Breast Cancer Resistance Protein In Cells Research Articles

High-throughput BCRP inhibitors screening system based on styrene maleic acid polymer membrane protein stabilization strategy and surface plasmon resonance biosensor

Multidrug resistance (MDR) is a dominant challenge in cancer chemotherapy failure. The over-expression of breast cancer resistance protein (BCRP) in tumorous cells, along with its extensive substrate profile, is a leading cause of tumor MDR. Herein, on the basis of styrene maleic acid (SMA) polymer membrane protein stabilization strategy and surface plasmon resonance (SPR) biosensor, a novel high-throughput screening (HTS) system for BCRP inhibitors has been established. Firstly, LLC-PK1 and LLC-PK1/BCRP cell membranes were co-incubated with SMA polymers to construct SMA lipid particles (SMALPs). PK1-SMALPs were thus immobilized in channel 1 of the L1 chip as the reference channel, and BCRP-SMALPs were immobilized in channel 2 as the detection channel to establish the BCRP-SMALPs-SPR screening system. The methodological investigation demonstrated that the screening system was highly specific and stable. Three active compounds were screened out from 26 natural products and their affinity constants with BCRP were determined. The KD of xanthotoxin, bergapten, and naringenin were 5.14 μM, 4.57 μM, and 3.72 μM, respectively. The in vitro cell verification experiments demonstrated that xanthotoxin, bergapten, and naringenin all significantly increased the sensitivity of LLC-PK1/BCRP cells to mitoxantrone with possessing reversal BCRP-mediated MDR activity. Collectively, the developed BCRP-SMALPs-SPR screening system in this study has the advantages of rapidity, efficiency, and specificity, providing a novel strategy for the in-depth screening of BCRP inhibitors with less side effects and higher efficacy.

Abstract 4674: Reserpine analog modulates transport function of ABCG2

Abstract Backgrounds: Reserpine is an agent that is used for the treatment of hypertension. ABCG2 also known as breast cancer resistance protein (BCRP) is a member of the ABC transporters associated with multidrug resistance (MDR). Our high-throughput screening assay has revealed that reserpine analog inhibits transport function of ABCG2. Aim: To investigate the effect of reserpine analog on ABCG2 function. Results: Flow cytometry assay showed that reserpine analog blocked the efflux of Pheophorbide a (PhA: fluorescent substrate of ABCG2) from ABCG2-expressing HCT-116/BCRP colon cancer cells in a concentration-dependent manner. Furthermore, cell proliferation assay (MTS assay) revealed that 5 and 10 μM reserpine analog sensitized HCT-116/BCRP cells to SN38 with IC50 value 0.61 + 0.12 μM and 0.22 + 0.036 μM, respectively. Finally, reserpine analog did not stimulate ABCG2-mediated ATP hydrolysis, but rather inhibited it (concentration required for 50% inhibition = 2.7 μM), suggesting that reserpine analog might prevent conformational changes required for ATP hydrolysis. Conclusion: Reserpine analog could be developed as a potent modulator to overcome ABCG2-mediated MDR in cancer cells. Citation Format: Shinobu Ohnuma, Shoji Kokubo, Norihiko Sugisawa, Megumi Murakami, Yuuri Hatsuzawa, Minoru Kobayashi, Taiki Kajiwara, Hideyuki Suzuki, Hideaki Karasawa, Kazuhiro Watanabe, Takayuki Doi, Suresh V. Ambudkar, Takashi Kamei, Michiaki Unno. Reserpine analog modulates transport function of ABCG2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4674.

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.

Rapamycin Antagonizes BCRP-Mediated Drug Resistance Through the PI3K/Akt/mTOR Signaling Pathway in mPRα-Positive Breast Cancer.

PurposeOverexpression of breast cancer (BCa) resistance protein (BCRP) is detected in approximately 30% of BCa cases. BCRP indicates a poor response to chemotherapy, and it has become a classic target to overcome drug-resistant tumor cells. In this study, we aimed to explore the mechanism of BCRP overexpression and a strategy to reverse this overexpression in invasive BCa.MethodsBCRP expression in BCa tissues was determined by immunohistochemistry. GSE25066 was downloaded from the NCBI GEO database. Western blot was used to determine the expression of key molecules in vitro. Cell counting kit-8 assays were used to assess the drug response of BCa cells.ResultsOur results suggested that BCRP is an independent risk factor for BCa. We further established that upon 17α-PG binding, membrane progesterone receptor α (mPRα) promoted BCRP expression via the PI3K/Akt/mTOR signaling pathway. mPRα physically interacted with p-Akt1 S473. Moreover, rapamycin, an inhibitor of mTOR complex 1 (mTORC1), downregulated BCRP expression and enhanced the effects of particular drugs, including doxorubicin and paclitaxel.ConclusionBCRP is a potential biomarker of poor prognosis in BCa. BCRP expression is regulated by 17α-PG in mPRα-positive BCa cells through the PI3K/Akt/mTOR signaling pathway. Rapamycin might enhance the therapeutic effect of chemotherapy agents in mPRα-positive MDA-MB-453/BCRP cells and might be a therapeutic option for mPRα-positive invasive BCa with BCRP overexpression.

Interactions of the major effective components in Shengmai formula with breast cancer resistance protein at the cellular and vesicular levels

Shengmai Formula (SMF) is one of the traditional Chinese medicine representative formulas and is widely used for the treatment of cardio- and cerebrovascular disease. Previous studies demonstrated that the major effective ingredients in SMF can interact with each other based on some uptake transporters. However, the role of the efflux transporter breast cancer resistance protein (BCRP) in these interactions involving SMF remains unclear. The purpose of this study was to investigate the interactions of the major active components of SMF with BCRP and the compatibility mechanism of these complex components in SMF based on BCRP. We selected 4 main fractions, including ginseng total saponins (GTS), ophiopogon total saponins (OTS), ophiopogon total flavonoids (OTF), and fructus schisandrae total lignans (STL), and 12 bioactive components, including ginsenosides Re, Rd, Rb1, and Rg1, ophiopogonins D and Dˊ, methylophiopogonanones A and B, schizandrins A and B, and schizandrols A and B to explore the interactions of SMF with BCRP in LLC-PK1 and LLC-PK1/BCRP cells and BCRP membrane vesicles. The results showed that ginsenosides Re and Rg1, methylophiopogonanone B, and schizandrin A can be transported by BCRP into LLC-PK1/BCRP cells. Schisandrol B exhibited a markedly inhibitory effect on the transport function of BCRP and can significantly inhibit the uptake of methylophiopogonanone B and schizandrin A into LLC-PK1/BCRP cells. In “Inside-Out” BCRP membrane vesicles, BCRP mediated the transport of ginsenosides Re and Rg1, methylophiopogonanone B, and schizandrin A, with Km values of 111.9 ± 31.26 μM, 82.01 ± 16.72 μM, 57.06 ± 8.789 μM, and 37.19 ± 6.512 μM, respectively. GTS, STL, ginsenosides Rd and Rb1, and schisandrol B were potent inhibitors of BCRP and showed different degrees of inhibition on the transport of ginsenosides Re and Rg1, methylophiopogonanone B, and schizandrin A via BCRP. In conclusion, GTS, STL, ginsenosides Rd and Rb1, and schizandrol B are potential inhibitors of BCRP. Ginsenosides Re and Rg1, methylophiopogonanone B, and schizandrin A are potential substrates of BCRP, and their transport, which is mediated by BCRP, may be inhibited by potential inhibitors in SMF. There are potential interactions of these main effective components of SMF at the cellular and vesicular levels that are mediated by BCRP. The interplay of these bioactive components based on BCRP may be an important compatibility mechanism in SMF.

Abstract 25: Identifying new inhibitors of ABCG2 by high-throughput screening

Abstract Background: ABCG2, also known as breast cancer resistance protein (BCRP), is a member of the ATP-binding cassette (ABC)-family of membrane transport proteins associated with multidrug resistance (MDR). No ABCG2 inhibitor has shown clinical significance yet. Aim: To identify novel inhibitors of ABCG2 by high-throughput screening. Material & Methods: After higher expression of ABCG2 in HCT-116/BCRP cells was confirmed by flow cytometry (FACSVerse) using 5D3 antibody, high-throughput fluorescent cell-based assay was performed with the original chemical library of Tohoku University containing about 6000 compounds using ABCG2-specific fluorescent substrate, Pheophorbide a (PhA) in HCT-116/BCRP cells overexpressing ABCG2. Ko143, a potent ABCG2 inhibitor, was used as positive control. Fluorescence intensity in the cells was read by a fluorescence plate reader (SpectraMax M2e) in bottom read mode, with 395 nm excitation, 670 nm emission. To validate the results, flow cytometry assay was performed with 10 µM PhA in HCT-116/BCRP cells. The fluorescence intensity was analyzed by flow cytometry (FACSVerse). Results: Fluorescenct cell-based assay with high-throughput screening could detect 23 candidate compounds, which showed higher fluorescence intensity as positive control Ko143 showed. Then, these 23 compounds were validated with flow cytometry assays. The flow cytometry assays revealed that 16 compounds increased fluorescence intensity of PhA in HCT-116/BCRP cells in dose dependent manner, suggesting that these candidate compounds inhibited transport function of ABCG2. Among 16 compounds, 6 compounds showed further inhibitory effect for transport function of ABCG2 as equivalent to that of Ko143. Conclusion: These 6 compounds may provide a basis for further investigation of ABCG2 function. Citation Format: Shoji Kokubo, Shinobu Ohnuma, Hideaki Karasawa, Akihiro Yamamura, Hideyuki Suzuki, Megumi Murakami, Norihiko Sugisawa, Keigo Kanehara, Keisuke Sato, Takanori Ishida, Takashi Kamei, Takeshi Naitoh, Michiaki Unno. Identifying new inhibitors of ABCG2 by high-throughput screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 25.

Paliperidone, a relatively novel atypical antipsychotic drug, is a substrate for breast cancer resistance protein.

Paliperidone (PAL) is a relatively novel atypical antipsychotic drug for schizophrenia that induces markedly varying responses. Breast cancer resistance protein (BCRP) is a recently discovered member of the ATP-binding cassette superfamily that has been used to control drug absorption, distribution and elimination, and especially to impede drug entry into the brain. To the best of our knowledge, the present study is the first to investigate the possibility of using PAL as a BCRP substrate. The intracellular accumulation and bidirectional transport were investigated using transfected 293 cell/BCRP and porcine renal endothelial cell (LLC-PK1)/BCRP cell monolayers and BCRP overexpression was confirmed by reverse transcription-quantitative polymerase chain reaction and western blot analysis. The in vitro affinity to BCRP was assessed in human BCRP (Arg482) membranes. The intracellular accumulation and bidirectional transport investigations demonstrated that BCRP can efflux PAL from cells and significantly decrease its cellular concentration over a concentration range of 0.1-50 µM. The in vitro affinity experiments indicated that PAL has a moderate affinity to BCRP at 0.1-100 µM. These results together suggest that PAL is a substrate for BCRP and that it can affect the blood-brain barrier penetration of PAL at therapeutic dosages.

Ochratoxin A transport by the human breast cancer resistance protein (BCRP), multidrug resistance protein 2 (MRP2), and organic anion-transporting polypeptides 1A2, 1B1 and 2B1

Ochratoxin A (OTA) is a fungal secondary metabolite that can contaminate various foods. OTA has several toxic effects like nephrotoxicity, hepatotoxicity, and neurotoxicity in different animal species, but its mechanisms of toxicity are still unclear. How OTA accumulates in kidney, liver, and brain is as yet unknown, but transmembrane transport proteins are likely involved. We studied transport of OTA in vitro, using polarized MDCKII cells transduced with cDNAs of the efflux transporters mouse (m)Bcrp, human (h)BCRP, mMrp2, or hMRP2, and HEK293 cells overexpressing cDNAs of the human uptake transporters OATP1A2, OATP1B1, OATP1B3, or OATP2B1 at pH7.4 and 6.4. MDCKII-mBcrp cells were more resistant to OTA toxicity than MDCKII parental and hBCRP-transduced cells. Transepithelial transport experiments showed some apically directed transport by MDCKII-mBcrp cells at pH7.4, whereas both mBcrp and hBCRP clearly transported OTA at pH6.4. There was modest transport of OTA by mMrp2 and hMRP2 only at pH6.4. OATP1A2 and OATP2B1 mediated uptake of OTA both at pH7.4 and 6.4, but OATP1B1 only at pH7.4. There was no detectable transport of OTA by OATP1B3. Our data indicate that human BCRP and MRP2 can mediate elimination of OTA from cells, thus reducing OTA toxicity. On the other hand, human OATP1A2, OATP1B1, and OATP2B1 can mediate cellular uptake of OTA, which could aggravate OTA toxicity.

Tariquidar Is an Inhibitor and Not a Substrate of Human and Mouse P-glycoprotein.

Since its development, tariquidar (TQR; XR9576; N-[2-[[4-[2-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]carbamoyl]-4,5-dimethoxyphenyl]quinoline-3-carboxamide) has been widely regarded as one of the more potent inhibitors of P-glycoprotein (P-gp), an efflux transporter of the ATP-binding cassette (ABC) transporter family. A third-generation inhibitor, TQR exhibits high affinity for P-gp, although it is also a substrate of another ABC transporter, breast cancer resistance protein (BCRP). Recently, several studies have questioned the mechanism by which TQR interfaces with P-gp, suggesting that TQR is a substrate for P-gp instead of a noncompetitive inhibitor. We investigated TQR and its interaction with human and mouse P-gp to determine if TQR is a substrate of P-gp in vitro. To address these questions, we used multiple in vitro transporter assays, including cytotoxicity, flow cytometry, accumulation, ATPase, and transwell assays. A newly generated BCRP cell line was used as a positive control that demonstrates TQR-mediated transport. Based on our results, we conclude that TQR is a potent inhibitor of both human and mouse P-gp and shows no signs of being a substrate at the concentrations tested. These in vitro data further support our position that the in vivo uptake of [(11)C]TQR into the brain can be explained by its high-affinity binding to P-gp and by it being a substrate of BCRP, followed by amplification of the brain signal by ionic trapping in acidic lysosomes.

The interaction between human breast cancer resistance protein (BCRP) and five bisbenzylisoquinoline alkaloids

BCRP is one of the key factors to drug absorption, distribution and elimination. Bisbenzylisoquinoline alkaloids are a large family of natural phytochemicals with great potential for clinical use. In this study, the interaction between BCRP and five bisbenzylisoquinoline alkaloids (neferine, isoliensinine, liensinine, dauricine and tetrandrine) were evaluated using LLC-PK1/BCRP cell model. The intracellular accumulation and bi-directional transport studies were conducted, and then molecular docking analysis was carried out employing a homology model of BCRP. Our study revealed that the permeability of these five alkaloids was not high, the Papp values were all less than 6.5 × 10(-6)cm/s. Liensinine and dauricine were substrates of BCRP: at lower concentration (10 μM), the net efflux ratios were 2.87 and 1.64 respectively. And their cellular accumulation was lower in LLC-PK1/BCRP cells than in LLC-PK1 cells. On the other hand, tetrandrine, isoliensinine and neferine were not substrates of BCRP. On the basis of docking studies, a direct hydrogen bond was formed between liensinine and arginine 482 which is a hot spot of BCRP for substrate specificity; and dauricine had hydrophobic interaction with BCRP. In conclusion, our study indicated that BCRP could mediate the excretion of liensinine and dauricine, thus influence their pharmacological activity and disposition.

Oleic acid decreases BCRP mediated efflux of mitoxantrone in Caco-2 cell monolayers

Breast cancer resistance protein (BCRP) efflux restricts intestinal absorption of substances like heterocyclic amines, mycotoxins and certain human and veterinary drugs. Fat rich meals seem to increase absorption of drugs which are BCRP substrates or inhibitors. We therefore hypothesize that absorption of toxicants normally effluxed by BCRP are increased by fatty acids in food. Transport across and accumulation of 3H-Mitoxantrone (MXR) in Caco-2 cell monolayers were measured after 60min exposure to emulsions of 3H-MXR (1μM) and oleic acid (0.5–5mM). In addition, BCRP gene expression (RT-PCR) and the amount of BCRP protein (Western blot) were measured in oleic acid exposed Caco-2 cells. Oleic acid increased transport of MXR in a concentration dependent manner and 2mM oleic acid or higher increased accumulation of MXR in cells, without any signs of cytotoxicity. Gene expression of BCRP was increased after exposure to oleic acid for 6h, but the amount of BCRP protein was not increased. In conclusion, oleic acid clearly induced BCRP gene expression and reduced BCRP mediated efflux, although the amount of BCRP in cells was not affected. Consequently, effects of fatty acids on BCRP mediated efflux are important to consider in risk assessment of toxicants in food.

Higher frequencies of BCRP+ cardiac resident cells in ischaemic human myocardium

Several cardiac resident progenitor cell types have been reported for the adult mammalian heart. Here we characterize their frequencies and distribution pattern in non-ischaemic human myocardial tissue and after ischaemic events. We obtained 55 biopsy samples from human atria and ventricles and used immunohistological analysis to investigate two cardiac cell types, characterized by the expression of breast cancer resistance protein (BCRP)/ABCG2 [for side population (SP) cells] or c-kit. Highest frequencies of BCRP+ cells were detected in the ischaemic right atria with a median of 5.40% (range: 2.48-11.1%) vs. 4.40% (1.79-7.75%) in the non-ischaemic right atria (P = 0.47). Significantly higher amounts were identified in ischaemic compared with non-ischaemic ventricles, viz. 5.44% (3.24-9.30%) vs. 0.74% (0-5.23%) (P = 0.016). Few numbers of BCRP+ cells co-expressed the cardiac markers titin, sarcomeric α-actinin, or Nkx2.5; no co-expression of BCRP and progenitor cell marker Sca-1 or pluripotency markers Oct-3/4, SSEA-3, and SSEA-4 was detected. C-kit+ cells displayed higher frequencies in ischaemic (ratio: 1:25 000 ± 2500 of cell counts) vs. non-ischaemic myocardium (1:105 000 ± 43 000). Breast cancer resistance protein+/c-kit+ cells were not identified. Following in vitro differentiation, BCRP+ cells isolated from human heart biopsy samples (n = 6) showed expression of cardiac troponin T and α-myosin heavy-chain, but no full differentiation into functional beating cardiomyocytes was observed. We were able to demonstrate that BCRP+/CD31- cells are more abundant in the heart than their c-kit+ counterparts. In the non-ischaemic hearts, they are preferentially located in the atria. Following ischaemia, their numbers are elevated significantly. Our data might provide a valuable snapshot at potential progenitor cells after acute ischaemia in vivo, and mapping of these easily accessible cells may influence future cell therapeutic strategies.

PDZK1 Regulates Breast Cancer Resistance Protein in Small Intestine

Transporter adaptor protein PDZK1 regulates several influx transporters for xenobiotics and nutrients in small intestine, and their expression on the apical membrane is diminished in pdzk1 gene knockout [pdzk1(-/-)] mice. In the present study, we initially attempted to use pdzk1(-/-) mice to functionally identify influx transporters responsible for intestinal absorption of cimetidine. Contrary to our expectation, the plasma concentration of cimetidine after oral administration to pdzk1(-/-) mice was higher than that in wild-type mice, and the double peaks of plasma concentration found in wild-type mice were not observed in pdzk1(-/-) mice. Western blot analysis of intestinal brush-border membranes revealed that expression of breast cancer resistance protein (BCRP) but not of P-glycoprotein is reduced in pdzk1(-/-) mice. This result was compatible with the reduction of apical localization of BCRP in pdzk1(-/-) mice assessed by immunohistochemical analysis. Transcellular transport of cimetidine in the basal-to-apical direction in Madin-Darby canine kidney II (MDCKII) cells stably expressing both BCRP and PDZK1 (MDCKII/BCRP/PDZK1) was higher than that in MDCKII cells stably expressing BCRP (MDCKII/BCRP) cells. Moreover, MDCKII/BCRP/PDZK1 cells are more resistant than MDCKII/BCRP cells to the cytotoxicity of the anticancer agent 7-ethyl-10-hydroxycamptothecin (SN-38), which is a substrate of BCRP. These results were consistent with the higher expression of BCRP on apical membranes in MDCKII/BCRP/PDZK1 cells. Pull-down and immunoprecipitation studies revealed a physical interaction between BCRP and PDZK1. Taken together, these findings demonstrate that PDZK1 plays a pivotal role in the apical localization of BCRP. This is the first identification of a regulatory protein that physically interacts with and regulates BCRP in small intestine in vivo.

Methylation status of breast cancer resistance protein detected by methylation‐specific polymerase chain reaction analysis is correlated inversely with its expression in drug‐resistant lung cancer cells

Breast cancer resistance protein (BCRP) functions as a drug efflux transporter that mediates drug resistance. Topoisomerase I inhibitors, including 7-ethyl-10-hydroxycamptothecin (SN-38), are substrates effluxed by BCRP. However, it remains unclear whether the overexpression of BCRP induces drug resistance during chemotherapy. The objectives of the current study were to examine a correlation of altered promoter methylation of BCRP with BCRP expression and to investigate the correlation between methylation status according to methylation-specific polymerase chain reaction (MSP) analysis and BCRP expression levels in several small cell and nonsmall cell lung cancer cells. Non-BCRP-expressing PC-6 cells, which were sensitive to SN-38, were treated with DNA methyltransferase inhibitor to induce BCRP re-expression by means of reverse transcriptase-polymersae chain reaction, Western blot, and flow cytometric analyses. Subsequently, bisulfite sequencing analysis in both PC-6 cells and SN-38-resistant PC-6/SN2-5H, highly expressing BCRP cells was performed to identify the methylated region in the BCRP promoter. Finally, the authors established an MSP method on the basis of methylated and unmethylated DNA sequences. DNA methyltransferase inhibitor treatment of PC-6 cells induced BCRP re-expression at the messenger RNA and protein levels. Bisulfite sequencing analysis revealed that both alleles at all CpG sites were methylated completely in PC-6 cells, whereas alleles at portions of CpG sites in PC-6/SN2-5H cells were unmethylated. There was an inverse correlation between promoter methylation of BCRP determined by MSP and BCRP expression in both small cell and nonsmall cell lung cancer cells. The current results indicated that demethylation of at least 1 allele is necessary for BCRP re-expression and that promoter demethylation of BCRP may be a mechanism of BCRP expression in lung cancer cells.

The Breast Cancer Resistance Protein (Bcrp1/Abcg2) Limits Fetal Distribution of Glyburide in the Pregnant Mouse: An Obstetric-Fetal Pharmacology Research Unit Network and University of Washington Specialized Center of Research Study

Breast cancer resistance protein (BCRP) is most abundantly expressed in the apical membrane of placental syncytiotrophoblasts, suggesting that it may protect the fetus by impeding drug penetration across the placental barrier. Glyburide (GLB) is an antidiabetic drug routinely used to treat gestational diabetes. In this study, we first determined whether GLB is a BCRP/Bcrp1 substrate. The intracellular [(3)H]GLB concentrations in Madin-Darby canine kidney (MDCK)/BCRP cells were significantly lower than those in MDCK/vector cells. The addition of 10 muM fumitremorgin C, a specific BCRP inhibitor, significantly increased the intracellular [(3)H]GLB concentrations approximately 2-fold in MDCK/BCRP cells, but it had no effect in MDCK/vector cells. Similar results were obtained using MDCKII parent and MDCKII/Bcrp1 cells. GLB was also shown to be a BCRP/Bcrp1 substrate in transwell transport experiments. We then examined whether Bcrp1 limits fetal distribution of GLB in the pregnant mouse. GLB was administered by retro-orbital injection to the wild-type and Bcrp1(-/-) pregnant mice. The maternal plasma samples and fetuses were collected at various times (0.5-240 min) after drug administration. The GLB concentrations in the maternal plasma samples and homogenates of fetal tissues were determined by high-performance liquid chromatography/mass spectrometry. Although the maternal plasma area under the concentration-time curves (AUCs) of GLB in the wild-type and Bcrp1(-/-) pregnant mice were comparable, the fetal AUC of GLB in the Bcrp1(-/-) pregnant mice was approximately 2 times greater than that in the wild-type pregnant mice. These results suggest that GLB is a BCRP/Bcrp1 substrate, and Bcrp1 significantly limits fetal distribution of GLB in the pregnant mouse, but it has only a minor effect on the systemic clearance of the drug.

Reversal of BCRP‐mediated multidrug resistance by stable expression of small interfering RNAs

Breast cancer resistance protein (BCRP) is an ATP-binding cassette multidrug transporter that confers resistance to various anticancer drugs like Mitoxantrone. Overexpression of BCRP confers multidrug resistance (MDR) in cancer cells and is a frequent impediment to successful chemotherapy. For stable reversal of BCRP-depending MDR by RNA interference technology, a hU6-RNA gene promoter-driven expression vector encoding anti-BCRP short hairpin RNA (shRNA) molecules was constructed. By treating endogenously and exogenously expresses high levels of BCRP cells with these constructs, expression of the targeted BCRP-encoding mRNA, and transport protein was inhibited completely. Furthermore, the accumulation of mitoxantrone in the anti-BCRP shRNA-treated cells increased. And the sensitivity to mitoxantrone of anti-BCRP shRNA-treated cells is increased 14.6-fold and 2.44-fold respectively compared to their control (P < 0.05). These data indicated that stable shRNA-mediated RNAi could be tremendously effective in reversing BCRP-mediated MDR and showed promises in overcoming MDR by gene therapeutic applications.

Transport of Glyburide by Placental ABC Transporters: Implications in Fetal Drug Exposure

Much evidence has demonstrated that a number of ATP-binding cassette (ABC) efflux transporters including P-glycoprotein (PGP), the multidrug resistance-associated proteins (MRPs) and the breast cancer resistance protein (BCRP) are highly expressed in placental tissues and are believed to profoundly limit the passage of therapeutic or toxic xenobiotics to the fetus. Recent studies indicate that the oral hypoglycemic glyburide does not cross the human placenta to an appreciable extent. Our objective was to identify placental transporters potentially involved in limiting the transplacental transfer of glyburide to the fetus. Thus, [ 3H]-glyburide transport was examined in BCRP, PGP, MRP1, MRP2 and MRP3 over-expressing cell lines in the presence or absence of specific inhibitors. Our results demonstrated significant increases in the intracellular accumulation of [ 3H]-glyburide in BCRP and MRP3 over-expressing cells in the presence of the inhibitors novobiocin and indomethacin, respectively. PGP inhibition with verapamil or MRP inhibition with indomethacin did not affect [ 3H]-glyburide accumulation in the PGP or MRP2 over-expressing cell lines and only limited changes were seen in the MRP1 over-expressing cell line. On the other hand, glyburide was found to significantly inhibit MRP1-, MRP2- and MRP3-mediated efflux of 5-carboxyfluorescein diacetate and PGP-mediated transport of rhodamine 123. Our evidence is the first to clearly indicate that glyburide is preferentially transported by BCRP and MRP3.

271 Embryonic hepatoblasts share patterns of gene expression with other stem cell populations

Much evidence has demonstrated that a number of ATP-binding cassette (ABC) efflux transporters including P-glycoprotein (PGP), the multidrug resistance-associated proteins (MRPs) and the breast cancer resistance protein (BCRP) are highly expressed in placental tissues and are believed to profoundly limit the passage of therapeutic or toxic xenobiotics to the fetus. Recent studies indicate that the oral hypoglycemic glyburide does not cross the human placenta to an appreciable extent. Our objective was to identify placental transporters potentially involved in limiting the transplacental transfer of glyburide to the fetus. Thus, [3H]-glyburide transport was examined in BCRP, PGP, MRP1, MRP2 and MRP3 over-expressing cell lines in the presence or absence of specific inhibitors. Our results demonstrated significant increases in the intracellular accumulation of [3H]-glyburide in BCRP and MRP3 over-expressing cells in the presence of the inhibitors novobiocin and indomethacin, respectively. PGP inhibition with verapamil or MRP inhibition with indomethacin did not affect [3H]-glyburide accumulation in the PGP or MRP2 over-expressing cell lines and only limited changes were seen in the MRP1 over-expressing cell line. On the other hand, glyburide was found to significantly inhibit MRP1-, MRP2- and MRP3-mediated efflux of 5-carboxyfluorescein diacetate and PGP-mediated transport of rhodamine 123. Our evidence is the first to clearly indicate that glyburide is preferentially transported by BCRP and MRP3.

Estrone and 17β‐Estradiol Reverse Breast Cancer Resistance Protein‐mediated Multidrug Resistance

Breast cancer resistance protein (BCRP), an adenosine triphosphate‐binding cassette transporter, confers resistance to a series of anticancer reagents, including mitoxantrone, SN‐38 and topotecan. In the present study, we found that estrone and l7β‐estradiol potentiated the cytotoxicity of mitoxantrone, SN‐38 and topotecan in BCRP‐transduced K562 cells (K562/BCRP). These estrogens showed only a marginal effect, or none, in parental K562 cells. Estrone and 17β‐estradiol increased the cellular accumulation of topotecan in K562/BCRP cells, but not in K562 cells, suggesting that these estrogens inhibit the BCRP‐mediated drug efflux and overcome drug resistance.

Breast cancer resistance protein expression and resistance to daunorubicin in blast cells from patients with acute myeloid leukaemia.

Breast cancer resistance protein (BCRP) is a recently described member of the ATP binding cassette transporter superfamily. It has been shown to confer resistance to mitoxantrone, topotecan, doxorubicin and daunorubicin in human tumour cell lines. We describe a study of BCRP expression in blast cells derived from 20 patients with acute myeloid leukaemia (AML). Twelve samples were from patients who had received previous cytotoxic therapy. BCRP expression was measured by immunocytochemistry using the BXP-34 monoclonal antibody. In vitro drug sensitivity was assessed using the methyl thiazol tetrazoliumbromide assay. BCRP expression varied between patients, and six out of 22 (27%) samples had > 10% cells staining positively (median 37%, range 13-95%). BCRP positivity was seen in both de novo samples and those from previously treated patients. There was a marked variation in the effect of all drugs tested between patients. Although there was no correlation between BCRP positivity and the effect of mitoxantrone, topotecan or doxorubicin, the median daunorubicin LC(50) value of BCRP(+) cells was fourfold higher than that of BCRP- cells (0.89 micromol/l compared with 0.21 micromol/l, P < 0.05). These results suggest that BCRP may be involved in resistance to the agents commonly used in AML and may explain some of the anomalous results found when studying other membrane transporters, such as P-gp or MRP.