Mitoxantrone Resistance Protein Research Articles

In Vivo Hepatic Enhancer Elements in the Human ABCG2 Locus.

ABCG2 encodes the mitoxantrone resistance protein (MXR; breast cancer resistance protein), an ATP-binding cassette (ABC) efflux membrane transporter. Computational analysis of the ∼300 kb region of DNA surrounding ABCG2 (chr4:88911376-89220011, hg19) identified 30 regions with potential cis-regulatory capabilities. These putative regulatory regions were tested for their enhancer and suppressor activity in a human liver cell line using luciferase reporter assays. The in vitro enhancer and suppressor assays identified four regions that decreased gene expression and five regions that increased expression >1.6-fold. Four of five human hepatic in vitro enhancers were confirmed as in vivo liver enhancers using the mouse hydrodynamic tail vein injection assay. Two of the in vivo liver enhancers (ABCG2RE1 and ABCG2RE9) responded to 17β-estradiol or rifampin in human cell lines, and ABCG2RE9 had ChIP-seq evidence to support the binding of several transcription factors and the transcriptional coactivator p300 in human hepatocytes. This study identified genomic regions surrounding human ABCG2 that can function as regulatory elements, some with the capacity to alter gene expression upon environmental stimulus. The results from this research will drive future investigations of interindividual variation in ABCG2 expression and function that contribute to differences in drug response.

Kinetic Characterization of Sulfasalazine Transport by Human ATP-Binding Cassette G2

The pharmacokinetics of sulfasalazine, an anti-inflammatory drug is influenced by ATP-binding cassette G2 (ABCG2) (breast cancer resistance protein (BCRP), mitoxantrone resistance protein (MXR)) both in vitro and clinically. Due to its low passive permeability, the intracellular concentration of sulfasalazine is dependent on uptake transporters, rendering the characterization of transporter specific interactions in cell based experimental systems difficult. Applying membrane assays a detailed kinetic analysis of sulfasalazine ABCG2 interaction was conducted and Km values of 0.70 +/- 0.03 microM and 0.66 +/- 0.08 microM were obtained at pH 7.0 and pH 5.5, respectively.

Real-Time Quantitative Polymerase Chain Reaction for BCRP, MDR1, and MRP1 mRNA Levels in Lymphocytes and Monocytes

The expression levels of mRNAs for MDR1 (P-glycoprotein), multidrug resistance-associated proteins (MRP1), and BCRP (breast cancer resistance protein; mitoxantrone resistance protein) were quantitatively determined in lymphocytes and monocytes. Monocytes and lymphocytes were obtained from 3 healthy male and 2 healthy female volunteers. BCRP, MDR1, and MRP1 mRNA levels were determined by real-time quantitative reverse transcriptase polymerase chain reaction. In lymphocytes, relative concentrations of mRNAs for target proteins (BCRP, MDR1, and MRP1) were 1.00 ± 0.478, 9.67 ± 5.53, and 0.116 ± 0.0613 respectively, and in monocytes 1.00 ± 0.854, 0.821 ± 0.263, and 0.090 ± 0.052, respectively. The MDR1 mRNA level was cell type dependent, whereas there was no difference in BCRP and MRP1 expression levels between lymphocytes and monocytes. Comparison of mRNA levels for the three major multidrug-resistant efflux pumps reveals that MDR1 is the predominant form in lymphocytes and BCRP is the predominant form in monocytes.

Modulation of function of three ABC drug transporters, P-glycoprotein (ABCB1), mitoxantrone resistance protein (ABCG2) and multidrug resistance protein 1 (ABCC1) by tetrahydrocurcumin, a major metabolite of curcumin

Many studies have been performed with the aim of developing effective resistance modulators to overcome the multidrug resistance (MDR) of human cancers. Potent MDR modulators are being investigated in clinical trials. Many current studies are focused on dietary herbs due to the fact that these have been used for centuries without producing any harmful side effects. In this study, the effect of tetrahydrocurcumin (THC) on three ABC drug transporter proteins, P-glycoprotein (P-gp or ABCB1), mitoxantrone resistance protein (MXR or ABCG2) and multidrug resistance protein 1 (MRP1 or ABCC1) was investigated, to assess whether an ultimate metabolite form of curcuminoids (THC) is able to modulate MDR in cancer cells. Two different types of cell lines were used for P-gp study, human cervical carcinoma KB-3-1 (wild type) and KB-V-1 and human breast cancer MCF-7 (wild type) and MCF-7 MDR, whereas, pcDNA3.1 and pcDNA3.1-MRP1 transfected HEK 293 and MXR overexpressing MCF7AdrVp3000 or MCF7FL1000 and its parental MCF-7 were used for MRP1 and MXR study, respectively. We report here for the first time that THC is able to inhibit the function of P-gp, MXR and MRP1. The results of flow cytometry assay indicated that THC is able to inhibit the function of P-gp and thereby significantly increase the accumulation of rhodamine and calcein AM in KB-V-1 cells. The result was confirmed by the effect of THC on [(3)H]-vinblastine accumulation and efflux in MCF-7 and MCF-7MDR. THC significantly increased the accumulation and inhibited the efflux of [(3)H]-vinblastine in MCF-7 MDR in a concentration-dependent manner. This effect was not found in wild type MCF-7 cell line. The interaction of THC with the P-gp molecule was clearly indicated by ATPase assay and photoaffinity labeling of P-gp with transport substrate. THC stimulated P-gp ATPase activity and inhibited the incorporation of [(125)I]-iodoarylazidoprazosin (IAAP) into P-gp in a concentration-dependent manner. The binding of [(125)I]-IAAP to MXR was also inhibited by THC suggesting that THC interacted with drug binding site of the transporter. THC dose dependently inhibited the efflux of mitoxantrone and pheophorbide A from MXR expressing cells (MCF7AdrVp3000 and MCF7FL1000). Similarly with MRP1, the efflux of a fluorescent substrate calcein AM was inhibited effectively by THC thereby the accumulation of calcein was increased in MRP1-HEK 293 and not its parental pcDNA3.1-HEK 293 cells. The MDR reversing properties of THC on P-gp, MRP1, and MXR were determined by MTT assay. THC significantly increased the sensitivity of vinblastine, mitoxantrone and etoposide in drug resistance KB-V-1, MCF7AdrVp3000 and MRP1-HEK 293 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Taken together, this study clearly showed that THC inhibits the efflux function of P-gp, MXR and MRP1 and it is able to extend the MDR reversing activity of curcuminoids in vivo.

Role for Drug Transporters Beyond Tumor Resistance: Hepatic Functional Imaging and Genotyping of Multidrug Resistance Transporters for the Prediction of Irinotecan Toxicity

The multigenic nature of drug response is increasingly recognized and underscores our current difficulties with adequately predicting interindividual variability in both the response to and toxicity of many chemotherapeutic agents. The clinical pharmacology of irinotecan nicely illustrates the complexity of drug response pathways and highlights the pressing need to develop easily measured markers that are predictive of drug disposition, toxicity, and efficacy. Until recently, the main focus has been on understanding the variation in irinotecan metabolism, since it was considered the primary factor controlling systemic exposure to this drug. Irinotecan is a prodrug that must be metabolized to the active compound SN-38, a potent inhibitor of topoisomerase I. Metabolism of irinotecan to SN-38 is catalyzed largely by carboxylesterase-2 (CES2), and CYP3A4/CYP3A5 catalyzes the formation of inactive metabolites. A significant fraction of SN-38 is converted to the glucuronide conjugate (SN-38G)—a conversion catalyzed largely by UGT1A1. More than 10 years of research have nicely illustrated the critical role of UGT1A1 activity in determining the pharmacologic response to irinotecan, as well as in the development of its rate-limiting toxicities of neutropenia and lateonset diarrhea. In vivo and genetic markers have been established for many drug metabolizing enzymes, and in the case of UGT1A1, genotyping alone is highly predictive of function. However, metabolism is only one factor controlling the systemic and cellular exposure to irinotecan and its metabolites. Drug transporters have also been implicated in the disposition of irinotecan, SN-38 and SN-38G, with evidence for transport by P-glycoprotein (Pgp; encoded by ABCB1), multidrug resistance-associated protein 1 (MRP1; ABCC1), MRP2 (ABCC2), and mitoxantrone resistance protein (MXR; ABCG2). More recently, an organic anion transporting polypeptide (OATP1B1; SLCO1B1) was shown to transport SN-38 but not irinotecan or SN-38G. An accurate prediction of those patients most likely to develop life-threatening toxicities during irinotecan therapy needs to consider variation in not only the metabolism of this important drug but also its transport into and out of both tumor cells and those cells associated with its disposition and toxicity. The article in this issue by Michael et al is an important contribution to the development of more comprehensive biomarkers of irinotecan disposition. Hepatic imaging was performed in advanced colorectal cancer patients using well-characterized substrates of the multidrug resistance transporters Pgp, MRP1 and MRP2, and hepatic functional imaging parameters were correlated with irinotecan pharmacokinetics. The area under the curve (AUC) of SN-38 was positively correlated with the hepatic retention of the imaging agents, a significant finding since SN-38 exposure is linked to the neutropenia and late-onset diarrhea associated with irinotecan therapy. Indeed, there was a trend toward an association between hepatic imaging parameters and toxicity. A preliminary analysis of ABCB1 genotypes was also performed and is consistent with the important role of Pgp in the disposition of the imaging agents. Hepatobiliary imaging was developed for the evaluation of hepatic dysfunction and to monitor the response to drugs and other interventions. The two classes of Tc-labeled agents that were used in these studies are rapidly taken up into the hepatocyte and eliminated into the bile by distinct active transport mechanisms. The iminodiacetic acid (IDA) analogs (Tc-DIDA and Tc-DISIDA) are organic anions and likely require uptake transporters for entry into the hepatocyte. Biliary excretion of TcDIDA and Tc-DISIDA is mediated by MRP2 while transport by MRP1 will eliminate drug from the hepatocyte back into the systemic circulation. Consistent with the significant role of MRP2 in the elimination of IDA agents, their biliary excretion is decreased in Dubin-Johnson syndrome patients who often carry polymorphisms in ABCC2 that result in a loss of MRP2 function. Tc-labeled sestamibi (Tc-MIBI) is a Pgp substrate and has been used to demonstrate Pgp inhibition by pharmacologic agents and genetic disruption. These imaging agents have great potential in the evaluation of drug transport function in the liver and fill a current void in this area. The lack of selective substrates that are metabolically stable has made it difficult to assess in vivo transporter function. Most substrates of Pgp, MRP1, and MRP2 undergo significant first pass and systemic metabolism and pharmacokinetic parameters for JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 24 NUMBER 26 SEPTEMBER 1

Modulation of multidrug resistance efflux pump activity to overcome chemoresistance in cancer

Early publications using cultured cancer cells immediately recognized the phenomenon of resistance to anticancer agents. However, it was not until 1973 that it was first demonstrated that a major factor in the resistance of cancer cells was that of reduced drug accumulation. This year marks the 30th anniversary of the discovery by Juliano and Ling that P-glycoprotein mediates this active efflux of chemotherapeutic drugs from cancer cells. Since this seminal finding, the investigation of P-glycoprotein (MDR1, ATP binding cassette [ABC]B1) has proceeded with great vigour. However, it soon became apparent that P-glycoprotein was not expressed in all drug-resistant cells that displayed an accumulation deficiency, which led to the discovery of other ABC transporters involved in drug efflux. In 1992, the multidrug resistance-associated protein (MRP1, ABCC1) was identified in small cell lung cancer followed by breast cancer resistance protein (mitoxantrone resistance protein, ABCG2) in 1999. After three decades of research, can we confidently define the contribution of multidrug resistance transporters to chemoresistance and do we have clinically useful drugs to sensitise cancers?

The multidrug resistance protein BMDP/ABCG2: A new and highly relevant efflux pump at the blood–brain barrier

Combinatorial chemistry is able to generate high numbers of compounds of pharmaceutical interest. An important aspect for a new drug is to know its availability in the nervous system, which means the ability to cross the barriers between blood and brain. Thus the permeability of thousands of compounds will have to be screened in the near future. This will not be possible in vivo, so that powerful in vitro models are demanded that closely mimic the in vivo system at least with respect to barrier properties. Two barrier systems are of main interest with respect to the passage of compounds into the brain: the blood–brain barrier (BBB) of the cerebral capillaries and the blood–liquor barrier (BLB) of the choroid plexus. In this paper main focus will be on the development of an in vitro model for the BBB. The brain capillary endothelial cells (BCEC) are isolated from freshly slaughtered pigs and cultivated on permeable filters to mimic the interfacial localization between blood and CNS. Cultivating the cells in serum-free medium improves their barrier properties. Hydrocortisone supplementation additionally reinforces the BBB properties. Under these conditions we observed an up-regulation of an efflux pump that has been found earlier in a breast cancer cell line, there named BCRP (Breast Cancer Resistance Protein). This efflux pump is also known as Mitoxantrone Resistance Protein (MXR) or Placenta-specific ABC Protein (ABCP). We identified this protein for the first time at the blood–brain barrier. mRNA expression studies of different brain cells showed that this efflux pump is mainly expressed in the brain capillary endothelial cells. With RT-PCR we showed a minor expression in pericytes as well as in epithelial cells from the choroid plexus and in spinal cord astrocytes. From cDNA sequence analysis we know that this efflux pump is a homologue to the ABCG2-transporter consisting of only one ATP-binding cassette and six membrane spanning helices. For this so-called half transporter, an oligomerization in the plasma membrane is discussed. Quantitative real-time PCR analysis indicates that this pump is highly relevant for the blood–brain barrier functionality since its expression level is about 3 fold higher compared to P-gp. Northern blot analysis yielded a 30 fold higher mRNA content. Immunocytochemistry revealed that in brain endothelial cells the protein is predominantly localized in the apical plasma membrane which compares to the blood side in vivo. The transport direction for known ABCG2 substrates consistently supports this finding. This paper will discuss the importance of this newly discovered ABC transporter at the blood–brain barrier in comparison to other members of ABC subfamilies.

Mechanisms of resistance to anticancer drugs: the role of the polymorphic ABC transporters ABCB1 and ABCG2

ATP-binding cassette (ABC) genes play a role in the resistance of malignant cells to anticancer agents. The ABC gene products, including ABCB1 (P-glycoprotein) and ABCG2 (breast cancer-resistance protein [BCRP], mitoxantrone-resistance protein [MXR], or ABC transporter in placenta [ABCP]), are also known to influence oral absorption and disposition of a wide variety of drugs. As a result, the expression levels of these proteins in humans have important consequences for an individual's susceptibility to certain drug-induced side effects, interactions, and treatment efficacy. Naturally occurring variants in ABC transporter genes have been identified that might affect the function and expression of the protein. This review focuses on recent advances in the pharmacogenetics of the ABC transporters ABCB1 and ABCG2, and discusses potential implications of genetic variants for the chemotherapeutic treatment of cancer.

Mitoxantrone Resistance protein (MXR) is down-regulated in colorectal cancer

Introduction. MXR knockout mice were recently shown to display a lethal photosensitive phenotype suggesting an inverse relationship between MXR and photosensitivity. Normal human colon expresses MXR, but colorectal cancer is susceptible to photodynamic therapy. We hypothesized that MXR is down regulated in colorectal cancer. Methods. Total RNA was extracted from paired normal and cancer tissues harvested from 13 patients with colorectal cancer. MXR mRNA expression was determined by RT-PCR using 18S RNA as an internal control and confirmed by Northern blot using β-actin mRNA as an internal control. MXR protein expression was determined by immunohistochemistry (IHC). The log of the ratio of relative expression by RT-PCR was analyzed using a linear mixed model that included subject and gel dates as random effects. Means and standard deviations were calculated, and t-tests were used as appropriate. Results. MXR mRNA was present in normal colon and showed a mean decrease of 6.6-fold in cancer by RT-PCR ( P < 0.0001) and a mean decrease of 4.8-fold by Northern blot ( P = 0.0036). The 95% lower confidence interval for relative expression was a 4.6-fold decrease. MXR protein expression was abundant in the brush border of normal colon on IHC and showed a marked decrease in colorectal cancer. Conclusions. MXR is significantly down regulated in human colorectal cancer. MXR effluxes the phototoxin, protoporphyrin IX. MXR knockout mice accumulate protoporphyrin IX and are photosensitive. Selective accumulation of protoporphyrin IX in cancer tissue after systemic administration of a photosensitizing agent is the biochemical basis of photodynamic therapy. This data showing down regulation of MXR in colorectal cancer may provide the molecular basis for colorectal cancer’s susceptibility to photodynamic therapy.

Characterization of Oligomeric Human Half-ABC Transporter ATP-binding Cassette G2

Human ATP-binding cassette G2 (ABCG2, also known as mitoxantrone resistance protein, breast cancer-resistance protein, ABC placenta) is a member of the superfamily of ATP-binding cassette (ABC) transporters that have a wide variety of substrates. Overexpression of human ABCG2 in model cancer cell lines causes multidrug resistance by actively effluxing anticancer drugs. Unlike most of the other ABC transporters which usually have two nucleotide-binding domains and two transmembrane domains, ABCG2 consists of only one nucleotide-binding domain followed by one transmembrane domain. Thus, ABCG2 has been thought to be a half-transporter that may function as a homodimer. In this study, we characterized the oligomeric feature of human ABCG2 using non-denaturing detergent perfluoro-octanoic acid and Triton X-100 in combination with gel filtration, sucrose density gradient sedimentation, and gel electrophoresis. Unexpectedly, we found that human ABCG2 exists mainly as a tetramer, with a possibility of a higher form of oligomerization. Monomeric and dimeric ABCG2 did not appear to be the major form of the protein. Further immunoprecipitation analysis showed that the oligomeric ABCG2 did not contain any other proteins. Taken together, we conclude that human ABCG2 likely exists and functions as a homotetramer.

Breast Cancer Resistance Protein (BCRP) in Acute Leukaemia

Multidrug resistance, cross-resistance to structurally and functionally unrelated drugs, is an important cause of treatment failure in acute leukemia. Multidrug resistance can result from the overexpression of ATP-dependent efflux pumps, such as P-glycoprotein and members of the multidrug resistance associated protein (MRP) family. Recently a novel transporter has been identified, which is called breast cancer resistance protein (BCRP), ABCG2 or mitoxantrone resistance protein. BCRP confers resistance to chemotherapeutic agents, such as mitoxantrone, doxorubicin and daunorubicin. This review describes BCRP detection techniques and the normal physiology of BCRP. The role of BCRP in the physiology of hematopoietic stem cells is addressed as well as the involvement of BCRP in multidrug resistance in acute leukemia. In AML and ALL, several studies showed that BCRP is expressed and functionally active at low, but variable levels. However, further studies are warranted to investigate its effect on clinical outcome, and explore whether patients could benefit from the combination of BCRP inhibitors and chemotherapy.

Monoclonal antibodies as a tool for structure-function studies of the MDR1-P-glycoprotein.

P-glycoprotein is considered one of the most important member of the rapidly growing superfamily of integral proteins known as the ATP-binding cassette (ABC) which in human also include several other multidrug resistance membrane proteins (i.e., MRP), the product of the cystic fibrosis gene, the TAP-1/TAP2 peptide transporters encoded by the major histocompatibility complex genes and the gene encoding for breast cancer resistance protein (BCRP) also known as MXR1 (mitoxantrone resistance protein). Many monoclonal antibodies (MAbs) reacting with distinct P-glycoprotein domains have been isolated and used to study the molecular organization and cellular functions of this ABC protein. MAbs have been used for multidrug resistance (mdr) gene cloning, delineation of the secondary and tertiary structure of P-glycoprotein and molecular analysis of the mechanisms involved in substrate recognition and transport. The immunodetection of the distinct products of the mdr gene family in normal and malignant cells and tissues has greatly contributed to the understanding of the physiological role of P-glycoprotein and its possible involvement in the refractory of tumors to chemotherapy. The present article deals with the immunological methods used for the structure-function studies of the P-glycoprotein. After introducing the basic structural features of this ABC transporter, the antibody based-approach is discussed with aiming to furnishing methodological perspectives for further investigations of the physiological role of P-glycoprotein and the multidrug resistance phenomenon.

BCRP gene expression is associated with a poor response to remission induction therapy in childhood acute myeloid leukemia.

Breast cancer resistance protein (BCRP), also known as mitoxantrone resistance protein (MRX) or placenta ABC protein (ABC-P), is the second member of the ABCG subfamily of ABC transport proteins (gene symbol ABCG2). Transfection and enforced expression of BCRP in drug-sensitive cells confers resistance to mitoxantrone, doxorubicin, daunorubicin and topotecan. In this study the expression of BCRP gene was measured using TaqMan real-time PCR in 59 children with newly diagnosed AML. Nine patients were also analyzed in relapse. The median of BCRP gene expression was more than 10 times higher in patients who did not achieve remission after the first phase of chemotherapy (n = 24) as compared to patients who did achieve remission at this stage (n = 21; P = 0.012). In first relapse the expression of the BCRP gene was higher than at diagnosis (P = 0.038). Although high levels of BCRP gene expression were more frequent in subtypes of AML with a favorable prognosis, we found that within both risk groups (high and low risk), patients who expressed high levels of BCRP had a worse prognosis (P = 0.023). Our results strongly suggest that the expression of the BCRP gene reduces the response to chemotherapy in AML and that BCRP expression is higher at the time of relapse.

Expression of the BCRP gene (ABCG2/MXR/ABCP) in childhood acute lymphoblastic leukaemia.

The breast cancer resistance protein (BCRP), also known as mitoxantrone resistance protein (MXR) or placenta ABC protein (ABC-P), is the second member of the ABCG subfamily of ABC transport proteins (gene symbol ABCG2). BCRP has been detected in acute myeloid leukaemia and in breast, colon and gastric cancer but there has been no reports regarding BCRP expression in acute lymphoblastic leukaemia (ALL). We report the first results of BCRP expression in childhood ALL. Sixty-seven children (47 initial stage, 20 relapses) with ALL were analysed for BCRP gene expression by TaqMan real-time polymerase chain reaction. The expression of BCRP in mononuclear cells obtained from the bone marrow (BM) and peripheral blood (PB) of healthy donors was also investigated. There was no relationship between BCRP expression and age, sex, initial blast cell count, prednisolone response or BM response on d 15 and 33. Patients with T-lineage ALL showed a lower expression of BCRP (P = 0.044). Kaplan-Meier analysis of the relapse-free interval showed no prognostic significance of BCRP expression when different levels of BCRP expression were used as cut-off points. No significant difference in expression of BCRP mRNA was measured between initial-stage and relapsed-stage ALL or between normal MNC obtained from BM and ALL patients. The results indicate a low expression of BCRP in childhood ALL. Relationships between BCRP and clinical, molecular or in vivo resistance characteristics of the patients were not observed.

A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2)

The fluorescent compounds rhodamine 123, LysoTracker Green DMD-26, mitoxantrone, and BODIPY-prazosin were used with the antagonist fumitremorgin C (FTC) in order to develop functional assays for the half-transporter, MXR/BCRP/ABCP1. A measure of FTC-inhibitable efflux was generated for each compound in a series of MXR-overexpressing drug-selected cell lines and in ten unselected cell lines which were used to determine if the four fluorescent compounds were sensitive enough to detect the low MXR levels found in drug-sensitive cell lines. FTC-inhibitable efflux of mitoxantrone and prazosin was found in four of the ten cell lines, SF295, KM12, NCI-H460, and A549, and low but detectable levels of MXR mRNA were also observed by Northern analysis in these cells. FTC-inhibitable mitoxantrone and prazosin efflux in both selected and unselected cell lines was found to correlate well with MXR levels as determined by Northern blotting, r 2=0.89 and r 2=0.70 respectively. In contrast, rhodamine and LysoTracker were not able to reliably detect MXR. Cytotoxicity assays performed on two of the four unselected cell lines confirmed increased sensitivity to mitoxantrone in the presence of FTC. FTC was found to be a specific inhibitor of MXR, with half-maximal inhibition of MXR-associated ATPase activity at 1 μM FTC. Short term selections of the SF295, KM12, NCI-H460 and A549 cell lines in mitoxantrone resulted in a small but measurable increase in MXR by both Northern blot and functional assay. These studies show that flow cytometric measurement of FTC-inhibitable mitoxantrone or prazosin efflux is a sensitive and specific method for measuring the function of the MXR half-transporter in both selected and unselected cell lines.