抗肿瘤细胞多药抗性基因MDR1和MRP1双靶位自剪切核酶的合成及其在体外活性研究

多药耐药基因MDR1和 (或 )MRP1过度表达是引起肿瘤细胞对化疗药物产生多重耐药性的重要原因 .在以往研究抗MDR1基因的核酶 (196MDR1 Rz)的基础上 ,合成了针对MRP1基因 2 10和 730编码子的核酶 (2 10MRP1 Rz ,730MRP1 Rz) ,同时利用RNA二级结构分析程序mfold 3 0设计合成了一种双靶位自剪切核酶体系 (Coat) .将 196MDR1 Rz和 2 10MRP1 Rz基因同时载入到该体系中 ,建立了抗MDR1 MRP1双靶位核酶 .在无细胞体系中对上述核酶进行的生物活性实验表明 ,2 10MRP1 Rz与 730MRP1 Rz相比能够更有效地切割底物 ;载入Coat的抗MDR1和MRP1核酶均能得以有效释放 ,同时切割各自的靶RNA序列 ,切割效率与单靶位核酶一致 ;串联核酶的先后顺序不影响切割活性     

Role of MDR1 and MRP1 in trophoblast cells,elucidated using retroviral gene transfer

Natural differences in expression and retroviral transduction techniques were used to test the hypothesis that MDR1 P-glycoprotein (P-gp) and MRP1 (multidrug resistance-related protein) contribute to xenobiotic handling by placental trophoblast. RT-PCR and Western blotting in placenta, primary cytotrophoblast cell cultures, and BeWo, JAr, and JEG choriocarcinoma cell lines showed that MRP1 was ubiquitously expressed, whereas MDR1 was absent or minimally expressed in BeWo and JEG cell lines. In syncytiotrophoblast, P-gp was localized predominantly to the microvillous, maternal facing plasma membrane, and MRP1 to the basal, fetal facing plasma membrane. Functional studies showed that cyclosporin A-sensitive accumulation of [³H]vinblastine by cells containing both transport proteins was significantly different from those expressing predominantly MRP1. Retroviral gene transfer of MDR1 to BeWo cells confirmed that this difference was due to the relative expression of MDR1. Therefore, both P-gp and MRP1 contribute to xenobiotic handling by the trophoblast. Localization of P-gp to the microvillous membrane suggests an essential role in preventing xenobiotic accumulation by the syncytiotrophoblast and, therefore, in protecting the fetus. placenta; multidrug resistance; xenobiotic     

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.     

Expression and localization of surfactant proteins in human nasal epithelium

Surfactant proteins (SPs), designated SP-A, SP-B, SP-C, and SP-D, play an important role in surfactant metabolism and host defense mechanisms in the lung. This study investigates expression of the different SP types in human nasal mucosa and cultured normal human nasal epithelial (NHNE) cells and whether the expression of SP mRNA is influenced by the degree of mucociliary differentiation. RT-PCR was performed with mRNA from cultured NHNE cells and nasal mucosa. Immunohistochemical staining for SPs was performed on nasal mucosa specimens. Western blot analysis was performed on cell lysates from cultured NHNE cells. SP-A2, SP-B, and SP-D mRNAs were expressed in normal NHNE cells and human nasal mucosa. SPs were localized in ciliated cells of the surface epithelium and serous acini of the submucosal glands. SP-A, SP-B, and SP-D proteins were expressed in cultured NHNE cells. The degree of mucociliary differentiation influenced expression of the SP gene. We demonstrate that SP-A, SP-B, and SP-D are expressed in human nasal mucosa and cultured NHNE cells. Further study of the functional role of SPs in the upper airway is required.     

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.     

Spatiotemporal coupling of cAMP transporter to CFTR chloride channel function in the gut epithelia

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized at apical cell membranes and exists in macromolecular complexes with a variety of signaling and transporter molecules. Here, we report that the multidrug resistance protein 4 (MRP4), a cAMP transporter, functionally and physically associates with CFTR. Adenosine-stimulated CFTR-mediated chloride currents are potentiated by MRP4 inhibition, and this potentiation is directly coupled to attenuated cAMP efflux through the apical cAMP transporter. CFTR single-channel recordings and FRET-based intracellular cAMP dynamics suggest that a compartmentalized coupling of cAMP transporter and CFTR occurs via the PDZ scaffolding protein, PDZK1, forming a macromolecular complex at apical surfaces of gut epithelia. Disrupting this complex abrogates the functional coupling of cAMP transporter activity to CFTR function. Mrp4 knockout mice are more prone to CFTR-mediated secretory diarrhea. Our findings have important implications for disorders such as inflammatory bowel disease and secretory diarrhea.     

Applicability of different antibodies for the immunohistochemical localization of CFTR in respiratory and intestinal tissues of human and murine origin.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, which has a major role as a chloride (Cl(-)) channel. Although perhaps all functions of CFTR are still not fully characterized, localization studies are necessary to understand the consequences of the more than 1000 mutations thus far identified. Our aim was to determine the histological localization of CFTR on respiratory and colon epithelia of human and murine origin with a panel of several antibodies produced against different CFTR epitopes, using an indirect immunofluorescence method. Our results on human tissues confirm the apical localization of CFTR in ciliated cells of the respiratory mucosa and show that in colon tissue CFTR is observed in both apical and basolateral membranes of epithelial cells from colon crypts. However, poor tissue preservation of colon biopsies after immunohistochemistry (IHC) raises doubts about the latter localization. Contrary to human, mouse colon epithelium (not biopsed) presents good tissue preservation and evidences many cylindrical surface cells with high apical expression of CFTR. For the antibodies' sensitivity, we demonstrate that MATG1061, 24-1, M3A7, and MPCT-1 give good results, allowing the histological localization of CFTR protein of both human and murine origin.     

Fibrinolytic components in nasal mucosa and nasal secretion

 We evaluated a possible role for fibrinolytic components in nasal secretion by tissue localization with immunohistochemical techniques and by measuring their antigen concentrations in nasal discharge by means of ELISA and fibrin autography. Nasal mucosa was obtained surgically from the inferior turbinate. Urokinase-type plasminogen activator (u-PA) specific staining was observed in pseudostratified ciliated epithelium and was predominant in mucous cells of the seromucinous gland, while serous cells were almost devoid of stain. The pattern of staining of plasminogen activator inhibitor-2 was similar to that of u-PA. In contrast, plasminogen activator inhibitor-1(PAI-1) immunoreactive material was localized exclusively in serous cells of seromucinous glands. Positive staining for tissue-type plasminogen activator (t-PA) was observed in endothelial cells and basal cells, which differentiate into either ciliated or goblet cells. Nasal secretions were partially fractionated by immunospecific antibody-immobilized Sepharose. Subsequent fibrin autography patterns indicated the presence of u-PA, PAI-1, and t-PA. After methacholine provocation, the level of t-PA increased transiently but decreased rapidly with subsequent challenges. These differential stainings of fibrinolytic components and the existence of PAs and PAI-1 in the nasal discharge suggest that the fibrinolytic system may play a role in the movement and fluidity of nasal secretion. Accepted: 25 May 1998     

Molecular docking studies and in vitro screening of new dihydropyridine derivatives as human MRP1 inhibitors

The overexpression of multidrug resistance protein 1 (MRP1) by tumor cells results in multidrug resistance (MDR) to structurally unrelated anticancer drugs. Circumvention of MDR by combination of chemosensitizers with antitumor compounds is a new field of investigation in cancer chemotherapy. Much effort has been put-in recently to identify the modulators/inhibitors of MRP1 to overcome the MDR. 1,4-Dihydropyridine (DHP) derivatives are indicated to be a new class of MRP1 inhibitors in cancer treatment. Molecular docking studies were carried out on 48 newly synthesized DHP derivatives with the crystal structure of MRP1 to gain some structural insights on the binding mode and possible interactions with the active site of MRP1 (NBD1). The 10 top-ranked molecules were selectively evaluated, experimentally for their MRP1 inhibitory effect using the insect cell membrane MRP1 ATPase assay. The inhibitory capacity (IC(50) concentrations) of the test compounds was compared with the reported IC(50)- or the K(i)-concentrations for benzbromarone, a standard MRP1 inhibitor. Amongst the compounds tested, compounds IA(1) and IIA(5) were found to exhibit a potent MRP1 inhibitory action with IC(50) values of 20±4 and 14±2 μM (mean±SD), respectively as compared to benzbromarone (IC(50)=4 μM). The compound IIA(5), in particular was found to be more potent than IA(1) in accordance with the docking results. These new DHP derivatives possess promising characteristics for their development as MDR reversal agents.     

Sulforaphane and erucin increase MRP1 and MRP2 in human carcinoma cell lines

Multidrug resistance (MDR) transporters have been termed the Phase III detoxification system because they not only export endogenous metabolites but provide protection from xenobiotic insult by actively secreting foreign compounds and their metabolites from tissues. However, MDR overexpression in tumors can lead to drug resistance, a major obstacle in the treatment of many cancers, including lung cancer. Isothiocyanates from cruciferous vegetables, such as sulforaphane (SF) and erucin (ER), are known to enhance the expression of Phase II detoxification enzymes. Here we evaluated the ability of SF and ER to modulate MDR mRNA and protein expressions, as well as transporter activity. The expression of P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1) and multidrug resistance protein 2 (MRP2) in liver (HepG2), colon (Caco-2) and lung (A549) cancer cells treated with ER or SF was analyzed by Western blotting. Neither SF nor ER affected P-gp expression in any of the cell lines tested. Both SF and ER increased the protein levels of MRP1 and MRP2 in HepG2 cells and of MRP2 in Caco-2 cells in a dose-dependent manner. In A549 lung cancer cells, SF increased MRP1 and MRP2 mRNA and protein levels; ER caused a similar yet smaller increase in MRP1 and MRP2 mRNA. In addition, SF and ER increased MRP1-dependent efflux of 5-carboxyfluorescein diacetate in A549 cells, although again the effect of SF was substantially greater than that of ER. The implication of these findings is that dietary components that modulate detoxification systems should be studied carefully before being recommended for use during chemotherapy, as these compounds may have additional influences on the disposition of chemotherapeutic drugs.     

Differential expression of Alpha,Mu, and Pi classes of glutathione S-transferases in chemosensory mucosae of rats during development

The expression of three classes of glutathione S-transferases (GSTs), Alpha, Mu, and Pi was investigated in the nasal mucosae of rats during development using immunohistochemical methods. GST Alpha and Mu were first detected in the supranuclear region of sustentacular cells on embryonic days 16. The Bowman's glands expressed differential patterns of immunoreactivity during development, beginning at postnatal day (P) 2 and P6 for Alpha and Mu classes, respectively and being greatest at P11 for both. The acinar cells of vomeronasal glands in the vomeronasal organ expressed Alpha and Mu classes of GSTs from P11 onwards. In the septal organ of Masera, the supranuclear region of sustentacular cells expressed GSTs from P11 with little or no variation during development. In the respiratory mucosa, Alpha and Mu classes of GSTs were detected at the brush borders of ciliated cells and in the acinar cells of posterior septal glands, but not in anterior septal or respiratory glands located on the turbinates. Compared to olfactory mucosa, the changes in immunoreactivity for GSTs were less pronounced in the respiratory mucosa during development. Specific GST Pi immunoreactivity was not detected in the nasal mucosae at any stage of development studied. The occurrence of GSTs in the nasal mucosa, including olfactory, vomeronasal, septal, and respiratory epithelia, suggests that the GSTs are actively involved in the biotransformation of xenobiotics including odorants and pheromones, and may also participate in perireceptor processes such as odorant clearance. In addition, we have developed a working model describing the cellular localization of certain phase I (e.g., cytochrome P-450s) and phase II (e.g., GSTs, -glutamyl transpeptidase) biotransformation enzymes in the olfactory mucosa and their proposed roles in xenobiotic metabolism.     

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.     

Human MDR1 and MRP1 Recognize Berberine as Their Transport Substrate

To examine whether human ATP-binding cassette (ABC) transporters play a role in the detoxification of plant alkaloid berberine, we investigated berberine transport using multidrug resistance protein1 (MDR1) and multidrug resistance-associated protein1 (MRP1). Cells expressing MDR1 or MRP1 accumulated less berberine. Berberine accumulation depended on the cellular ATP level, and was reversed by typical inhibitors of MDR1, suggesting that human MDR1 and MRP1 directly efflux berberine as their substrate.     

Human MDR1 and MRP1 recognize berberine as their transport substrate

To examine whether human ATP-binding cassette (ABC) transporters play a role in the detoxification of plant alkaloid berberine, we investigated berberine transport using multidrug resistance protein1 (MDR1) and multidrug resistance-associated protein1 (MRP1). Cells expressing MDR1 or MRP1 accumulated less berberine. Berberine accumulation depended on the cellular ATP level, and was reversed by typical inhibitors of MDR1, suggesting that human MDR1 and MRP1 directly efflux berberine as their substrate.     

Iodination of verapamil for a stronger induction of death,through GSH efflux,of cancer cells overexpressing MRP1

The multidrug resistance protein 1 (MRP1), involved in multidrug resistance (MDR) of cancer cells, was found to be modulated by verapamil, through stimulation of GSH transport, leading to apoptosis of MRP1-overexpressing cells. In this study, various iodinated derivatives of verapamil were synthesized, including iodination on the B ring, known to be involved in verapamil cardiotoxicity, and assayed for the stimulation of GSH efflux by MRP1. The iodination, for nearly all compounds, led to a higher stimulation of GSH efflux. However, determination of concomitant cytotoxicity is also important for selecting the best compound, which was found to be 10-fold more potent than verapamil. This will then allow us to design original anti-cancer compounds which could specifically kill the resistant cancer cells.     

Functional comparison between YCF1 and MRP1 expressed in Sf21 insect cells

YCF1 is a yeast vacuole membrane transporter involved in resistance to Cd(2+) and to exogenous glutathione S-conjugate precursors. MRP1 contributes to multidrug resistance (MDR) in tumor cells. MRP1 and YCF1 have extensive amino acid sequence homology (63% amino acid similarity). We expressed MRP1 or YCF1 in insect cell membranes and compared their functions to know more about their structure-function relationships. YCF1 and MRP1 with His epitopes were expressed in Sf21 insect cells; both of them in the plasma membrane. The ATP-dependent transport of [(3)H]LTC(4) in Sf/YCF1-His vesicles was osmotically sensitive and showed saturable kinetics with an apparent K(m) of 758 nM for LTC(4) and 94 microM for ATP which were similar to those in yeast cells. The K(m) of YCF1 for LTC(4) (758 nM) was sevenfold higher than that of MRP1 (108 nM). MK-571 and ONO-1078, reversing agents for MRP1-mediated MDR, considerably inhibited the transport of LTC(4) by both YCF1 and MRP1. However, PAK-104P, a pyridine analog that reverses MDR associated with P-gp and MRP1, inhibited the transporting activity of MRP1 stronger than that of YCF1. KE1, another MDR reversing agent, moderately inhibited the transport of LTC(4) by MRP1 but not that of YCF1. In conclusion, we successfully expressed yeast YCF1 in Sf21 insect cells and found that the localization of the protein was different from that in yeast. The function of YCF1 in Sf21 insect cells was similar but not identical to that of MRP1.     

Morphology of the nasal fossae and associated structures of the hamster (Mesocricetus auratus)

The hamster nasal cavity consists of vestibular, non-olfactory and olfactory portions. Much of the non-olfactory nasal cavity surface is lined by cuboidal, stratified cuboidal, and low columnar epithelia, devoid of cilia. Goblet cells and ciliated respiratory epithelium are present over only a small portion of the nasal cavity surface. The largest glandular masses in the hamster nose are the maxillary recess glands, the vomeronasal glands and the lateral nasal gland 1; these three glands contain neutral mucopolysaccharides (PAS-positive). Other nasal glands contain both acidic and neutral mucopolysaccharides; the staining reaction for acidic mucopolysaccharide is stronger in goblet cells and olfactory glands than in the other nasal glands. The ducts which open into the nasal vestibule are the excretory ducts of compound tubuloacinar serous glands. The one major PAS-positive gland whose duct opens into the nasal vestibule is the lateral nasal gland 1. The ducts of the compound tubuloacinar vomeronasal glands open into the lumen of the vomeronasal organ, which is connected to the ventral nasal meatus by means of the vomeronasal duct. The ducts of the branched tubuloacinar maxillary recess glands open into the maxillary recess. Few ducts open into the caudal half of the nasal cavity.     

Histological and carbohydrate histochemical studies of the nasal mucosa of sheep in Saudi Arabia with special reference to its glandular tissue

The histology and carbohydrate histochemistry of the nasal mucosa with attention to glandular tissue had been studied in 7 heads of sheep. Tissues were taken from vestibular region, septum at level of the alar fold, rostral portion of nasal conchae, caudal portion of nasal conchae, middle portion of septum and ethmoidal conchae region. Stratified squamous nonkeratinized epithelium was observed covering the vestibular region. The propria-submucosa of the nasal vestibule was richly permeated with glands having affinity for PAS and non-alcianophilic. The post-vestibular portion of the nasal cavity was lined by transitional epithelium and caudal to it, stratified columnar nonciliated epithelium was noticed. The respiratory epithelium covered the caudal half of the nasal conchae and the major portion of the septum as well as the recesses of the ethmoidal conchae. The glands associated with the respiratory mucosa were thick, coiled and tubular, containing both nonalcianophilic PAS positive and alcianophilic PAS positive cells. The olfactory mucosa covered the ethmoidal conchae and showed predominant serous glands. The results were discussed with that given for other mammals and in regard to the respiratory functions of the nasal mucosa.