Abundance of P-glycoprotein and Breast Cancer Resistance Protein Measured by Targeted Proteomics in Human Epileptogenic Brain Tissue.

Our goal was to measure the absolute differential abundance of key drug transporters in human epileptogenic brain tissue and to compare them between patients and at various distances from the epileptogenic zone within the same patient. Transporter protein abundance was quantified in brain tissue homogenates from patients who underwent epilepsy surgery, using targeted proteomics, and correlations with clinical and tissue characteristics were assessed. Fourteen brain samples (including four epileptogenic hippocampal samples) were collected from nine patients. Among the quantifiable drug transporters, the abundance (median, range) ranked: breast cancer resistance protein (ABCG2/BCRP; 0.55, 0.01-3.26 pmol/g tissue) > P-glycoprotein (ABCB1/MDR1; 0.30, 0.02-1.15 pmol/g tissue) > equilibrative nucleoside transporter 1 (SLC29A1/ENT1; 0.06, 0.001-0.35 pmol/g tissue). The ABCB1/ABCG2 ratio (mean 0.27, range 0.08-0.47) was comparable with literature values from nonepileptogenic brain tissue (mean 0.5-0.8). Transporter abundance was lower in the hippocampi than in the less epileptogenic neocortex of the same patients. ABCG2/BCRP and ABCB1/MDR1 expression strongly correlated with that of glucose transporter 1 (SLC2A1/GLUT1) (r = 0.97, p < 0.001; r = 0.90, p < 0.01, respectively). Low transporter abundance was found in patients with overt vascular pathology, whereas the highest abundance was seen in a sample with normally appearing blood vessels. In conclusion, drug transporter abundance highly varies across patients and between epileptogenic and less epileptogenic brain tissue of the same patient. The strong correlation in abundance of ABCB1/MDR1, ABCG2/BCRP, and SLC2A1/GLUT1 suggests variation in the content of the functional vasculature within the tissue samples. The epileptogenic tissue can be depleted of key drug transport mechanisms, warranting consideration when selecting treatments for patients with drug-resistant epilepsy.

ABCG2 Protein Levels and Association to Response to First-Line Irinotecan-Based Therapy for Patients with Metastatic Colorectal Cancer.

In this study we investigated the use of cancer cell protein expression of ABCG2 to predict efficacy of systemic first-line irinotecan containing therapy in patients with metastatic colorectal cancer (mCRC). From a Danish national cohort, we identified 119 mCRC patients treated with irinotecan containing therapy in first-line setting. Among these, 108 were eligible for analyses. Immunohistochemistry (IHC) analyses were performed on the primary tumor tissue in order to classify samples as high or low presence of ABCG2 protein. Data were then associated with patient outcome (objective response (OR), progression free survival (PFS) and overall survival (OS)). ABCG2 protein expression in the basolateral membrane was high (score 3+) in 33% of the patients. Exploratory analyses revealed a significant interaction between ABCG2 score, adjuvant treatment and OR (p = 0.041) in the 101 patients with evaluable disease. Patients with low ABCG2 (score 0-2) and no prior adjuvant therapy had a significantly higher odds ratio of 5.6 (Confidence Interval (CI) 1.68-18.7; p = 0.005) for obtaining OR. In contrast, no significant associations between ABCG2 expression and PFS or OS were found. These results suggest that measurement of the ABCG2 drug efflux pump might be used to select patients with mCRC for irinotecan treatment. However, additional studies are warranted before conclusions regarding a clinical use can be made. Moreover, patients with high ABCG2 immunoreactivity could be candidates for specific ABCG2 inhibition treatment in combination with irinotecan.

Expression and Cellular Distribution of P-Glycoprotein and Breast Cancer Resistance Protein in Amyotrophic Lateral Sclerosis Patients.

For amyotrophic lateral sclerosis (ALS), achieving and maintaining effective drug levels in the brain is challenging due to the activity of ATP-binding cassette (ABC) transporters which efflux drugs that affect drug exposure and response in the brain. We investigated the expression and cellular distribution of the ABC transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) using immunohistochemistry in spinal cord (SC), motor cortex, and cerebellum from a large cohort of genetically well characterized ALS patients (n = 25) and controls (n = 14). The ALS group included 17 sporadic (sALS) and 8 familial (fALS) patients. Strong P-gp expression was observed in endothelial cells in both control and ALS specimens. Immunohistochemical analysis showed higher P-gp expression in reactive astroglial cells in both gray (ventral horn) and white matter of the SC, as well as in the motor cortex of all ALS patients, as compared with controls. BCRP expression was higher in glia in the SC and in blood vessels and glia in the motor cortex of ALS patients, as compared with controls. P-gp and BCRP immunoreactivity did not differ between sALS and fALS cases. The upregulation of both ABC transporters in the brain may explain multidrug resistance in ALS patients and has implications for the use of both approved and experimental therapeutics.

Role of Elevated SFLT-1 on the Regulation of Placental Transporters in Women With Pre-Eclampsia.

Pre-eclampsia (PE) is an obstetric complication associated with elevated levels of fms-like tyrosine kinase 1 (sFlt-1) and dysregulated trophoblast differentiation. However, limited information exists on the expression and regulation of placental drug transporters in PE. Transporter mRNA and protein expression were analyzed in human placentas diagnosed with PE (n = 34) and gestational age-matched controls (n = 24), whereas placental BeWo cells were treated with angiogenic factors in vitro. Significant downregulation of breast cancer resistance protein (BCRP) and several other transporters were seen in placentas complicated by PE compared with controls, whereas mRNA levels of sFlt-1 were induced by 2.5-fold in PE placentas (P < 0.01). Treatment of BeWo cells with sFlt-1 resulted in an 85-90% downregulation of BCRP, which was attenuated by vascular endothelial growth factor. Our findings suggest that placental function is compromised during PE due to altered expression of clinically important transporters. Furthermore, our in vitro results show that sFlt-1 is involved in the regulation of BCRP.

Itraconazole Alters the Stem Cell Characteristics of A549 and NCI-H460 Human Lung Cancer Cells by Suppressing Wnt Signaling.

BACKGROUND Cancer stem cells (CSCs) behave as their malignant counterparts, but persist after treatment, and possess properties that allow them to interact with their environment. Itraconazole, an antifungal agent, also has a role in suppressing tumor progression, but its effects in regulating tumor cell stemness remain unclear. This study aimed to evaluate the effects of itraconazole on A549 and NCI-H460 human lung cancer cell stemness in vitro. MATERIAL AND METHODS A549 and NCI-H460 human lung cancer cells and BEAS-2B normal bronchial epithelial cells were cultured with and without itraconazole. Cell viability was evaluated. The expression of stem cell markers, CD133, ATP binding cassette subfamily G member 2 (ABCG2), and aldehyde dehydrogenase 1 (ALDH1), were measured by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR). Sphere-forming cells were evaluated in vitro. RESULTS Itraconazole reduced the expression of stemness molecules CD133, ABCG2, and ALDH1 in A549 and NCI-H460 human lung cancer cells, and the numbers of sphere-forming cells were reduced. However, itraconazole had little effect on cell viability but enhanced the chemosensitivity of A549 and NCI-H460 cells. Itraconazole inhibited Wnt signaling. Re-activation of Wnt signaling restored itraconazole-mediated inhibition on A549 and NCI-H460 cell stemness. CONCLUSIONS Itraconazole altered the stemness characteristics of A549 and NCI-H460 human lung cancer cells by suppressing Wnt signaling but did not affect cell viability.

Direct Measurement of Kinetic Parameters of ABCG2-Dependent Transport of Natural Flavonoids Using a Fluorogenic Substrate.

Flavonoids are an important part of the human diet since plant-derived polyphenols and the mechanisms governing their pharmacokinetics are important both due to their own nutriceutical activity and the potential for food-drug interactions. A central determinant of absorption and distribution of flavonoids in the human body is the ATP-binding cassette transporter ABCG2, expressed in gut epithelium and other barrier tissues. While flavonoids were previously identified as substrates and/or inhibitors of this protein, precise enzyme kinetic calculations of affinity and activity parameters are rare due to the lack of suitable experimental models. We present a novel method that allows the direct measurement of kinetic constants for ABCG2-mediated cellular efflux of natural flavonoids thanks to the application of fluorogenic 2-aminoethyl diphenylborinate, which reacts with intracellular flavonoids forming a fluorescent, nonmembrane-permeable conjugate, thus making it possible to measure the intracellular substrate concentration throughout the experiment. Our studies were performed in Madin-Darby canine kidney II-derived cell lines expressing human ABCG2 and involve substrate efflux from whole, unmodified cells, precluding the need for plasma membrane vesicle preparation. We present methods for calculation of enzyme kinetic constants by measuring substrate concentration at efflux-influx equilibrium or during efflux from preloaded cells, and we obtained K m values of 137 µM for quercetin, 36 µM for kaempferol, and 348 µM for luteolin. Our method also allows direct verification of the transport inhibition mechanism and potentially the structure-activity relationship in substrates. SIGNIFICANCE STATEMENT: The study presents the first direct calculation of kinetic constants for enzyme-mediated active transport of natural flavonoids in a whole-cell assay, using a fluorogenic compound to measure intracellular substrate concentrations at specific time points. It has implications for nutriceutical use of polyphenols, mechanisms of food-drug interactions, and studies on absorption/distribution-determining membrane transporters, allowing a quantitative approach to pharmacokinetics of flavonoid transport across barrier tissues.

Optimization of sample preparation for transporter protein quantification in tissues by LC-MS/MS.

BACKGROUND: Reproducible quantification of drug transporter protein expression in tissues is important for predicting transporter mediated drug disposition. Many mass-spectrometry based transporter protein quantification methods result in high variability of the estimated transporter quantities. Therefore, we aimed to evaluate and optimize mass spectrometry-based quantification method for drug transporter proteins in tissues. MATERIALS AND METHODS: Plasma membrane (PM) proteins from mouse tissues were isolated by applying three extraction protocols: commercial plasma membrane extraction kit, tissue homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation, and PM enrichment with Tween 40. Moreover, five different protein digestion protocols were applied on the same PM fraction. PM isolation and digestion protocols were evaluated by measuring the amount of transporter proteins by liquid chromatography-tandem mass spectrometry in selected reaction monitoring mode. RESULTS: Mouse liver homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation and PM enrichment with Tween 40 resulted in two times higher transporter protein quantity (Breast cancer resistance protein (Bcrp) 18.0 fmol/µg protein) in comparison with the PM samples isolated by extraction kit (Bcrp 9.8 fmol/µg protein). The evaluation of protein digestion protocols revealed that the most optimal protocol for PM protein digestion is with Lys-C and trypsin, in combination with trypsin enhancer and heat denaturation. Overall, quantities of Bcrp and Na+/K + ATPase proteins evaluated in mouse liver and kidney cortex by using our optimized PM isolation method, as well as, established digestion protocol were two to three times higher than previously reported and coefficient of variation (CV) for technical replicates was below 10%. CONCLUSION: We have established an improved transporter protein quantification methodology by optimizing PM isolation and protein digestion procedures. The optimized procedure resulted in a higher transporter protein yield and improved precision.

Co-detection of ALDH1A1, ABCG2, ALCAM and CD133 in three A549 subpopulations at the single cell level by one-step digital RT-PCR.

Cancer stem-like cells (CSCs) displaying the properties of normal stem cells have become the main culprit associated with cancer transportation and recurrence. As of now, various CSC functions and marker genes have been identified due to the heterogeneity of cancer, such as aldehyde dehydrogenase (ALDH), the second member of the ABC transporter G-subfamily (ABCG2), activated leukocyte cell adhesion molecule (ALCAM) and CD133. To investigate these markers, most conventional approaches are bulk-based strategies, which may veil the disparity of single cells' gene expression. In this study, one-step digital RT-PCR at the single cell level was developed to co-determine the expression of ALDH1A1, ABCG2, ALCAM and CD133 genes in A549 cancer stem cells that perform high ALDH activities (ALDH+ A549 cells), as well as in ALDH- A549 cells and A549 cells, with 36, 20 and 20 cell samples each. The results demonstrated that, when compared to single ALDH- or A549 cells, the majority of single ALDH+ A549 cells displayed a 1.5- and 2.0-fold increase in the gene expression of ALDH1A1 and ALCAM (P < 0.001), respectively. However, for ABCG2 and CD133, there was no significant difference (P > 0.05), which means that they are not appropriate as co-indicated markers to identify ALDH+ A549 cells. Conclusively, as a single cell level approach, one-step digital RT-PCR has potential in exploring efficient co-detection markers for the classification and identification of CSCs.

Evidence of progenitor cells in the adult human cochlea: sphere formation and identification of ABCG2.

OBJECTIVES: The aim of this study was to search for evidence of stem or progenitor cells in the adult human cochlea by testing for sphere formation capacity and the presence of the stem cell marker ABCG2. METHODS: Cochleas removed from patients undergoing vestibular schwannoma resection (n=2) and from brain-dead organ donors (n=4) were dissociated for either flow cytometry analysis for the stem cell marker ABCG2 or a sphere formation assay that is widely used to test the sphere-forming capacity of cells from mouse inner ear tissue. RESULTS: Spheres were identified after 2-5 days in vitro, and the stem cell marker ABCG2 was detected using flow cytometric analysis after cochlear dissociation. CONCLUSIONS: Evidence suggests that there may be progenitor cells in the adult human cochlea, although further studies are required.

Evidence of progenitor cells in the adult human cochlea: sphere formation and identification of ABCG2

OBJECTIVES: The aim of this study was to search for evidence of stem or progenitor cells in the adult human cochlea by testing for sphere formation capacity and the presence of the stem cell marker ABCG2. METHODS: Cochleas removed from patients undergoing vestibular schwannoma resection (n=2) and from brain-dead organ donors (n=4) were dissociated for either flow cytometry analysis for the stem cell marker ABCG2 or a sphere formation assay that is widely used to test the sphere-forming capacity of cells from mouse inner ear tissue. RESULTS: Spheres were identified after 2-5 days in vitro, and the stem cell marker ABCG2 was detected using flow cytometric analysis after cochlear dissociation. CONCLUSIONS: Evidence suggests that there may be progenitor cells in the adult human cochlea, although further studies are required.

Optimization of Western blotting analysis for the isolation and detection of membrane xenobiotic transporter ABCG2.

All organisms are exposed to numerous stress factors, which include harmful xenobiotics. The diversity of these compounds is enormous, thus in the course of evolution diverse biological defense mechanisms at various levels of organization have developed. One of them engages an evolutionarily conserved family of transporters from the ABC superfamily, found in most species - from bacteria to humans. An important example of such a transporter is the breast cancer resistance protein (BCRP/ABCG2), a typical integral membrane protein. It plays a key role in the absorption, distribution and elimination of a wide variety of xenobiotics, including drugs used in chemotherapy, and is involved in multidrug resistance. It also protects against phototoxic chlorophyll derivatives of dietary origin. BCRP is a hemitransporter which consists of one transmembrane domain, made of six alpha-helices forming a characteristic pore structure, and one ATP-binding domain, which provides the energy from ATP hydrolysis, required for active transport of the substrates. The isolation of BCRP is still not an easy task, because its insolubility in water and the presence of membrane rafts pose serious methodological and technical challenges during the purification. The aim of this study was to optimize the methods for detection and isolation of BCRP-enriched fractions obtained from animal tissue samples. In this report we describe an optimization of isolation of a BCRP-enriched membrane fraction, which is suitable for further protein quantitative and qualitative analysis using the molecular biology tools.

An easy and fast adenosine 5'-diphosphate quantification procedure based on hydrophilic interaction liquid chromatography-high resolution tandem mass spectrometry for determination of the in vitro adenosine 5'-triphosphatase activity of the human breast cancer resistance protein ABCG2.

Interactions with the human breast cancer resistance protein (hBCRP) significantly influence the pharmacokinetic properties of a drug and can even lead to drug-drug interactions. As efflux pump from the ABC superfamily, hBCRP utilized energy gained by adenosine 5'-triphosphate (ATP) hydrolysis for the transmembrane movement of its substrates, while adenosine 5'-diphosphate (ADP) and inorganic phosphate were released. The ADP liberation can be used to detect interactions with the hBCRP ATPase. An ADP quantification method based on hydrophilic interaction liquid chromatography (HILIC) coupled to high resolution tandem mass spectrometry (HR-MS/MS) was developed and successfully validated in accordance to the criteria of the guideline on bioanalytical method validation by the European Medicines Agency. ATP and adenosine 5'-monophosphate were qualitatively included to prevent interferences. Furthermore, a setup consisting of six sample sets was evolved that allowed detection of hBCRP substrate or inhibitor properties of the test compound. The hBCRP substrate sulfasalazine and the hBCRP inhibitor orthovanadate were used as controls. To prove the applicability of the procedure, the effect of amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir on the hBCRP ATPase activity was tested. Nelfinavir, ritonavir, and saquinavir were identified as hBCRP ATPase inhibitors and none of the five HIV protease inhibitors turned out to be an hBCRP substrate. These findings were in line with a pervious publication.

Augmenter of liver regeneration potentiates doxorubicin anticancer efficacy by reducing the expression of ABCB1 and ABCG2 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is highly chemoresistant and therefore challenges both physicians and patients. Augmenter of liver regeneration (ALR), previously also known as 'hepatic stimulator substance', is reported to inhibit the epithelial-mesenchymal transition (EMT) in HCC, one of the frequent events that occur in cancer metastasis, suggesting that ALR is involved in HCC. In this study, we report for the first time that the transfection of ALR enhances the antitumor effect of chemotherapy with doxorubicin, a typical anticancer drug, on HCC in vitro and in vivo. The efflux of doxorubicin from ALR-transfected HCC cells is efficiently suppressed. This implies the intracellular retention of doxorubicin in tumor cells, which is at least partly attributable to the effective inhibition of ABCB1 and ABCG2 transporter expression in ALR-expressing cells. The downregulation of ALR expression by short hairpin RNA diminishes the antitumor effect of ALR. We further demonstrate that ALR inhibits the AKT/Snail signaling pathway, resulting in the downregulation of ABCB1 and ABCG2 expression. In conclusion, our results suggest that ALR is a potential chemotherapeutic agent against HCC.

Expression and Activity of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Distal Lung Epithelial Cells In Vitro.

PURPOSE: Breast cancer resistance protein (BCRP/ABCG2) has previously been identified with high expression levels in human lung. The subcellular localisation and functional activity of the transporter in lung epithelia, however, remains poorly investigated. The aim of this project was to study BCRP expression and activity in freshly isolated human alveolar epithelial type 2 (AT2) and type 1-like (AT1-like) cells in primary culture, and to compare these findings with data obtained from the NCI-H441 cell line. METHODS: BCRP expression levels in AT2 and AT1-like cells and in different passages of NCI-H441 cells were determined using q-PCR and immunoblot. Transporter localisation was confirmed by confocal laser scanning microscopy. Efflux and transport studies using the BCRP substrate BODIPY FL prazosin and the inhibitor Ko143 were carried out to assess BCRP activity in the different cell models. RESULTS: BCRP expression decreased during transdifferentiation from AT2 to AT1-like phenotype. Culturing NCI-H441 cells at an air-liquid interface or submersed did not change BCRP abundance, however, BCRP levels increased with passage number. BCRP was localised to the apical membrane and cytosol in NCI-H441 cells. In primary cells, the protein was found predominantly in the nucleus. Functional studies were consistent with expression data. CONCLUSIONS: BCRP is differently expressed in AT2 and AT1-like cells with lower abundance and activity in the latter ones. Nuclear BCRP might play a transcriptional role in distal lung epithelium. In NCI-H441 cells, BCRP is expressed in apical cell membranes and its activity is consistent with the localisation pattern.

Expression of MRP1 and ABCG2 is associated with adverse clinical outcomes of papillary thyroid carcinoma with a solid component.

Solid variant of papillary thyroid carcinoma (PTC) is characterized by a solid component (SC) retaining classical cytological features of PTC. Despite some controversies, PTC with SC (PTCSC) cases have poor prognosis compared with well-differentiated PTC. We investigated if cancer stem cells (CSCs) may have a role in pathogenesis of PTCSC. PTCSC tumors (n=27) were histologically represented by a mixture of papillary component (PC) and varying degrees of SC involving 10% to 85% of the tumor. Immunohistochemical expression of CSC markers ABCG2 and MRP1, and HBME1 and CK19 was compared between SC and PC within each tumor in association with clinicopathological parameters. ABCG2 and MRP1 were highly expressed in SC, whereas their expression was limited or absent in PC (P=.04 and .002, respectively). In contrast, expression of HBME1 and CK19 appeared higher in PC than in SC (P=.08 and .02, respectively). Higher expression of ABCG2 was associated with higher incidence of large-sized SC (P=.01). Higher expression of MRP1 was associated with higher incidence of lymphovascular invasion (P=.049). Higher expression of ABCG2 and MRP1, and lower expression of CK19 in SC were associated with higher tumor recurrence rate (P=.02, .01, and .02, respectively), and shorter disease-free survival (P<.001 for all the variables). Our findings indicate that the tumor cells harboring CSC-like characteristics in SC could contribute to the pathogenesis of PTCSC and might account for the poor disease prognosis.

P-gp/ABCB1 exerts differential impacts on brain and fetal exposure to norbuprenorphine.

Norbuprenorphine is the major active metabolite of buprenorphine which is commonly used to treat opiate addiction during pregnancy. Norbuprenorphine produces marked respiratory depression and was 10 times more potent than buprenorphine. Therefore, it is important to understand the mechanism that controls fetal exposure to norbuprenorphine, as exposure to this compound may pose a significant risk to the developing fetus. P-gp/ABCB1 and BCRP/ABCG2 are two major efflux transporters regulating tissue distribution of drugs. Previous studies have shown that norbuprenorphine, but not buprenorphine, is a P-gp substrate. In this study, we systematically examined and compared the roles of P-gp and BCRP in determining maternal brain and fetal distribution of norbuprenorphine using transporter knockout mouse models. We administered 1mg/kg norbuprenorphine by retro-orbital injection to pregnant FVB wild-type, Abcb1a-/-/1b-/-, and Abcb1a-/-/1b-/-/Abcg2-/- mice on gestation day 15. The fetal AUC of norbuprenorphine was ∼64% of the maternal plasma AUC in wild-type mice, suggesting substantial fetal exposure to norbuprenorphine. The maternal plasma AUCs of norbuprenorphine in Abcb1a-/-/1b-/- and Abcb1a-/-/1b-/-/Abcg2-/- mice were ∼2 times greater than that in wild-type mice. Fetal AUCs in Abcb1a-/-/1b-/- and Abcb1a-/-/1b-/-/Abcg2-/- mice were also increased compared to wild-type mice; however, the fetal-to-maternal plasma AUC ratio remained relatively unchanged by the knockout of Abcb1a/1b or Abcb1a/1b/Abcg2. In contrast, the maternal brain-to-maternal plasma AUC ratio in Abcb1a-/-/1b-/- or Abcb1a-/-/1b-/-/Abcg2-/- mice was increased ∼30-fold compared to wild-type mice. Protein quantification by LC-MS/MS proteomics revealed significantly higher amounts of P-gp protein in the wild-type mice brain than that in the placenta. These results indicate that fetal exposure to norbuprenorphine is substantial and that P-gp has a minor impact on fetal exposure to norbuprenorphine, but plays a significant role in restricting its brain distribution. The differential impacts of P-gp on norbuprenorphine distribution into the brain and fetus are likely, at least in part, due to the differences in amounts of P-gp protein expressed in the blood-brain and blood-placental barriers. BCRP is not as important as P-gp in determining both the systemic and tissue exposure to norbuprenorphine. Finally, fetal AUCs of the metabolite norbuprenorphine-ß-d-glucuronide were 3-7 times greater than maternal plasma AUCs, while the maternal brain AUCs were <50% of maternal plasma AUCs, suggesting that a reversible pool of conjugated metabolite in the fetus may contribute to the high fetal exposure to norbuprenorphine.

Immunohistochemical Expression of P-glycoprotein and Breast Cancer Resistance Protein in Canine Mammary Hyperplasia, Neoplasia and Supporting Stroma.

The ability of a tumour to become simultaneously resistant to different drugs is known as multidrug resistance and is often due to the expression of ATP-dependent binding cassette transporters (ABC-transporters) such as P-glycoprotein (PGP) and breast cancer resistance protein (BCRP). In this study, the expression of PGP and BCRP was determined in the components of hyperplastic and neoplastic canine mammary glands, including the supporting stroma. The variation of expression of these molecules in carcinomas was evaluated between lesions of different histological stage and grade of malignancy. Samples included 47 hyperplastic tissues and 10 benign and 46 malignant neoplasms. Tumours were classified into histological subtype, histological stage and grade. Immunohistochemical evaluation of PGP and BCRP expression showed that both markers are potentially expressed by epithelial cells, myoepithelial cells in complex tumours and mesenchymal cells in mixed tumours, but expression of both proteins was significantly higher in malignant epithelial cells versus hyperplastic epithelium or the epithelium of benign tumours. BCRP showed significantly higher expression in epithelial cells of simple carcinomas versus those of complex and mixed carcinomas. Grade II and III carcinomas had higher epithelial PGP expression than grade I tumours. The positivity of stromal fibroblasts was higher in histological stage II versus I carcinomas, and in histological grade II versus I carcinomas. Malignant and invasive tumours were more likely to express PGP and/or BCRP in luminal and stromal components and evaluation of these markers could provide valuable information for the identification of tumours characterized by an aggressive and chemoresistant phenotype.

ABCG2 Overexpression Confers Poor Outcomes in Hepatocellular Carcinoma of Elderly Patients.

UNLABELLED: Backgroung/Aim: Breast cancer resistance protein [BCRP/ATP-binding cassette subfamily G member 2 (ABCG2)] is a member of the ATP-binding cassette transporter family, used as a maker of cancer stem cells (CSCs) and thought to be responsible for drug resistance by pumping them out of cells. However, its precise role in various cancer types has been controversial, and the aim of this study was to investigate the expression of ABCG2 in hepacellular carcinoma (HCC) and relate the results to established prognostic factors. PATIENTS AND METHODS: We conducted analysis of 181 HCC and paired-match adjacent normal liver tissue by immunohistochemistry from tissue array of slides. RESULTS: The mean score for ABCG2 expression was higher in tumor than in adjacent normal liver tissue of HCC patients (p<0.001). There was a statistically significant correlation between ABCG2 expression and age, differentiation status and hepatitis B surface antigen (p=0.031, p=0.015 and p=0.033, respectively). Additionally, increased expression of ABCG2 in HCC and its statistically significant correlation with hepatitis B surface antigen was found in elderly (p=0.039), not in younger patients (p=0.518). Importantly, by using Kaplan-Meier and Cox regression analysis, overall survival in patients with high expression of ABCG2 was found reduced in elderly patients (p=0.029 and p=0.081, respectively). CONCLUSION: ABCG2 can be used as a target for the development of novel therapies in HCC.

ABCG2, Cytogenetics, and Age Predict Relapse after Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia in Complete Remission.

Recent studies have shown that ABGG2 protein overexpression in acute myeloid leukemia (AML) may be associated with poor response to therapy and increased relapse risk. Few data are available in patients with AML undergoing allogeneic stem cell transplantation (SCT), particularly when in complete remission (CR). We analyzed 105 patients with AML who underwent allogeneic SCT in CR evaluating the role of ABCG2 and other pretransplantation features on subsequent transplantation outcomes. Factors negatively associated with leukemia-free survival (LFS) were unfavorable cytogenetics (3-year LFS 48% versus 80%, P = .0035) and ABCG2 positivity (65% versus 80%, P = .045). Three-year cumulative incidence of relapse (CIR) in the whole population was 20%; a higher incidence of relapse was associated with adverse cytogenetics (41% versus 16%, P = .018), ABCG2 overexpression (29% versus 15%, P = .04), and, marginally, age > 50 years (30% versus 14%, P = .06). We grouped patients according to the combination of these 3 risk factors: no patient relapsed within 3 years from SCT in the group without risk factors, whereas the 3-year CIR was 12% (95% confidence interval [CI], 2% to 25%) in the group with 1 risk factor and 47% (95% CI, 31% to 70%) in patients with 2 or 3 risk factors (P = .00005). In conclusion, allogeneic SCT does not seem to abrogate the negative prognosis associated with ABCG2 overexpression at diagnosis, specifically in terms of a higher relapse risk. ABCG2, age, and cytogenetics can predict AML relapse after SCT in patients who undergo transplantation while in CR.

Expression levels of ABCG2 on cord red blood cells and study of fetal anemia associated with anti-Jr(a).

BACKGROUND: The Jr(a) antigen of JR blood group systems is located on ABCG2 and Jr(a-) subjects whose red blood cells (RBCs) lack ABCG2 have been identified mostly among the Japanese. Although anti-Jr(a) can cause fetal anemia, little is known regarding its mechanism. STUDY DESIGN AND METHODS: We reviewed clinical courses of all reported cases with fetal anemia due to anti-Jr(a) . We analyzed the ABCG2 expressions of cord RBCs at various gestational ages. We examined the effects of sera containing anti-Jr(a) from three pregnancies with fetal anemia or monoclonal anti-Jr(a) on erythropoiesis and phagocytosis. We also examined epitopes of anti-Jr(a) . RESULTS: Case series suggested that the majority of fetal anemia with anti-Jr(a) may not be progressive in the later gestational ages. ABCG2 expression levels of cord RBCs were significantly higher than those of adults and neonates with high individual variation and gradually decreased with advancing gestational ages. Anti-Jr(a) did not significantly impact erythroid colony formation, although we detected a tendency toward the suppression of erythroid burst-forming unit formation by anti-Jr(a) using feline marrow cells. Anti-Jr(a) did not induce phagocytosis of sensitized RBCs by monocytes. While many anti-Jr(a) recognized the same regions as a monoclonal anti-ABCG2, 5D3, epitopes of anti-Jr(a) did not correlate with the incidence of fetal anemia. CONCLUSION: ABCG2 expression levels in cord RBCs are higher than those of adults, and the change of ABCG2 expression in erythroid lineage cells may influence the clinical course of fetal anemia with anti-Jr(a) , although we could not detect significant effects of anti-Jr(a) on erythroid colony formation or phagocytosis.