Metalloprobes: synthesis, characterization, and potency of a novel gallium(III) complex in human epidermal carcinoma cells

Multidrug resistance (MDR) mediated by overexpression of the MDR1 gene product, P-glycoprotein (Pgp), represents one of the best characterized barriers to chemotherapeutic treatment in cancer and may be a pivotal factor in progression of Alzheimer's disease (AD). Thus, agents capable of probing Pgp-mediated transport could be beneficial in biomedical imaging. Herein, we synthesized and structurally characterized a gallium(III) complex (5) of the naphthol-Schiff base ligand. The crystal structure revealed octahedral geometry for the metallodrug. Cytotoxicity profiles of 5 were evaluated in KB-3-1 (Pgp-) and KB-8-5 (Pgp+) human epidermal carcinoma cell lines. Compared with an LC(50) (the half-maximal cytotoxic concentration) value of 1.93 microM in drug-sensitive (Pgp-) cells, the gallium(III) complex 5 demonstrated an LC(50) value>100 microM in drug-resistant (Pgp+) cells, thus indicating that 5 was recognized by the Pgp as its substrate, thereby extruded from the cells and sequestered away from their cytotoxic targets. Radiolabeled analogues of 5 could be beneficial in noninvasive imaging of Pgp-mediated transport in vivo.     

Imaging multidrug resistance P-glycoprotein transport function using microPET with technetium-94m-sestamibi

The best characterized mechanism of multidrug resistance (MDR) in cancer involves the MDR1 efflux transporter P-glycoprotein (Pgp). The positron-emitting radiotracer hexakis(2-methoxyisobutylisonitrile)-(94m)Tc ((94m)Tc-MIBI) was synthesized and validated in cell transport studies as a substrate for MDR1 Pgp. In vivo small-scale PET imaging and biodistribution studies of mdr1a/1b (-/-) gene deleted and wild-type mice demonstrated the use of (94m)Tc-MIBI to detect Pgp function. The reversal effect of a Pgp modulator was shown in tissue distribution studies of KB 3-1 (Pgp-) and KB 8-5 (Pgp+) tumor-bearing nude mice. The current (94m)Tc-MIBI experiments parallel previous studies employing (99m)Tc-MIBI, showing essentially identical performance of the two technetium radiotracers and providing biological validation of (94m)Tc-MIBI for PET imaging of multidrug resistance.     

Annexin-I expression modulates drug resistance in tumor cells

The use of anti-cancer chemotherapy often leads to the rise of multidrug-resistant (MDR) tumors. We have previously reported the overexpression of a 40kDa protein (P-40) in several MDR tumor cell lines. In this report we describe the cloning of a 1.4kb cDNA with an open reading frame of 344 amino acids that encodes the P-40 protein. Analysis of the P-40 amino acid sequence showed it is identical to the human annexin I (Anx-I) protein. The identity of the isolated P-40 cDNA as Anx-I was confirmed by the specific binding of IPM96 mAb to a 40kDa protein following the in vitro expression of P-40 full-length cDNA. Northern blot analysis of total RNA from drug-sensitive and -resistant cells revealed an increase in P-40 (or Anx-I) mRNA in drug-resistant cells relative to drug-sensitive cells. Transfection of Anx-I cDNA into drug-sensitive MCF-7 cells was carried out without further drug selection and showed 2- to 5-fold increase in resistance of transfected cells to adriamycin, melphalan, and etoposide. Conversely, transfection of reverse Anx-I cDNA into SKOV-3 cells decreased the expression of Anx-I without affecting the expression of other members of the annexin family and showed a 3- to 8-fold increase in sensitivity to these drugs. Of interest was the correlation between the presence of Anx-I and MDR in MDA-MB-231 cells when compared to MCF-7 cells. MDA-MB-231 cells show 3- to 20-fold increase in resistance to adriamycin, melphalan, and etoposide in the absence of detectable levels of P-glycoprotein (P-gp1), the multidrug resistance protein (MRP1) or the breast cancer resistance protein (BCRP). Taken together, these results provide the first direct evidence for the role of Anx-I in MDR of tumor cells.     

Infection of H69AR cells with retroviral particles harboring interfering RNAi significantly reduced the multidrug resistance of these small cell lung cancer cells

Incubation of the drug-sensitive H69, a small cell lung cancer cell line, with increased concentrations of adriamycin yielded multidrug resistant (MDR) H69AR cells that over-express multidrug resistance-associated protein (MRP1). MRP1 co-transports its substrate with glutathione (GSH), leading to lower intracellular GSH. In this report we tested whether depleting intracellular GSH in MRP1-expressing cells could hyper-sensitize them to anticancer drugs or not. We have found that the GSH contents in MRP1-expressing cells are significantly lower than their corresponding control cells. The treatment with MRP1 substrate verapamil or the GSH synthetase inhibitor buthionine sulfoxi-mine significantly reduced the intracellular GSH contents in MRP1-expressing cells. Interestingly, depleting intracellular GSH contents can hyper-sensitize the MRP1-cDNA transfected BHK cells to daunomycin, but not the adriamycin-selected H69AR cells. Further analyses indicated that anti-apoptotic factor Bcl2 might be a factor responsible for the fact that depleting intracellular GSH could not hyper-sensitize H69AR cells to daunomycin. We hypothesized that knocking down the expression of Bcl2 could hyper-sensitize H69AR cells to daunomycin. Interestingly, infection of H69AR cells with retroviral particles harboring Bcl2 interfering RNAi not only reduced the expression of Bcl2, but also many factors that contribute to MDR, such as Bcl-xl, MRP1 and ABCC3, etc., leading to the MDR H69AR cells more sensitive to daunomycin than the parental H69 cell. Thus, although the mechanisms of the down-regulation of the genes contributing to MDR remain to be elucidated, retroviral particles harboring Bcl2 interfering RNAi could be used as an alternative way to sensitize the MDR cancer cells to anticancer drugs.     

Folate deprivation results in the loss of breast cancer resistance protein (BCRP/ABCG2) expression. A role for BCRP in cellular folate homeostasis

Breast cancer resistance protein (BCRP/ABCG2) is currently the only ABC transporter that exports mono- and polyglutamates of folates and methotrexate (MTX). Here we explored the relationship between cellular folate status and BCRP expression. Toward this end, MCF-7 breast cancer cells, with low BCRP and moderate multidrug resistance protein 1 (MRP1/ABCC1) levels, and their mitoxantrone (MR)-resistant MCF-7/MR subline, with BCRP overexpression and low MRP1 levels, were gradually deprived of folic acid from 2.3 microm to 3 nm resulting in the sublines MCF-7/LF and MCF-7/MR-LF. These cell lines expressed only residual BCRP mRNA and protein levels and retained a poor MRP2 (ABCC2) through MRP5 (ABCC5) expression. Furthermore, MCF-7/MR-LF cells also displayed 5-fold decreased MRP1 levels relative to MCF-7/MR cells. In contrast, BCRP overexpression was largely retained in MCF-7/MR cells grown in MR-free medium containing 2.3 microm folic acid. Loss of BCRP expression in MCF-7/LF and MCF-7/MR-LF cells resulted in the following: (a) a prominent decrease in the efflux of Hoechst 33342, a BCRP substrate; (b) an approximately 2-fold increase in MR accumulation as revealed by flow cytometry; this was accompanied by a 2.5- and approximately 84-fold increased MR sensitivity in these cell lines, respectively. Consistently, Ko143, a specific BCRP inhibitor, rendered MCF-7 and MCF-7/MR cells 2.1- and approximately 16.4-fold more sensitive to MR, respectively. Loss of BCRP expression also resulted in the following: (c) an identical MTX sensitivity in these cell lines thereby losing the approximately 28-fold MTX resistance of the MCF-7/MR cells; (d) an approximately 2-fold increase in the 4- and 24-h accumulation of [(3)H]folic acid. Furthermore, MCF-7/MR-LF cells displayed a significant increase in folylpoly-gamma-glutamate synthetase activity. Hence, consistent with the mono- and polyglutamate folate exporter function of BCRP, down-regulation of BCRP and increased folylpoly-gamma-glutamate synthetase activity appear to be crucial components of cellular adaptation to folate deficiency conditions. This is the first evidence for the possible role of BCRP in the maintenance of cellular folate homeostasis.     

99mTc-labeling of colchicine using [99mTc(CO)3(H2O)3]+ and [99mTc triple bond N]2+ core for the preparation of potential tumor-targeting agents

Multidrug resistance (MDR) mediated by over-expression of P-glycoprotein (Pgp) is one of the major causes of failure of chemotherapy in cancer treatment. Colchicine, a naturally occurring alkaloid, is a Pgp substrate and acts as an antimitotic agent by binding to microtubules. Hence, Colchicine and its analogues radiolabeled with 99mTc may have potential for visualization of MDR in tumors. Here we report 99mTc-labeling of colchicine derivatives using [99mTc(CO)3(H2O)3]+ and [99mTc triple bond N]2+ cores. Trimethylcolchicinic acid synthesized from colchicine was used as the precursor to prepare iminodiacetic acid and dithiocarbamate derivatives which were then radiolabeled with [99mTc(CO)3(H2O)3]+ and [99mTc triple bond N]2+ cores, respectively. Radiolabeling yield for both the complexes was > 98% as observed by HPLC and TLC patterns. In vitro studies in tumor cell lines showed significant uptake for 99mTc-carbonyl as well as for 99mTc-nitrido colchicine complexes. Biodistribution studies in Swiss mice bearing fibrosarcoma tumor showed 4.1 +/- 1.2% ID/g of uptake at 30 min pi for 99mTc(CO)3-complex as against 0.42 +/- 0.24% ID/g for the 99mTcN-complex. 99mTc(CO)3-colchicine complex exhibited better pharmacokinetics with lower liver accumulation as compared to the 99mTcN-complex. Thus, colchicine radiolabeled with [99mTc(CO)3(H2O)3]+ core is more promising with respect to in vivo distribution characteristics in tumor model.     

MDR1 P-glycoprotein reduces influx of substrates without affecting membrane potential

MDR1 (multidrug resistance) P-glycoprotein (Pgp; ABCB1) decreases intracellular concentrations of structurally diverse drugs. Although Pgp is generally thought to be an efflux transporter, the mechanism of action remains elusive. To determine whether Pgp confers drug resistance through changes in transmembrane potential (E(m)) or ion conductance, we studied electrical currents and drug transport in Pgp-negative MCF-7 cells and MCF-7/MDR1 stable transfectants that were established and maintained without chemotherapeutic drugs. Although E(m) and total membrane conductance did not differ between MCF-7 and MCF-7/MDR1 cells, Pgp reduced unidirectional influx and steady-state cellular content of Tc-Sestamibi, a substrate for MDR1 Pgp, without affecting unidirectional efflux of substrate from cells. Depolarization of membrane potentials with various concentrations of extracellular K(+) in the presence of valinomycin did not inhibit the ability of Pgp to reduce intracellular concentration of Tc-Sestamibi, strongly suggesting that the drug transport activity of MDR1 Pgp is independent of changes in E(m) or total ion conductance. Tetraphenyl borate, a lipophilic anion, enhanced unidirectional influx of Tc-Sestamibi to a greater extent in MCF-7/MDR1 cells than in control cells, suggesting that Pgp may, directly or indirectly, increase the positive dipole potential within the plasma membrane bilayer. Overall, these data demonstrate that changes in E(m) or macroscopic conductance are not coupled with function of Pgp in multidrug resistance. The dominant effect of MDR1 Pgp in this system is reduction of drug influx, possibly through an increase in intramembranous dipole potential.     

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 [³H]-vinblastine accumulation and efflux in MCF-7 and MCF-7MDR. THC significantly increased the accumulation and inhibited the efflux of [³H]-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 [¹²⁵I]-iodoarylazidoprazosin (IAAP) into P-gp in a concentration-dependent manner. The binding of [¹²⁵I]-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.     

长春新碱诱导建立人结肠癌多药耐受性小鼠模型及MDR1和MRP1基因表达

目的建立人结肠癌多药耐受性动物模型并初步探索其耐药机制。方法结合体内外诱导方法建立人结肠癌多药耐受性动物模型,利用VCR和CTX的肿瘤抑制实验评价其MDR特性;利用real-time PCR和West-ern blotting等方法分析其P-gp/MDR1和MRP1基因和蛋白的表达。结果肿瘤抑制实验结果显示,MDR和敏感型结肠癌模型的肿瘤生长速度差异不显著,MDR结肠癌动物模型对于VCR和CTX的耐药性均有较大程度的提高;表达分析结果显示,人结肠癌MDR动物模型的P-gp/MDR1表达水平有较大提高,而MRP1表达没有显著变化。结论人结肠癌多药耐受性动物模型具有较好的多药耐受性,其多药耐受性表型主要是由于P-gp/MDR1过量表达所导致。     

Di-2-pyridylketone 4,4-Dimethyl-3-thiosemicarbazone (Dp44mT) Overcomes Multidrug Resistance by a Novel Mechanism Involving the Hijacking of Lysosomal P-Glycoprotein (Pgp)

Multidrug resistance (MDR) is a major obstacle in cancer treatment. More than half of human cancers express multidrug-resistant P-glycoprotein (Pgp), which correlates with a poor prognosis. Intriguingly, through an unknown mechanism, some drugs have greater activity in drug-resistant tumor cells than their drug-sensitive counterparts. Herein, we investigate how the novel anti-tumor agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes MDR. Four different cell types were utilized to evaluate the effect of Pgp-potentiated lysosomal targeting of drugs to overcome MDR. To assess the mechanism of how Dp44mT overcomes drug resistance, cellular studies utilized Pgp inhibitors, Pgp silencing, lysosomotropic agents, proliferation assays, immunoblotting, a Pgp-ATPase activity assay, radiolabeled drug uptake/efflux, a rhodamine 123 retention assay, lysosomal membrane permeability assessment, and DCF (2′,7′-dichlorofluorescin) redox studies. Anti-tumor activity and selectivity of Dp44mT in Pgp-expressing, MDR cells versus drug-sensitive cells were studied using a BALB/c nu/nu xenograft mouse model. We demonstrate that Dp44mT is transported by the lysosomal Pgp drug pump, causing lysosomal targeting of Dp44mT and resulting in enhanced cytotoxicity in MDR cells. Lysosomal Pgp and pH were shown to be crucial for increasing Dp44mT-mediated lysosomal damage and subsequent cytotoxicity in drug-resistant cells, with Dp44mT being demonstrated to be a Pgp substrate. Indeed, Pgp-dependent lysosomal damage and cytotoxicity of Dp44mT were abrogated by Pgp inhibitors, Pgp silencing, or increasing lysosomal pH using lysosomotropic bases. In vivo, Dp44mT potently targeted chemotherapy-resistant human Pgp-expressing xenografted tumors relative to non-Pgp-expressing tumors in mice. This study highlights a novel Pgp hijacking strategy of the unique dipyridylthiosemicarbazone series of thiosemicarbazones that overcome MDR via utilization of lysosomal Pgp transport activity.     

Biophysical characterization of MDR breast cancer cell lines reveals the cytoplasm is critical in determining drug sensitivity

Dielectrophoresis (DEP) was used to examine a panel of MCF-7 cell lines comprising parental MCF-7 cells and MDR derivatives: MCF-7TaxR (paclitaxel-resistant, P-glycoprotein (P-gp) positive), MCF-7DoxR (doxorubicin-resistant MRP2 positive) plus MCF-7MDR1 (MDR1 transfected, P-gp positive). MCF-7DoxR and MCF-7MDR1 were broadly cross-resistant to natural product anticancer agents, whereas MCF-7TaxR cells were not, contrary to P-gp expression. Whilst DEP revealed modest membrane changes in MDR sub-lines, we saw significant changes in their cytoplasmic conductivity: MCF-7TaxR相似文献   

Drug concentration-dependent expression of multidrug resistance-associated protein and P-glycoprotein in the doxorubicin-resistant acute myelogenous leukemia sublines.

The multidrug resistance of cancer cells can be mediated by an overexpression of the human MDR1 and MRP genes, which encode the transmembrane efflux pumps, the 170 kDa P-glycoprotein (Pgp) and the 190 kDa multidrug resistance-associated protein (MRP), respectively. In this study, we investigate which protein is preferentially overexpressed in the function of doxorubicin concentrations in the acute myelogenous leukemia cell line (OCI/AML-2). Multidrug-resistant AML-2 sublines were isolated in doxorubicin concentrations of 20, 100, 250, and 500 ng/ml. MRP was at first expressed at low concentrations of less than 5 x IC50 (100 ng/ml) of doxorubicin followed by the overexpression of Pgp with concentrations of more than 12.5 x IC50 (250 ng/ml) of doxorubicin. In addition, it appeared that increased amounts of MRP and its mRNA in AML-2/DX20 and /DX100 decreased gradually in both AML-2/DX250 and /DX500 overexpressing Pgp. In conclusion, it is thought that the overexpression of MRP or Pgp is dependent upon drug concentrations. It could be implicated that the overexpression of MRP might be negatively related to that of Pgp.     

Cytoplasmic retraction of the amino terminus of human multidrug resistance protein 1

Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette (ABC) transport superfamily which also includes human multidrug resistance 1 (MDR1) gene product P-glycoprotein (Pgp). Overexpression of MRP1 or Pgp causes multidrug resistance in cancer cells. Different from Pgp, MRP1 contains an extra membrane-spanning domain (MSD1) with a putative extracellular amino terminus in addition to the core structure of two MSDs and two NBDs (nucleotide-binding domains). The structural and functional significance of the additional MSD1 in MRP1 remains elusive. In this study, we generated an IgG1 subclass monoclonal antibody, IU2H10, specific to the amino terminus of human MRP1 and mapped its epitope to 10 amino acids (S8ADGSDPLWD17). It can be used for Western blot, immunoprecipitation, and indirect immunofluorescence studies of human MRP1. However, surprisingly we found that IU2H10 cannot react with MRP1 unless cells are permeabilized. Furthermore, the IU2H10 epitope is exposed extracellularly when the carboxyl-terminal core domain of human MRP1 is deleted. Examination of the amino-terminal sequence of human MRP1 suggests that it consist of mainly coiled structures. These observations provide evidence for a model that is different from the prevailing extracellular location of the amino terminus of human MRP1. It is possible that part of the amino terminus of human MRP1, following exposure to the lumen of the endoplasmic reticulum, is retracted to the cytoplasm.     

Upregulation of the expression of endogenous Mdr1 P-glycoprotein enhances lipid translocation in MDCK cells transfected with human MRP2

Various ABC transporters can translocate lipid molecules from the cytoplasmic into the exoplasmic leaflet of the plasma membrane bilayer. Two of these, MDR1 P-glycoprotein (Pgp) and MRP1, are multidrug transporters responsible for the resistance of various cancers against chemotherapy. We wanted to study whether MRP2, an ABC transporter of the bile canalicular membrane with a substrate specificity very similar to that of MRP1, is capable of translocating lipids. The translocation of short-chain lipids across the apical membrane of MDCK cells transfected with MRP2 was significantly higher than that in untransfected controls. However, the characteristics of the lipid translocation were similar to substrate transport by MDR1 and not MRP2: transport was strongly inhibited by classic MDR1 Pgp inhibitors, was independent of cellular glutathione, and was insensitive to a drug known to inhibit MRP2 activity. When tested by immunoblot, the MRP2-transfected cells expressed high levels of MRP2 but also of endogenous Mdr1. The expression of Mdr1 was unstable during maintenance of the cell line and correlated with the rate of lipid translocation across the apical membrane. We conclude that the observed increase in lipid transport in the MDCK cells transfected with MRP2 is the consequence of the upregulation of the expression of endogenous Mdr1 and that careful characterization of endogenous Mdr1 expression is needed in studies aimed to identify substrates of plasma membrane transporters.     

Non-canonical functions of the cellular transporter P-glycoprotein

P-glycoprotein/ABCB1 (Pgp) is a well known protein of cell defense system. It is localized in cell membrane and pumps different drugs out of various cells using ATP energy. Its overexpression is associated with the development of multidrug resistance (MDR) in cancer cells. The data showing that Pgp also has other functions appeared recently, and this review surveys these data. In particular, (1) Pgp can protect cells from apoptosis; it suppresses the expression of endogenous protein TRAIL and decreases the activity of caspases 8 and 3; (2) Pgp is able to act as an outwardly directed flippase; (3) Pgp participates in a proper development of the innate immune response to intracellular pathogens and in the development of inflammation; (4) functionally active Pgp can be transferred from drug-resistant to drug-sensitive cells by microvesicles (MV). This is a new way of the Pgp-mediated MDR emergence in populations of tumor cells. Thus, Pgp functions as a regulator of some cellular processes. Molecular mechanisms of the Pgp influence on tumor cell viability are related not only with the drug efflux but also with some other functions.     

Increasing drug resistance in human lung cancer cells by mutant-type p53 gene mediated by retrovirus

Human mutant-type (mt) p53 cDNA was synthesized and cloned from human lung cancer cell line GL containing mt-p53 gene by using polymerase chain reaction (PCR). It was confirmed that the mt-p53 cDNA con-tained the complete coding sequence of p53 gene but mutated at codon 245 (G→T) and resulted in glycine to cysteine by sequencing analysis. The retroviral vector pD53M of the mt-p53 was constructed and introduced into the drug-sen-sitive human lung cancer cells GAO in which p53 gene did not mutate. The transfected GAO cells strongly expressed mutant-type p53 protein by immunohistochemistry, showing that pD53M vector could steadily express in GAO cells. The drug resistance to several anticancer agents of GAO cells infected by pD53M increased in varying degrees, with the highest increase of 4-fold, in vitro and in vivo. By quantitative PCR and flow cytometry (FCM) analyses, the expression of MDR1 gene and the activity of P-glycoprotein (Pgp) did not increase, the expression of MRP gene and the activity of m     

Transport of fluorescein in MDCKII-MRP1 transfected cells and mrp1-knockout mice.

The multidrug resistant-associated protein 1 (MRP1) is a membrane-bound transport protein that is involved in the efflux of organic anions and has been implicated in multidrug resistance in cancer. MRP1 has also been reported to be ubiquitously expressed in normal tissues, including the brain. The presence of functional organic anion transporters in the blood-brain and blood-CSF barriers that influence the distribution of various compounds to the brain has long been known. The purpose of this study was to examine the role of MRP1 in the brain distribution of a model organic anion, fluorescein. The substrate specificity of MRP1 for fluorescein was initially determined by examining the accumulation of fluorescein in MDCKII MRP1-transfected cells. The distribution of fluorescein in the brain was then examined in wild-type and mrp1 gene knockout mice. The results show that in MDCKII MRP1-transfected cells, the accumulation of fluorescein was significantly lower (about 40% lower) than that in wild-type MDCKII cells. MRP1 inhibitors such as probenecid, MK-571, and LY402913 enhanced fluorescein accumulation in MDCKII MRP1-transfected cells to a greater extent than in wild-type MDCKII cells. In an in vivo study, after intravenous injection of fluorescein, the fluorescein brain-to-plasma concentration ratio in mrp1 knockout mice was not significantly different than that in wild-type mice. However, when probenecid was co-administered with fluorescein in wild-type mice, the fluorescein brain-to-plasma ratio was significantly increased (1.5-fold). These findings suggest that fluorescein is a substrate for MRP1. Furthermore, the in vivo study also suggests that MRP1 has a limited role in the transport and distribution of fluorescein in the brain. Therefore, other organic anion transport proteins, including the various isoforms of the MRP family, may be responsible for the accumulation and transport of organic anions in the brain.     

Oligonucleotides blocking glucosylceramide synthase expression selectively reverse drug resistance in cancer cells

High glucosylceramide synthase (GCS) activity is one factor contributing to multidrug resistance (MDR) in breast cancer. Enforced GCS overexpression has been shown to disrupt ceramide-induced apoptosis and to confer resistance to doxorubicin. To examine whether GCS is a target for cancer therapy, we have designed and tested the effects of antisense oligodeoxyribonucleotides (ODNs) to GCS on gene expression and chemosensitivity in multidrug-resistant cancer cells. Here, we demonstrate that antisense GCS (asGCS) ODN-7 blocked cellular GCS expression and selectively increased the cytotoxicity of anticancer agents. Pretreatment with asGCS ODN-7 increased doxorubicin sensitivity by 17-fold in MCF-7-AdrR (doxorubicin-resistant) breast cancer cells and by 10-fold in A2780-AD (doxorubicin-resistant) ovarian cancer cells. In MCF-7 drug-sensitive breast cancer cells, asGCS ODN-7 only increased doxorubicin sensitivity by 3-fold, and it did not influence doxorubicin cytotoxicity in normal human mammary epithelial cells. asGCS ODN-7 was shown to be more efficient in reversing drug resistance than either the GCS chemical inhibitor d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol or the P-glycoprotein blocking agents verapamil and cyclosporin A. Experiments defining drug transport and lipid metabolism parameters showed that asGCS ODN-7 overcomes drug resistance mainly by enhancing drug uptake and ceramide-induced apoptosis. This study demonstrates that a 20-mer asGCS oligonucleotide effectively reverses MDR in human cancer cells.     

Tumor necrosis factor alpha induces stronger cytotoxicity in ABCG2-overexpressing resistant breast cancer cells compared with their drug-sensitive parental line

Tumor necrosis factor alpha (TNF-α) has been reported to modulate the multidrug resistance (MDR) phenotype in vitro and in vivo. Multidrug-resistant cells overexpressing the ABCB1 transporter are more susceptible to inhibition of proliferation and induction of apoptosis by TNF-α than their drug-sensitive counterparts. This study was aimed to investigate TNF-α modulatory and antiproliferative effects on drug-resistant cells overexpressing ABCG2. The effects of TNF-α on viability and proliferation rate of MCF-7 breast cancer cells and their ABCG2-overexpressing sublines MCF-7/mitoxantrone (MX) cells were studied using dye exclusion assay, dimethylthiazolyl-2,5-diphenyl tetrazolium bromide technique, and flow cytometric analysis of cell cycle. TNF-α influence on MX accumulation was investigated by flow cytometry. ABCG2-overexpressing cells were more susceptible to antiproliferative and cytotoxic effects of TNF-α than their parental cells. TNF-α increased accumulation of MX in both parental and resistant cells. Higher sensitivity of MDR cells to TNF-α cytotoxicity would help in characterization of its complex modulatory effects on cancer cells and benefit us in designing new approaches to overcome MDR.