Breast Cancer Resistance Protein Levels Research Articles

Early in vitro evidence indicates that deacetylated sialic acids modulate multi-drug resistance in colon and lung cancers via breast cancer resistance protein.

Cancers utilize sugar residues to engage in multidrug resistance. The underlying mechanism of action involving glycans, specifically the glycan sialic acid (Sia) and its various functional group alterations, has not been explored. ATP-binding cassette (ABC) transporter proteins, key proteins utilized by cancers to engage in multidrug resistant (MDR) pathways, contain Sias in their extracellular domains. The core structure of Sia can contain a variety of functional groups, including O-acetylation on the C6 tail. Modulating the expression of acetylated-Sias on Breast Cancer Resistance Protein (BCRP), a significant ABC transporter implicated in MDR, in lung and colon cancer cells directly impacted the ability of cancer cells to either retain or efflux chemotherapeutics. Via CRISPR-Cas-9 gene editing, acetylation was modulated by the removal of CAS1 Domain-containing protein (CASD1) and Sialate O-Acetyl esterase (SIAE) genes. Using western blot, immunofluorescence, gene expression, and drug sensitivity analysis, we confirmed that deacetylated Sias regulated a MDR pathway in colon and lung cancer in early in vitro models. When deacetylated Sias were expressed on BCRP, colon and lung cancer cells were able to export high levels of BCRP to the cell's surface, resulting in an increased BCRP efflux activity, reduced sensitivity to the anticancer drug Mitoxantrone, and high proliferation relative to control cells. These observations correlated with increased levels of cell survival proteins, BcL-2 and PARP1. Further studies also implicated the lysosomal pathway for the observed variation in BCRP levels among the cell variants. RNASeq data analysis of clinical samples revealed higher CASD1 expression as a favorable marker of survival in lung adenocarcinoma. Collectively, our findings indicate that deacetylated Sia is utilized by colon and lung cancers to engage in MDR via overexpression and efflux action of BCRP.

Transporter and metabolic enzyme-mediated intra-enteric circulation of SN-38, an active metabolite of irinotecan: A new concept

SN-38, an active metabolite of irinotecan (CPT-11), is thought to circulate enterohepatically via organic anion-transporting polypeptides (OATPs), UDP-glucuronyl transferases (UGTs), multidrug resistance-related protein 2 (MRP2), and breast cancer resistance protein (BCRP). These transporters and enzymes are expressed in not only hepatocytes but also enterocytes. Therefore, we hypothesized that SN-38 circulates between the intestinal lumen and the enterocytes via these transporters and metabolic enzymes. To test this hypothesis, metabolic and transport studies of SN-38 and its glucuronide (SN-38G) were conducted in Caco-2 cells. The mRNA levels of UGTs, MRP2, BCRP, and OATP2B1 were confirmed in Caco-2 cells. SN-38 was converted to SN-38G in Caco-2 cells. The efflux of intracellularly generated SN-38G across the apical (digestive tract) membranes was significantly higher than the efflux across the basolateral (blood, portal vein) membranes of Caco-2 cells cultured on polycarbonate membranes. SN-38G efflux to the apical side was significantly reduced in the presence of MRP2 and BCRP inhibitors, suggesting that SN-38G is transported across the apical membrane by MRP2 and BCRP. Treatment of Caco-2 cells with OATP2B1 siRNA increased the SN-38 residue on the apical side, confirming that OATP2B1 is involved in the uptake of SN-38 into enterocytes. No SN-38 was detected on the basolateral side with or without siRNA treatment, suggesting that the enterohepatic circulation of SN-38 is limited, contrary to previous reports. These results suggest that SN-38 is absorbed into the enterocytes via OATP2B1, glucuronidated by UGTs to SN-38G, and excreted into the digestive tract lumen by MRP2 and BCRP. SN-38G can be deconjugated by β-glucuronidase from intestinal bacteria in the digestive tract lumen to regenerate SN-38. We named this new concept of local drug circulation “intra-enteric circulation.” This mechanism may allow SN-38 to circulate in the intestine and cause the development of delayed diarrhea, a serious side effect of CPT-11.

Clinical Significance of ABCG2/BCRP Quantified by Fluorescent Nanoparticles in Breast Cancer Patients Undergoing Neoadjuvant Chemotherapy

Breast cancer resistance protein (BCRP), also known as ATP-binding cassette transporter G2 (ABCG2), is associated with chemotherapy resistance. BCRP is also implicated in breast cancer stem cells, and is reported as a poor prognostic factor. However, the relationship of BCRP levels in breast cancer tissues with chemotherapy resistance and prognosis has not been clarified. We aimed to evaluate the correlation between BCRP expression and prognosis in breast cancer using immunohistochemistry with fluorescent phosphor-integrated dots (IHC-PIDs). A total of 37 breast cancer patients with residual cancer in the primary tumor and axillary lymph nodes were evaluated. BCRP levels in breast cancer tissue and metastatic lymph nodes were quantitatively detected after neoadjuvant chemotherapy (NAC). Among these 37 patients, 24 had corresponding core needle biopsies obtained before NAC. Biomarker assay with IHC-PIDs showed high accuracy for the quantitative assessment of BCRP with low expression. High BCRP expression in the primary tumor and metastatic lymph nodes after preoperative chemotherapy was associated with worse overall survival. In conclusion, high BCRP levels may be associated with poor prognosis in patients with breast cancer, having residual tumors within the primary tumor and lymph nodes after preoperative chemotherapy. These findings provide a basis for further appropriate adjuvant therapy in these patients.

Abstract P3-05-15: Clinical significance of breast cancer resistance protein (BCRP) quantified by fluorescence nanoparticle in breast cancer patients undergoing neoadjuvant chemotherapy

Abstract Breast cancer resistance protein (BCRP), also known as ATP-binding cassette transporter G2 (ABCG2), is associated with chemotherapy resistance. BCRP is also implicated in breast cancer stem cells and has been reported as a poor prognostic factor. However, the relationship of BCRP levels in breast cancer tissues with chemotherapy resistance and prognosis has not been clarified to date. Our laboratory recently developed nanoparticles suitable for the quantitative immunohistochemical (IHC) method called phosphor-integrated dots (PIDs). Using PID-based IHC (IHC-PIDs), many biomarker proteins can be visualized and quantitatively analyzed at the single-particle level in paraffine-embedded, formalin-fixed tumor samples. We aimed to evaluate the correlation between quantitatively analyzed BCRP expression and prognosis in breast cancer tissue samples using immunohistochemistry with IHC-PIDs. Thirty-seven Japanese patients diagnosed with invasive ductal carcinoma of the breast who underwent mastectomy and lymph node dissection at Tohoku University Hospital (Sendai, Japan) between 2004 and 2010 were included. All patients had undergone NAC and were pathologically diagnosed with non-pathological complete response or progressive disease and lymph node metastasis postoperatively. The patients were followed up within a median of 10.1 years (range, 1.1–18.2 years). Among the thirty-seven patients, twenty-eight patients had core needle biopsy specimens taken before NAC. BCRP levels in breast cancer tissue and metastatic lymph nodes were quantitatively detected after preoperative chemotherapy and core needle biopsy. Biomarker assay with IHC-PIDs showed high accuracy for quantitative assessment of BCRP with low expression. The optimal PT+LN BCRP cut-off score for predicting survival was 27.4, with a sensitivity of 60.6% and a specificity of 77.3%. BCRP expression in PT+LN samples was dichotomized into high and low groups at this cutoff value, and OS was compared between these group. The results showed that OS was significantly worse in the high BCRP-expression group (log-rank p=0.0089). In conclusion, high BCRP levels are associated with poor prognosis in patients with breast cancer with residual tumors within the primary tumor and lymph nodes after preoperative chemotherapy. These findings provide a basis for further appropriate adjuvant therapy in these patients. Citation Format: Hiroshi Tada, Kohsuke Gonda, Narufumi Kitamura, Minoru Miyashita, Narumi Harada-Shoji, Yohei Hamanaka, Akiko Ebata, Takanori Ishida. Clinical significance of breast cancer resistance protein (BCRP) quantified by fluorescence nanoparticle in breast cancer patients undergoing neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-15.

Correlation between the mRNA levels of BCRP and LUNX genes and pathological types and stages of patients with non-small cell lung cancer

To analyze the correlation between the mRNA levels of breast cancer resistance protein (BCRP) and lung-specific X protein (LUNX) genes with pathological types and stages of patients with non-small cell lung cancer (NSCLC) and their significance for prognosis. Eighty nine patients with NSCLC admitted to Huaihe Hospital of Henan University between June 2015 and June 2018 were recruited, with 55 patients with benign lung lesions admitted during the same period of time selected as the control group. The mRNA levels of BCRP and LUNX genes were detected in the peripheral blood samples from the two groups, and their correlation with the clinicopathological characteristics and prognosis of the patients was analyzed. The expression rates of BCRP and LUNX mRNA in the NSCLC group were significantly higher compared with the control group (P < 0.05). The level of BCRP mRNA of the NSCLC patients has correlated with the degree of differentiation and TNM staging (P < 0.05), but not with gender, age, smoking, pathological types and lymph node metastasis (P > 0.05). The level of LUNX mRNA of them has correlated with the degree of differentiation, TNM staging and lymph node metastasis (P < 0.05), but not with gender, age, smoking, and pathological types (P > 0.05). Compared with those with no expression, the overall survival rate of patients with BCRP and LUNX expression was significantly lower (P < 0.05). The degree of differentiation, TNM staging, lymph node metastasis, and expression of the BCRP and LUNX mRNA may all affect the prognosis of the patients. The levels of BCRP and LUNX mRNA in the peripheral blood of patients with NSCLC are significantly increased. The expression of BCRP mRNA is correlated with the degree of differentiation and TNM staging, whilst the expression of LUNX mRNA is correlated with the differentiation degree, TNM staging and lymph node metastasis. Both may be used as independent predictors for the prognosis of patients with NSCLC.

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.

Cranberry Ingestion Modulated Drug Transporters and Metabolizing Enzymes: Gefitinib Used as a Probe Substrate in Rats.

Cranberry, a polyphenol-rich functional food, is commonly used for the prophylaxis of urinary tract infections. Gefitinib, an anticancer agent clinically prescribed to treat non-small-cell lung cancer, is a substrate of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), and metabolized mainly by cytochrome P450 (CYP) 3A4 and CYP2D6. This study used gefitinib as a probe substrate to investigate the modulation of cranberry on P-gp, BCRP, CYP3A4 and CYP2D6. Rats were administered gefitinib with and without 5.0 g/kg of cranberry as juice (CJ). The concentration of gefitinib in serum was determined by LC-MS/MS. The results showed that CJ significantly increased the Cmax and AUC0-t of gefitinib by 28% and 55%, respectively. Mechanism studies indicated that CJ activated P-gp, and cranberry metabolites (CM) inhibited CYP2D6. Moreover, the protein level of P-gp in rat enterocytes was decreased, whereas that in hepatocytes was increased. In addition, the protein levels of BCRP, CYP3A4 and CYP2D6 in enterocytes and hepatocytes were decreased. In conclusion, CJ ingestion affected the activities and protein levels of P-gp, BCRP, CYP3A4 and CYP2D6.

Evaluation of Potential Herb-Drug Interactions Between Shengmai Injection and Losartan Potassium in Rat and In Vitro

Aim: The present study aimed to explore the potential herb-drug interactions (HDI) between Shengmai injection (SMI) and losartan potassium (LOS) based on the expression profiles of cytochromes P450 (CYP450) and drug transporters in rat and in vitro. Methods: Different concentrations of SMI were used to explore the influence of SMI on the antihypertensive efficacy of LOS in the hypertension rat model established by N (omega)-nitro-L-arginine methyl ester (L-NAME) for 4 weeks. Subsequently, the serum concentration levels of LOS and losartan carboxylic acid (EXP3174) were determined by Liquid Chromatography Mass Spectrometry (LC-MS) and pharmacokinetic analysis. Human liver microsomes, human multidrug resistance protein 1 (MDR1/P-gp), and breast cancer resistance protein (BCRP) vesicles, human embryonic kidney 293 cell line with stable expression of the organic anion transporting polypeptide 1B1 (HEK293-OATP1B1 cells) and mock-transfected HEK293 (HEK293-MOCK) cells were used to verify the effects of SMI on CYP450 enzymes and drug transporters in vitro. Results: Low, medium, and high concentrations of SMI increased the antihypertensive efficacy of LOS to varying degrees. The high dose SMI increased the half-life (t 1/2 ), the maximum plasma concentration (C max), the area under the plasma concentration-time curve (AUC) from time zero to the time of the last measurable plasma concentration (AUC 0-t ), AUC from time zero to infinity (AUC 0-∞ ), and mean residence time (MRT) values of LOS and decreased its apparent volume of distribution (Vd) and clearance (CL) values. The AUC 0-t , AUC 0-∞ , and MRT of LOS were increased, whereas the CL was decreased by the medium concentration of SMI. In addition, the high, medium, and low doses of SMI increased the relative bioavailability (Frel) of LOS. SMI exhibited no significant effects on the pharmacokinetics of EXP3174. In vitro, SMI exhibited different suppressive effects on the enzyme activity levels of CYP1A2 (6.12%), CYP2B6 (2.72%), CYP2C9 (14.31%), CYP2C19 (12.96%), CYP2D6 (12.26%), CYP3A4 (3.72%), CYP2C8 (10.00–30.00%), MDR1 (0.75%), OATP1B1(2.03%), and BCRP (0.15%). Conclusion: In conclusion, SMI improved the antihypertensive efficacy of LOS in the L-NAME-induced hypertension rat model by increasing the concentration of LOS, while leaving the concentration of EXP3174 intact. SMI affected the pharmacokinetic properties of LOS by decreasing the elimination of LOS. These effects might partly be attributed to the inhibition of the activities of CYP3A4, CYP2C9, and of the drug transporters (P-gp, BCRP, and OATP1B1) by SMI, which need further scrutiny.

A nanotherapeutic strategy to overcome chemoresistance to irinotecan/7-ethyl-10-hydroxy-camptothecin in colorectal cancer

Clinical development of 7-ethyl-10‑hydroxy-camptothecin (SN38), the active metabolite of irinotecan (CPT-11), is hindered by its insolubility and poor stability. Another obstacle is that tumors could become resistant to SN38/CPT-11 through multiple mechanisms involving breast cancer resistance protein (BCRP). Herein one of the most potent and selective BCRP inhibitors, Ko143, is encapsulated into a recently constructed prodrug PEG-S-S-SN38 displaying a high and fixed drug loading, multiple intratumoral stimuli (oxidative stress, GSH and esterase)-responsive drug release and significant in vitro and in vivo superiorities over CPT-11. The obtained “combo” for simultaneous delivery of SN38 and Ko143, named as BI@PEG-SN38, has a high SN38 loading efficacy (14.85 wt.%) and a good Ko143 encapsulation efficacy (3.79%). Through generating panels of human colorectal cancer models expressing altered levels of BCRP via lentiviral transfection and CRISPR-Cas9, characteristics of different drug formulations are carefully evaluated. Impressively, BI@PEG-SN38 nanoparticles effectively reverse chemoresistance to CPT-11 (resistance index dropping from ∼274.00–456.00 to ∼1.70–4.68) and PEG-S-S-SN38 (resistance index dropping from ∼5.83–14.00 to ∼1.70–4.68) in three BCRP-overexpressing cancer cell lines. More importantly, reversal of BCRP-mediated chemoresistance to CPT-11 (P values lower than 0.001–0.0001) and PEG-S-S-SN38 (P values lower than 0.01–0.001) by BI@PEG-SN38 nanoparticles are further confirmed with two panels of colorectal cancer xenograft models in vivo. As the first nano-formulation of Ko143 and the first systemic co-delivery vehicle of SN38/CPT-11 and a BCRP inhibitor, BI@PEG-SN38 provides a new approach for resolving the bottlenecks for clinical translation of SN38 and numerous “chemosensitizers” like Ko143, and exhibits promising applicability in precision cancer medicine. Statement of significanceTo resolve the bottlenecks in clinical application of anticancer agents SN38/CPT-11 and the most potent breast cancer resistant protein (BCRP) inhibitor Ko143, a “combo” nanotherapeutic simultaneously delivering SN38 and Ko143 was constructed and named as BI@PEG-SN38. By generating panels of colorectal cancer models, we demonstrate that BI@PEG-SN38 nanoparticles effectively and selectively reversed BCRP-mediated tumor resistance to SN38/CPT-11 in vitro and in vivo. As the first nano-formulation of Ko143 and the first systemic co-delivery vehicle of SN38/CPT-11 and a BCRP inhibitor, BI@PEG-SN38 provides a new strategy for clinical development of SN38 and numerous “chemosensitizers”, and exhibits promising applicability in precision cancer medicine. Panels of cancer cell lines established here provides a useful platform for BCRP- and cancer-related research and technology development.

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.

Exposure to High Altitude Environment Is Associated With Drug Metabolism Change: miRNA-873-5p-Mediated Alteration of Function and Expression Levels of Drug Transporters Under Hypoxia

Background: Hypoxia is the main characteristic of a high-altitude environment, affecting drug metabolism. However, little is known about the role of microRNA (miRNA) in drug metabolism under high-altitude hypoxia. This study aims to investigate the functions and the expression levels of multidrug resistance mutation 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 vivo and in vitro after exposure to high-altitude hypoxia. Methods: Western blot and qPCR analyses were used to analyze the protein and mRNA expression levels of MDR1, MRP2, BCRP, PEPT1, and OATP2B1. 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 after exposure to high-altitude hypoxia, and uptake and transport studies were performed in Caco-2 cells. To clarify whether miRNA-873-5p regulates MDR1 and pregnane X receptor (PXR) under hypoxia, Caco-2 cells were transfected with mimics or inhibitors of miRNA-873-5p and negative control (NC). Findings: Hypoxia increased the expression and activity of most of the drug transporters in vivo and in vitro. We found that miRNA-873-5p regulated MDR1 and PXR expression, thereby affecting drug metabolism. Interpretation: Herein, it was shown that miRNA might affect the expression of drug transporter and nuclear receptor under hypoxia. miRNA-873-5p play important roles in the process of drug metabolism under hypoxia. Funding Information: This work was supported by the National Natural Science Foundation of China (No. 81760673 and 81460568), the Qinghai Innovation Platform Construction Project (No. 2021-ZJ-T03), and the Key Research Foundation of Development and Transformation of Qinghai Province (2021-SF-158). Declaration of Interests: None of the authors has any potential conflict of interest or financial interests to disclose. Ethics Approval Statement: All experimental procedures were performed strictly according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Animal Ethics Committee of The Qinghai University.

Abundant Expression of OCT2, MATE1, OAT1, OAT3, PEPT2, BCRP, MDR1, and xCT Transporters in Blood-Arachnoid Barrier of Pig and Polarized Localizations at CSF- and Blood-Facing Plasma Membranes.

The physiologic and pharmacologic roles of the blood-arachnoid barrier (BAB) remain unclear. Therefore, the purpose of the present study was to comprehensively evaluate and compare the absolute protein expression levels of transporters in the leptomeninges and plexus per cerebrum, and to determine the localizations of transporters at the cerebrospinal fluid (CSF)-facing and blood (dura)-facing plasma membranes of the BAB in pig. Using multidrug resistance protein 1 (MDR1) and organic anion transporter (OAT) 1 as blood (dura)-facing and CSF-facing plasma membrane marker proteins, respectively, we established that breast cancer resistance protein (BCRP), multidrug resistance-associated protein (MRP) 4, organic anion-transporting polypeptide (OATP) 2B1, multidrug and toxin extrusion protein 1 (MATE1), and glucose transporter 1 (GLUT1) are localized at the blood-facing plasma membrane, and OAT3, peptide transporter (PEPT) 2, MRP3, organic cation transporter (OCT) 2, xCT, monocarboxylate transporter (MCT) 1, MCT4, and MCT8 are localized at the CSF-facing plasma membrane of the BAB. The absolute protein expression levels of OAT1, OAT3, MDR1, BCRP, PEPT2, xCT, MATE1, OCT2, and 4f2hc in the whole BAB surrounding the entire cerebrum were much larger than those in the total of the choroid plexuses forming the blood-cerebrospinal fluid barrier (BCSFB). Although MRP4, OATP2B1, MCT8, GLUT1, and MCT1 were also statistically significantly more abundant in the BAB than in the choroid plexuses per porcine cerebrum, these transporters were nevertheless almost equally distributed between the two barriers. In contrast, OATP1A2, MRP1, OATP3A1, and OCTN2 were specifically expressed in the choroid plexus. These results should be helpful in understanding the relative overall importance of transport at the BAB compared with that at the BCSFB, as well as the rank order of transport capacities among different transporters at the BAB, and the directions of transport mediated by individual transporters. SIGNIFICANCE STATEMENT: We found that BCRP, MRP4, OATP2B1, MATE1, and GLUT1 localize at the blood-facing plasma membrane of the blood-arachnoid barrier (BAB), while OAT3, PEPT2, MRP3, OCT2, xCT, MCT1, MCT4, and MCT8 localize at the CSF-facing plasma membrane. 4F2hc is expressed in both membranes. For OAT1, OAT3, MDR1, BCRP, PEPT2, xCT, MATE1, OCT2, and 4f2hc, the absolute protein expression levels in the whole BAB surrounding the entire cerebrum are much greater than the total amounts in the choroid plexuses.

Relevance of Breast Cancer Resistance Protein to Pharmacokinetics of Florfenicol in Chickens: A Perspective from In Vivo and In Vitro Studies.

Florfenicol (FFC) is a valuable synthetic fluorinated derivative of thiamphenicol widely used to treat infectious diseases in food animals. The aims of the study were to investigate whether FFC is a substrate for the breast cancer resistance protein (BCRP) and whether the transporter influences oral availability of FFC. In vitro transport assays using MDCK-chAbcg2 cells were conducted to assess chicken BCRP-mediated transport of FFC, while in vivo pharmacokinetic experiments with single or combined BCRP inhibitor gefitinib were employed to study the role of BCRP in oral FFC disposition. According to U.S. Food and Drug Administration (FDA) criteria, FFC was found to be a potential BCRP substrate due to the net efflux ratio being over 2.0 (2.37) in MDCK cells stably transfected with chicken BCRP and the efflux completely reversed by a BCRP inhibitor (Gefitinib). The molecular docking results indicated that florfenicol can form favorable interactions with the binding pocket of homology modeled chicken BCRP. Pharmacokinetic studies of FFC in different aged broilers with different expression levels of BCRP showed that higher BCRP expression would cause a lower Area Under Curve (AUC) and a higher clearance of FFC. In addition, more extensive absorption of florfenicol after the co-administration with gefitinib (a BCRP inhibitor) was observed. The overall results demonstrated that florfenicol is a substrate of the chicken breast cancer resistant protein which in turn affects its pharmacokinetic behavior.

Impact of Breast Cancer Resistance Protein Expression on the In Vitro Efficacy of Anticancer Drugs in Pancreatic Cancer Cell Lines.

Breast cancer resistance protein (BCRP) overexpression confers multidrug resistance to cancer cells, and the efficacy of anticancer drugs has been reported to be significantly affected by BCRP in cell lines transfected with BCRP or selected with drugs. It is unclear whether the in vitro efficacy of anticancer drugs is affected by endogenous BCRP, although cancer cell line panels consisting of defined tumor cell lines with endogenous BCRP have been used to screen for anticancer drugs in the pharmaceutical industry. We assessed the impact of BCRP expression on efficacy of anticancer drugs using pancreatic cancer cell lines expressing varying levels of endogenous BCRP. Pancreatic cancer cell lines were selected from the Cancer Cell Line Encyclopedia (CCLE). The EC50 of 7-ethyl-10-hydroxycamptothecin (SN-38), topotecan, and mitoxantrone decreased in the presence of a BCRP inhibitor in PANC-1 and AsPC-1 cells, which exhibit high BCRP expression. However, no significant alterations in EC50 were observed in HPAF-II, SW 1990, and MIA PaCa-2, which show moderate or low BCRP expression. The shift of EC50 of anticancer drugs with and without a BCRP inhibitor increased with an increase of BCRP mRNA expression levels; however, the shift was obvious only in cells highly expressing BCRP. Thus, the in vitro efficacy of anticancer drugs on cell proliferation may be minimally affected by BCRP in most pancreatic cancer cell lines, considering that 72% of pancreatic cancer cell lines in CCLE show moderate or low BCRP expression. The effect of BCRP should be carefully evaluated in pancreatic cell lines that highly express BCRP.

Correlation between Membrane Protein Expression Levels and Transcellular Transport Activity for Breast Cancer Resistance Protein.

Emerging evidence indicates an important role for the breast cancer resistance protein (BCRP) in limiting brain penetration of substrate drugs. While in vitro transwell assays can provide an indication of BCRP substrate potential, the predictability of these assays in relation to in vivo brain penetration is still under debate. The present study examined the correlation of BCRP membrane protein expression level and transcellular transport activity across Madin-Darby canine kidney (MDCK) II monolayers. We expressed human BCRP or murine BCRP1 in MDCKII wild-type cells using BacMam2 virus transduction. The selective P-glycoprotein (P-gp) inhibitor LY335979 (1 μM) was included in the transport medium to measure BCRP-mediated transcellular transport for P-gp and BCRP cosubstrates. The BCRP levels in membrane extracts from MDCKII-BCRP or MDCKII-Bcrp1 cells were quantified by liquid chromatography-tandem mass spectrometry. The results are summarized as follows: 1) the membrane protein expression levels correlate with the corrected efflux ratios of substrates for human BCRP and murine BCRP1 within the efflux ratios investigated; 2) we demonstrate good concordance in rank order between the BCRP and BCRP1-mediated efflux ratios for 12 drugs; and 3) we propose an approach to contextualize in vitro BCRP transport data of discovery compounds by comparing them to the in vitro and in vivo transport data of the reference drug dantrolene and taking into account interbatch variation in BCRP expression. This approach correctly predicted compromised brain penetration for 25 discovery compounds in rodents, which were BCRP substrates but not P-gp or weak P-gp substrates. These results suggest that BCRP-expressing MDCKII cells are useful in predicting the in vivo role of BCRP in brain penetration.

Involvement of protoporphyrin IX accumulation in the pathogenesis of isoniazid/rifampicin-induced liver injury: the prevention of curcumin

Combination of isoniazid (INH) and rifampicin (RFP) causes liver injury frequently among tuberculosis patients. However, mechanisms of the hepatotoxicity are not entirely understood. Protoporphyrin IX (PPIX) accumulation, as an endogenous hepatotoxin, resulting from isoniazid and rifampicin co-therapy (INH/RFP) has been reported in PXR–humanized mice. Aminolevulinic acid synthase1 (ALAS1), ferrochelatase (FECH) and breast cancer resistance protein (BCRP) play crucial roles in PPIX synthesis, metabolism and transport, respectively. Herein, this study focused on the role of INH/RFP in these processes. We observed PPIX accumulation in human hepatocytes (L-02) and mouse livers. FECH expression was initially found downregulated both in L-02 cells and mouse livers and expression levels of ALAS1 and BCRP were elevated in L-02 cells after INH/RFP treatment, indicating FECH inhibition and ALAS1 induction might confer a synergistic effect on PPIX accumulation. Additionally, our results revealed that curcumin alleviated INH/RFP-induced liver injury, declined PPIX levels and induced FECH expression in both L-02 cells and mice. In conclusion, our data provide a novel insight in the mechanism of INH/RFP-induced PPIX accumulation and evidence for understanding pathogenesis of INH/RFP-induced liver injury, and suggest that amelioration of PPIX accumulation might be involved in the protective effect of curcumin on INH/RFP-induced liver injury.

Selective induction of P-glycoprotein at the CNS barriers during symptomatic stage of an ALS animal model

P-glycoprotein (P-gp), Breast cancer resistance protein (BCRP) and Multidrug resistance-associated protein 2 (MRP2) residing at the blood-brain barrier (BBB) and the blood-spinal cord barrier (BSCB) are major obstacles for drug delivery to the Central Nervous System (CNS). Disease-induced changes of these xenobiotic transporters at the CNS barriers have been previously documented. Changes in the functional expression of these transporters at the CNS barriers would limit the clinical efficacy of therapeutic agents targeting the CNS. In this study, we characterized the changes in expression and efflux activity of P-gp, BCRP and MRP2 at the BBB and BSCB of an amyotrophic lateral sclerosis (ALS) SOD1-G93A transgenic rat model across the three stages of disease progression: pre-onset, onset and symptomatic. Up-regulation of P-gp and BCRP at the BBB and BSCB during disease progression of ALS would reduce drug entry to the CNS, while any decreases in transport activity would increase drug entry. In SOD rats at the ALS symptomatic stage, we observed increases in both P-gp transport activity and expression compared to age-matched wildtypes. BCRP and MRP2 levels were unchanged in these animals. Immunohistochemical analysis in brain and spinal cord capillaries of SOD rats from all three ALS stages and age-matched wildtypes showed no differences in nuclear localization of a known P-gp regulator, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). It suggests that NFκB may have a limited role during P-gp induction observed in our study and additional signaling pathways could be responsible for this response. Our observations imply that novel pharmacological approaches for treating ALS require selecting drugs that are not P-gp substrates in order to improve therapeutic efficacy in the CNS during ALS progression.

Unconjugated bilirubin elevation impairs the function and expression of breast cancer resistance protein (BCRP) at the blood-brain barrier in bile duct-ligated rats.

Liver failure is associated with dyshomeostasis of efflux transporters at the blood-brain barrier (BBB), which contributes to hepatic encephalopathy. In this study we examined whether breast cancer resistance protein (BCRP), a major efflux transporter at the BBB, was altered during liver failure in rats. Rats underwent bile duct ligation (BDL) surgery, and then were sacrificed after intravenous injection of prazosin on d3, d7 and d14. The brains and blood samples were collected. BCRP function at the BBB was assessed by the brain-to-plasma prazosin concentration ratio; Evans Blue extravasation in the brain tissues was used as an indicator of BBB integrity. The protein levels of BCRP in the brain tissues were detected. Human cerebral microvessel endothelial cells (HCMEC/D3) and Madin-Darby canine kidney cells expressing human BCRP (MDCK-BCRP) were tested in vitro. In addition, hyperbilirubinemia (HB) was induced in rats by intravenous injection of unconjugated bilirubin (UCB). BDL rats exhibited progressive decline of liver function and HB from d3 to d14. In the brain tissues of BDL rats, both the function and protein levels of BCRP were progressively decreased, whereas the BBB integrity was intact. Furthermore, BDL rat serum significantly decreased BCRP function and protein levels in HCMEC/D3 cells. Among the abnormally altered components in BDL rat serum tested, UCB (10, 25 μmol/L) dose-dependently inhibit BCRP function and protein levels in HCMEC/D3 cells, whereas 3 bile acids (CDCA, UDCA and DCA) had no effect. Similar results were obtained in MDCK-BCRP cells and in the brains of HB rats. Correlation analysis revealed that UCB levels were negatively correlated with BCRP expression in the brain tissues of BDL rats and HB rats as well as in two types of cells tested in vitro. UCB elevation in BDL rats impairs the function and expression of BCRP at the BBB, thus contributing to hepatic encephalopathy.

Genetic and Dietary Regulation of Glyburide Efflux by the Human Placental Breast Cancer Resistance Protein Transporter.

Glyburide is frequently used to treat gestational diabetes owing to its low fetal accumulation resulting from placental efflux by the breast cancer resistance protein (BCRP)/ABCG2 transporter. Here we sought to determine how exposure to the dietary phytoestrogen genistein and expression of a loss-of-function polymorphism in the ABCG2 gene (C421A) impacted the transport of glyburide by BCRP using stably transfected human embryonic kidney 293 (HEK) cells, human placental choriocarcinoma BeWo cells, and human placental explants. Genistein competitively inhibited the BCRP-mediated transport of (3)H-glyburide in both wild-type (WT) and C421A-BCRP HEK-expressing cells, with greater accumulation of (3)H-glyburide in cells expressing the C421A variant. In BeWo cells, exposure to genistein for 60 minutes increased the accumulation of (3)H-glyburide 30%-70% at concentrations relevant to dietary exposure (IC50 ∼180 nM). Continuous exposure of BeWo cells to genistein for 48 hours reduced the expression of BCRP mRNA and protein by up to 40%, which impaired BCRP transport activity. Pharmacologic antagonism of the estrogen receptor attenuated the genistein-mediated downregulation of BCRP expression, suggesting that phytoestrogens may reduce BCRP levels through this hormone receptor pathway in BeWo cells. Interestingly, genistein treatment for 48 hours did not alter BCRP protein expression in explants dissected from healthy term placentas. These data suggest that whereas genistein can act as a competitive inhibitor of BCRP-mediated transport, its ability to downregulate placental BCRP expression may only occur in choriocarcinoma cells. Overall, this research provides important mechanistic data regarding how the environment (dietary genistein) and a frequent genetic variant (ABCG2, C421A) may alter the maternal-fetal disposition of glyburide.

Breast cancer resistance protein (BCRP)-containing circulating microvesicles contribute to chemoresistance in breast cancer.

At present, one of the major problems of cancer therapy is drug resistance. Breast cancer resistance protein (BCRP), a marker of the multidrug-resistant phenotype, affects drug absorption, distribution, metabolism, and excretion in normal tissues. Meanwhile, extracellular vesicles (EVs) have attracted increasing attention as a medium of cell-to-cell communication. However, the association between BCRP and circulating EVs remains unclear. The present study demonstrated that patients who did not respond or had progressive/stable disease following chemotherapy had markedly higher BCRP levels compared to those that did not receive chemotherapy. Moreover, BCRP was upregulated at the mRNA and protein levels in tumor-derived circulating EVs from patients with a poor response to chemotherapy. Interestingly, the results also demonstrated that BCRP was co-expressed with MUC1, which is frequently expressed in breast cancer and is exported via EVs, and both BCRP and MUC1 were up-regulated after chemotherapy. In conclusion, the present study indicates that tumor-derived circulating EVs that carry BCRP may serve as a predictive biomarker of the response to chemotherapy for breast cancer. In addition, the results provide a window for individualized treatment to overcome resistance to chemotherapeutic drugs.