetal membrane, primarily in the chorion trophoblast cell layer and scarcely in the amnion epithelial layer (AEC), and primarily localized on both AEC and CTC cell surfaces. Efflux assay data showed that FM cells have similar drug resistance activity as BeWo cells, suggesting that FM also have drug transportation capabilities. BeWo- and CTC-derived exosomes expressed limited BCRP protein on the surface, so it was predominantly contained in the exosomal lumen. As far as we are aware, this is the first study to report BCRP expression in fetal membrane cells and as cargo in fetal membrane-derived exosomes. We report that fetal membrane cells are capable of drug transportation. Based on these results, investigational drug trials should include the FM and its exosomes as possible drug transportation routes in pregnancy.

Novel microtubule inhibitor SQ overcomes multidrug resistance in MCF-7/ADR cells by inhibiting BCRP function and mediating apoptosis

The occurrence of multidrug resistance (MDR) is one of the impediments in the clinical treatment of breast cancer, and MDR breast cancer has abnormally high breast cancer resistance protein (BCRP/ABCG2) expression. However, there are currently no clinical drugs that inhibit this target. Our previous study found that 2-Methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol (SQ0814061/SQ), a small molecule drug with low toxicity to normal tissues, could target microtubules, inhibit the proliferation of breast cancer, and reduce its migration and invasion abilities. However, the effect and the underlying mechanism of SQ on MDR breast cancers are still unknown. Therefore, in this study, we investigated the effect of SQ on adriamycin-resistant MCF-7 (MCF-7/ADR) cells and explored the underlying mechanism. The MTT assay showed that SQ had potent cytotoxicity to MCF-7/ADR cells. In particular, the results of western blot and flow cytometry proved that SQ could effectively inhibit the expression of BCRP in MCF-7/ADR cells to decrease its drug delivery activity. In addition, SQ could block the cell cycle at G2/M phase in parental and MCF-7/ADR cells, thereby mediating cell apoptosis, which was related with the inhibition of PI3K-Akt-MDM2 pathway. Taken together, our findings indicate that SQ overcomes multidrug resistance in MCF-7/ADR cells by inhibiting BCRP function and mediating apoptosis through PI3K-Akt-MDM2 pathway inhibition.

Sunitinib induced resistance of endothelial cells by up-regulating P-glycoprotein and PI3K/Akt pathway

Drug resistance is a crucial obstacle to achieve satisfactory chemotherapeutic effects. Numerous studies have shown that the PI3K/Akt signaling pathway plays a significant role in various processes of cellular events and tumor progression, while few studies have focused on the PI3K/Akt signaling pathway in drug resistance of endothelial cells. The present study aims to explore the relationship of PI3K/Akt signaling and cellular resistance to anticancer drugs in human microvessel endothelial cells (HMEC-1). We established stable sunitinib-resiatant human microvessel endothelial cells (HMEC-su) after long-term exposure to sunitinib (a small-molecule tyrosine kinase receptor inhibitor) for 12 months. HMEC-su showed significant alternations of cell morphology and exhibited a 2.32-fold higher IC50 of sunitinib than parental HMEC-1 cells. Expression of P-glycoprotein (P-gp) and breast cancer-resistance protein (ABCG2) which mediates drug efflux, increased significantly in HMEC-su lines compared with HMEC-1 cells by western blots assay. Our study further demonstrates that LY294002 (blocking the PI3K/Akt pathway) enhances the sensibility of HMEC-su to suntinib and inhibits the gene transcription and protein expression of P-gp, ABCG2 in HMEC-su cells. In conclusion, these results indicate that LY294002 could reverse P-gp and ABCG2 mediated-drug resistance to sunitinib in HMEC-su cells by inhibiting PI3K/Akt signaling.

Magnolol and Honokiol Inhibited the Function and Expression of BCRP with Mechanism Exploration.

Breast cancer resistance protein (BCRP), one of the ATP-binding cassette (ABC) transporters, was associated with the multidrug resistance (MDR) of chemotherapy. Magnolol (MN) and honokiol (HK) are major bioactive polyphenols of Magnolia officinalis. This study investigated the effects of MN and HK on the function and expression of BCRP for the purpose of developing BCRP inhibitor to overcome MDR. Cell lines including MDCKII-BCRP and MDCKII-WT were used for evaluating the function and expression of BCRP. The results showed that MN (100–12.5 µM) and HK (100–12.5 µM) significantly decreased the function of BCRP by 80~12% and 67~14%, respectively. In addition, MN and HK were verified as substrates of BCRP. Furthermore, MN and HK reduced the protein expression of BCRP, and inhibited the phosphorylation of epidermal growth factor receptor (EGFR) and phosphatidylinositol 3-kinase (PI3K). In conclusion, both MN and HK decreased the function and expression of BCRP via EGFR/PI3K signaling pathway. Therefore, both compounds were promising candidates for reversing the MDR of chemotherapy.

Protection of Hesperidin against Methotrexate- Induced Nephrotoxicity may be Mediated by Nrf2/HO-1 Pathway

Abstract: Background: Methotrexate (MTX), a successfully used chemotherapeutic in the treatment of various malignancies and autoimmune diseases, might cause severe nephrotoxicity. Here, we aimed at investigating possible nephroprotective effects of hesperidin (HES), a flavanone present in citrus fruits, against MTX-induced toxicity. Methods: Rats were divided into control, HES, MTX, and MTX/HES groups, where HES was administered in a dose of 100 mg/kg/day orally for 8 days and MTX in a single i.p. dose of 20 mg/kg on day 5 of the experiment. Results: Pretreatment with HES significantly improved MTXinduced deteriorated kidney function and structure, as well as reversed MTX effects on renal tumor necrosis factor (TNF)-α level and caspase 3 expression. MTX upregulated renal breast cancer resistance protein (BCRP); an efflux transporter that extrudes MTX from the kidney. Unfortunately, MTX/HES did not show a further increase in BCRP expression but rather showed downregulation. MTX also caused downregulation of renal nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1) expressions, whereas HES reversed the MTX effect and upregulated renal Nrf2/HO-1. Conclusion: HES conferred protection against MTX-mediated nephrotoxicity, at least in part via anti-inflammatory and anti-apoptotic mechanisms. Nrf2/HO-1 pathway, but not BCRP, may have a role in HES-induced nephroprotection against MTX toxicity. Key words: Methotrexate, Hesperidin, Nrf2, HO-1, TNF-α, Caspase 3, BCRP.

Exposure to High-Altitude Environment Is Associated with Drug Transporters Change: microRNA-873-5p-Mediated Alteration of Function and Expression Levels of Drug Transporters under Hypoxia.

Hypoxia is the main characteristic of a high-altitude environment, affecting drug metabolism. However, so far, the mechanism of microRNA (miRNA) involved in the regulation of drug metabolism and transporters under high-altitude hypoxia is still unclear. This study aims to investigate the functions and expression levels of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 2 (MRP2), breast cancer resistance protein (BCRP), peptide transport 1 (PEPT1), and organic anion-transporting polypeptides 2B1 (OATP2B1) in rats and colon cancer (Caco-2) cells after exposure to high-altitude hypoxia. The protein and mRNA expression of MDR1, MRP2, BCRP, PEPT1, and OATP2B1 were determined by Western blot and qPCR. The functions of MDR1, MRP2, BCRP, PEPT1, and OATP2B1 were evaluated by determining the effective intestinal permeability and absorption rate constants of their specific substrates in rats under high-altitude hypoxia, and uptake and transport studies were performed on Caco-2 cells. To screen the miRNA associated with hypoxia, Caco-2 cells were examined by high throughput sequencing. We observed that the miR-873-5p was significantly decreased under hypoxia and might target MDR1 and pregnane X receptor (PXR). To clarify whether miR-873-5p regulates MDR1 and PXR under hypoxia, Caco-2 cells were transfected with mimics or inhibitors of miR-873-5p and negative control (NC). The function and expression of drug transporters were found to be significantly increased in rats and Caco-2 cells under hypoxia. We found that miR-873-5p regulated MDR1 and PXR expression. Herein, it is shown that miRNA may affect the expression of drug transporter and nuclear receptor under hypoxia. SIGNIFICANCE STATEMENT: This study explores if alterations to the microRNAs (miRNAs), induced by high-altitude hypoxia, can be translated to altered drug transporters. Among miRNAs, which show a significant change in a hypoxic environment, miR-873-5p can act on the multidrug resistance protein 1 (MDR1) gene; however, there are multiple miRNAs that can act on the pregnane X receptor (PXR). This study speculates that the miRNA-PXR-drug transporter axis is important in the physiological disposition of drugs.

Negligible Effect of Quercetin in the Pharmacokinetics of Sulfasalazine in Rats and Beagles: Metabolic Inactivation of the Interaction Potential of Quercetin with BCRP.

Breast cancer resistance protein (BCRP) mediates pharmacokinetic drug interactions. This study evaluated the potential of quercetin to inhibit and induce BCRP in vitro and in vivo. The inhibition of BCRP was investigated for quercetin and its metabolites using BCRP/mBcrp1-overexpressing MDCKII cells by flow cytometry. The induction of BCRP was investigated in LS174T cells using quantitative PCR. The expression of rat BCRP in rat small intestine, liver, and kidney was also measured after multiple administrations of quercetin in rats (50, 100, and 250 mg/kg, seven days). The in vivo pharmacokinetic changes of sulfasalazine following single or multiple administration of quercetin in rats and beagles were investigated. Although the induction effect of quercetin on BCRP was observed in vitro, the in vivo expression of rat BCRP was not changed by multiple quercetin administrations. Oral administration of quercetin did not affect the plasma concentration or pharmacokinetic parameters of sulfasalazine, regardless of dose and dosing period in either rats or beagles. In addition, the inhibitory effect of quercetin metabolites on BCRP/mBcrp1 was not observed. These results suggest that the in vivo drug interaction caused by quercetin via BCRP was negligible, and it may be related to the metabolic inactivation of quercetin for the inhibition of BCRP.

Risk of Hematologic Events With Coadministration of Methotrexate and the Breast Cancer Resistance Protein Inhibitor Febuxostat.

The breast cancer resistance protein (BCRP) is a key drug transporter found in the liver, kidney, central nervous system, and gastrointestinal tract. Due to the wide expression of BCRP, interactions of other drugs with methotrexate (MTX) may differ in oral and intravenous MTX users, and understanding of these interactions may be useful in preventing severe adverse events. Febuxostat, a urate-lowering drug, inhibits BCRP. The objective of this study was to clarify the differences in the drug-drug interaction profiles of oral and intravenous methotrexate, associated with BCRP. We analyzed the Japanese Adverse Drug Event Report database and compared the frequency of hematologic events in patients taking oral and intravenous MTX, with or without the concomitant use of febuxostat or allopurinol. Hematologic events were defined as pancytopenia and neutropenia. Multiple logistic regression analysis was then used to identify the risk factors for hematologic events in oral and intravenous MTX users. We identified 8 453 oral and 810 intravenous MTX users with 546 and 126 cases of hematologic events, respectively. Compared with those not using febuxostat, a disproportionate number of hematologic events was observed in intravenous MTX users concomitantly using febuxostat (P < 0.01). The multivariate logistic analysis of intravenous MTX users showed that hematologic events were significantly associated with febuxostat use (P < 0.01) and age ≥ 60 years (P < 0.01). Our findings suggest that patients being treated with intravenous MTX who concomitantly use febuxostat may be at an increased risk of hematologic events, presumably due to BCRP-mediated drug-drug interaction.

BCRP Expression with Lipofuscin Accumulation in Abnormal Neurons from a Child with Transmantle Cortical Dysplasia (TMCD) and Refractory Epilepsy

Cerebral cortical development’s malformations, including the transmantel cortical dysplasia (TMCD), have been associated with refractory epilepsy (RE). Several ABC-transporters as “P-glycoprotein (P-gp), Multidrug resistance proteins (MRP-1) and breast Cancer Resistant Protein (BCRP)” are up-regulated in human epileptogenic brain lesions of RE, however they have not been explored in Transmantle cortical dysplasia (TMCD). We describe a 13 years old boy with Refractory Epilepsy (RE) and abnormal Magneic Resonance Image (MRI) (T1, FLAIR and T2) compatible with TMCD. Clinical follow-up, images and pathologic studies were developed by routinely methods. Epilepsy surgical treatment included total lesion resection with complete seizures remission at date. Deeper brain areas related with images findings, showed features of TMCD with abnormal ballooned neurons with high accumulation of PAS+, sudanophilic and autoflourescent lipopigment (LP). Immunohistochemistry, using primary monoclonal antibodies for P-gp, MVP and BCRP proteins, showed high expression of BCRP in several ballooned-LP+ cells. In contrast, P-gp and MVP were negative and MRP-1 has not been investigated. The links of BCRP with LP and AEDs are not known, however, the expression of BCRP in these LP+ ballooned neurons from the epileptogenic brain area, with P-gp/MVP negative results, suggest that BCRP could be associated to refractory epileptic phenotype.

Maternal exposure to bisphenol A induces fetal growth restriction via upregulating the expression of estrogen receptors.

Bisphenol A (BPA) accumulation in the placenta leads to fetal growth restriction (FGR). Here we aimed to explore the effect and the underlying mechanism of BPA exposure on fetal development. ELISA was performed to measure estrogen levels in human placenta and BeWo cells. qRT-PCR and Western blotting were conducted to determine the expression of estrogen receptors (ERs), breast cancer resistance protein (BCRP), the key enzymes for ER synthesis, and DNA methyltransferases (DNMTs). Bisulfite-sequencing PCR analysis was performed to measure CpG methylation in ER genes. Flow cytometry was used to examine cell apoptosis. We found that human FGR placentae had significantly increased BPA and estrogen levels and decreased BCRP levels compared with healthy placentae. BPA downregulated BCRP expression via ERs, and BCRP silencing promoted ER expression in BeWo cells. Compared with vehicle treatment, BPA treatment significantly enhanced the expression of key enzymes for estrogen synthesis and ERs in BeWo cells. BPA treatment inhibited CpG methylation in ER genes, along with downregulated DNMT1 expression and upregulated DNMT3a and DNMT3b expression. BPA treatment significantly promoted BeWo cell apoptosis compared with vehicle treatment. Importantly, ER inhibitor ICI-182780 significantly reversed all the BPA-induced effects on BeWo cells. In conclusion, BPA promotes estrogen production and cell apoptosis in BeWo cells via upregulating ER expression, leading to FGR.

Curcuma zedoaria petroleum ether extract reverses the resistance of triple-negative breast cancer to docetaxel via pregnane X receptor.

BackgroundTriple-negative breast cancer (TNBC) is characterized by its aggressiveness and poor prognosis. Docetaxel is the common chemotherapeutic drug used in the treatment of TNBC. However, resistance to docetaxel has limited the effectiveness of TNBC treatment. Petroleum ether extracts of Curcuma zedoaria (PECZ) can inhibit the proliferation of MDA-MB-231 cells. However, the effect of PECZ on docetaxel resistance is not clear.MethodsA docetaxel-resistant MDA-MB-231 (MDA-MB-231/docetaxel) cell line was established, and Cell Counting Kit-8 (CCK-8), quantitative real-time PCR (qRT-PCR), and western blotting assays were used to evaluate the effect of docetaxel resistance in MDA-MB-231 cells. Next, CCK-8 was also performed to detect the effect of docetaxel or the combination treatment of docetaxel and PECZ on the proliferation of MDA-MB-231/docetaxel cells. Thereafter, MDA-MB-231/docetaxel cells were subcutaneously injected into nude mice to induce a TNBC xenograft model, and the mice were divided into a model group, docetaxel group, PECZ group, and combination of docetaxel and PECZ group. Subsequently, hematoxylin and eosin (HE) staining, immunohistochemical, qRT-PCR, and western blotting were used to estimate the effect of pre-treatment with PECZ on docetaxel tolerance reversal.ResultsPECZ significantly inhibited the expression of pregnane X receptor (PXR), multidrug resistance 1 (MDR1), breast cancer resistance protein (BCRP), and cytochrome P-450 (CYP3A4) in MDA-MB-231/docetaxel cells. Only higher concentrations of docetaxel could inhibit the viability of MDA-MB-231/docetaxel cells. When pre-treated with PECZ, lower concentrations of docetaxel could significantly inhibit cell viability. Meanwhile, combination treatment also reduced the tumor volume, ameliorated the pathological change of tumor tissues, and down-regulated the expressions of PXR, MDR1, BCRP, and CYP3A4 (according to HE staining, immunohistochemical, qRT-PCR and western blotting results in vivo).ConclusionsOur research showed that PECZ reversed docetaxel resistance in TNBC by PXR both in vitro and in vivo, which provides the basis for further investigations into the potential therapeutic impact of docetaxel resistance in TNBC.

Altered Umbilical Cord Blood Nutrient Levels, Placental Cell Turnover and Transporter Expression in Human Term Pregnancies Conceived by Intracytoplasmic Sperm Injection (ICSI).

Assisted reproductive technologies (ART) may increase risk for abnormal placental development, preterm delivery and low birthweight. We investigated placental morphology, transporter expression and paired maternal/umbilical fasting blood nutrient levels in human term pregnancies conceived naturally (n = 10) or by intracytoplasmic sperm injection (ICSI; n = 11). Maternal and umbilical vein blood from singleton term (>37 weeks) C-section pregnancies were assessed for levels of free amino acids, glucose, free fatty acids (FFA), cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), very low-density lipoprotein (VLDL) and triglycerides. We quantified placental expression of GLUT1 (glucose), SNAT2 (amino acids), P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) (drug) transporters, and placental morphology and pathology. Following ICSI, placental SNAT2 protein expression was downregulated and umbilical cord blood levels of citrulline were increased, while FFA levels were decreased at term (p < 0.05). Placental proliferation and apoptotic rates were increased in ICSI placentae (p < 0.05). No changes in maternal blood nutrient levels, placental GLUT1, P-gp and BCRP expression, or placental histopathology were observed. In term pregnancies, ICSI impairs placental SNAT2 transporter expression and cell turnover, and alters umbilical vein levels of specific nutrients without changing placental morphology. These may represent mechanisms through which ICSI impacts pregnancy outcomes and programs disease risk trajectories in offspring across the life course.

Ganciclovir and Its Hemocompatible More Lipophilic Derivative Can Enhance the Apoptotic Effects of Methotrexate by Inhibiting Breast Cancer Resistance Protein (BCRP)

Efflux transporters, namely ATP-binding cassette (ABC), are one of the primary reasons for cancer chemoresistance and the clinical failure of chemotherapy. Ganciclovir (GCV) is an antiviral agent used in herpes simplex virus thymidine kinase (HSV-TK) gene therapy. In this therapy, HSV-TK gene is delivered together with GCV into cancer cells to activate the phosphorylation process of GCV to active GCV-triphosphate, a DNA polymerase inhibitor. However, GCV interacts with efflux transporters that are responsible for the resistance of HSV-TK/GCV therapy. In the present study, it was explored whether GCV and its more lipophilic derivative (1) could inhibit effluxing of another chemotherapeutic, methotrexate (MTX), out of the human breast cancer cells. Firstly, it was found that the combination of GCV and MTX was more hemocompatible than the corresponding combination with compound 1. Secondly, both GCV and compound 1 enhanced the cellular accumulation of MTX in MCF-7 cells, the MTX exposure being 13–21 times greater compared to the MTX uptake alone. Subsequently, this also reduced the number of viable cells (41–56%) and increased the number of late apoptotic cells (46–55%). Moreover, both GCV and compound 1 were found to interact with breast cancer resistant protein (BCRP) more effectively than multidrug-resistant proteins (MRPs) in these cells. Since the expression of BCRP was higher in MCF-7 cells than in MDA-MB-231 cells, and the cellular uptake of GCV and compound 1 was smaller but increased in the presence of BCRP-selective inhibitor (Fumitremorgin C) in MCF-7 cells, we concluded that the improved apoptotic effects of higher MTX exposure were raised mainly from the inhibition of BCRP-mediated efflux of MTX. However, the effects of GCV and its derivatives on MTX metabolism and the quantitative expression of MTX metabolizing enzymes in various cancer cells need to be studied more thoroughly in the future.