Interaction of phenothiazines, stilbenes and flavonoids with multidrug resistance-associated transporters, P-glycoprotein and MRP1

Multidrug resistance (MDR) of cancer cells poses a serious obstacle to successful chemotherapy. The overexpression of multispecific ATP-binding cassette transporters appears to be the main mechanism of MDR. A search for MDR-reversing agents able to sensitize resistant cells to chemotherapy is ongoing in the hope of their possible clinical use. Studies of MDR modulators, although they have not produced clinically beneficial effects yet, may greatly enrich our knowledge about MDR transporters, their specificity and mechanism of action, especially substrate and/or inhibitor recognition. In the present review, interactions of three groups of modulators: phenothiazines, flavonoids and stilbenes with both P-glycoprotein and MRP1 are discussed. Each group of compounds is likely to interact with the MDR transporters by a different mechanism. Phenothiazines probably interact with drug binding sites, but they also could indirectly affect the transporter's activity by perturbing lipid bilayers. Flavonoids mainly interact with ABC proteins within their nucleotide-binding domains, though the more hydrophobic flavonoids may bind to regions within transmembrane domains. The possible mechanism of MDR reversal by stilbenes may result from their direct interaction with the transporter (possibly within substrate recognition sites) but some indirect effects such as stilbene-induced changes in gene expression pattern and in apoptotic pathways should also be considered. Literature data as well as some of our recent results are discussed. Special emphasis is put on cases when the interactions of a given compound with both P-glycoprotein and MRP1 have been studied simultaneously.     

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.     

ABC transporters in lipid transport

Since it was found that the P-glycoproteins encoded by the MDR3 (MDR2) gene in humans and the Mdr2 gene in mice are primarily phosphatidylcholine translocators, there has been increasing interest in the possibility that other ATP binding cassette (ABC) transporters are involved in lipid transport. The evidence reviewed here shows that the MDR1 P-glycoprotein and the multidrug resistance (-associated) transporter 1 (MRP1) are able to transport lipid analogues, but probably not major natural membrane lipids. Both transporters can transport a wide range of hydrophobic drugs and may see lipid analogues as just another drug. The MDR3 gene probably arose in evolution from a drug-transporting P-glycoprotein gene. Recent work has shown that the phosphatidylcholine translocator has retained significant drug transport activity and that this transport is inhibited by inhibitors of drug-transporting P-glycoproteins. Whether the phosphatidylcholine translocator also functions as a transporter of some drugs in vivo remains to be seen. Three other ABC transporters were recently shown to be involved in lipid transport: ABCR, also called Rim protein, was shown to be defective in Stargardt's macular dystrophy; this protein probably transports a complex of retinaldehyde and phosphatidylethanolamine in the retina of the eye. ABC1 was shown to be essential for the exit of cholesterol from cells and is probably a cholesterol transporter. A third example, the ABC transporter involved in the import of long-chain fatty acids into peroxisomes, is discussed in the chapter by Hettema and Tabak in this volume.     

Structural requirements of flavonoids for inhibition of antigen-Induced degranulation,TNF-alpha and IL-4 production from RBL-2H3 cells

To clarify the structure-activity relationships of flavonoids for antiallergic activity, the inhibitory effects of various flavonoids on the release of beta-hexosaminidase, as a marker of degranulation of RBL-2H3 cells, were examined. Among them, luteolin (IC(50)=3.0 microM), diosmetin (2.1 microM), and fisetin (3.0 microM) were found to show potent inhibitory activity, and the results suggested the following structural requirements of flavonoids: (1) the 2-3 double bond of flavones and flavonols is essential for the activity; (2) the 3- or 7-glycoside moiety reduced the activity; (3) as the hydroxyl groups at the 3'-, 4'-, 5-, 6-, and 7-positions increased in number, the inhibitory activities become stronger; (4) the flavonols with a pyrogallol type moiety (the 3',4',5'-trihydroxyl groups) at the B ring exhibited less activity than those with a phenol type moiety (the 4'-hydroxyl group) or catechol type moiety (the 3',4'-dihydroxyl groups) at the B ring; (5) the activities of flavones were stronger than those of flavonols; and (6) methylation of flavonols at the 3-position reduced the activity. However, (7) several flavones and flavonols with the 4'- and/or 7-methoxyl groups did not obey rules (3), (4), and (5). In addition, several flavonoids, that is apigenin, luteolin, diosmetin, fisetin, and quercetin, inhibited the antigen-IgE-mediated TNF-alpha and IL-4 production from RBL-2H3 cells, both of which participate in the late phase of type I allergic reactions.     

Structural requirements of flavonoids for inhibition of protein glycation and radical scavenging activities

To clarify the structural requirements of flavonoids for formation of advanced glycation end-products (AGEs), various flavonoids were examined. The results suggested the following structural requirements of flavonoids for the inhibition of AGEs formation: (1) as the hydroxyl groups at the 3′-, 4′-, 5-, and 7-positions increased in number, the inhibitory activities became stronger; (2) the activities of flavones were stronger than those of corresponding flavonols, flavanones, and isoflavones; (3) methylation or glucosylation of the 4′-hydroxyl group of flavones, flavonols, and flavanones reduced activity; (4) methylation or glycosylation of the 3-hydroxyl group of flavonols tended to increase activity; (5) glycosylation of the 7-hydroxyl group of flavones and isoflavones reduced activity. In addition, various flavonoids with strong AGEs formation inhibitory activity tended to exhibit strong scavenging activity for 1,1-diphenyl-2-picrylhydrazyl and superoxide anion radicals, with several exceptions.     

Flavonoid systematics of the genus Perideridia (Umbelliferae)

A survey of flavonoids in sixteen of the seventeen taxa in the genusPerideridia (Umbelliferae) showed the presence of thirteen glycosides of the flavonols kaempferol, quercetin, and isorhamnetin, and seven glycosides of the flavones apigenin, luteolin and chrysoeriol. An anthocyanin and four other flavonoids also occur, but remain unidentified dueto their low concentration. Several species characteristically produce speciesspecific compounds. The majority of species, however, produce flavonoids common to one or more taxa, but each taxon can be distinguished by its own specific complement of these flavonoids. Based on classes of flavonoids the genus can be divided into three groups: (1) those species which produce only flavonols; (2) those which produce mainly flavonols and a few flavones; and (3) those which produce predominantly flavones with flavonols absent or present only in trace amounts. Geographically, the flavonol-producing species are centered in California, extending northeastward to Idaho and eastward into Arizona. The flavonol/flavone producers are concentrated more towards the Pacific Northwest and eastward through the Rocky Mountains to the midwestern United States.     

Structural requirements of flavonoids for nitric oxide production inhibitory activity and mechanism of action

To clarify the structure-activity relationships of flavonoids for nitric oxide (NO) production inhibitory activity, we examined the inhibitory effects of 73 flavonoids on NO production in lipopolysaccharide-activated mouse peritoneal macrophages. Among those flavonoids, apigenin (IC(50)=7.7 microM), diosmetin (8.9 microM), and tetra-O-methylluteolin (2.4 microM), and hexa-O-methylmyricetin (7.4 microM) were found to show potent inhibitory activity, and the results suggested the following structural requirements of flavonoids: (1) the activities of flavones were stronger than those of corresponding flavonols; (2) the glycoside moiety reduced the activity; (3) the activities of flavones were stronger than those of corresponding flavanones; (4) the flavones and flavonols having the 4'-hydroxyl group showed stronger activities than those lacking the hydroxyl group at the B ring and having the 3',4'-dihydroxyl group; (5) the flavonols having the 3',4'-dihydroxyl group (catechol type) showed stronger activities than those having the 3',4',5'-trihydroxyl group (pyrogallol type); (6) the 5-hydroxyl group tended to enhance the activity; (7) methylation of the 3-, 5-, or 4'-hydroxyl group enhanced the activity; (8) the activities of isoflavones were weaker than those of corresponding flavones; (9) methylation of the 3-hydroxyl group reduced the cytotoxicity. In addition, potent NO production inhibitors were found to inhibit induction of inducible nitric oxide synthase (iNOS) without iNOS enzymatic inhibitory activity.     

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.     

Regulation of Hepatic Drug Transporter Activity and Expression by Organochlorine Pesticides

Organochlorine (OC) pesticides constitute a major class of persistent and toxic organic pollutants, known to modulate drug‐detoxifying enzymes. In the present study, OCs were demonstrated to also alter the activity and expression of human hepatic drug transporters. Activity of the sinusoidal influx transporter OCT1 (organic cation transporter 1) was thus inhibited by endosulfan, chlordane, heptachlor, lindane, and dieldrine, but not by dichlorodiphenyltrichloroethane isomers, whereas those of the canalicular efflux pumps MRP2 (multidrug resistance‐associated protein 2) and BCRP (breast cancer resistance protein) were blocked by endosulfan, chlordane, heptachlor, and chlordecone; this latter OC additionally inhibited the multidrug resistance gene 1 (MDR1)/P‐glycoprotein (P‐gp) activity. OCs, except endosulfan, were next found to induce MDR1/P‐gp and MRP2 mRNA expressions in hepatoma HepaRG cells; some of them also upregulated BCRP. By contrast, expression of sinusoidal transporters was not impaired (organic anion‐transporting polypeptide (OATP) 1B1 and OATP2B1) or was downregulated (sodium taurocholate co‐transporting polypeptide (NTCP) and OCT1). Such regulations of drug transporter activity and expression, depending on the respective nature of OCs and transporters, may contribute to the toxicity of OC pesticides.     

Metabolic O-demethylation does not alter the influence of isoflavones on the biophysical properties of membranes and MRP1-like protein transport activity

Multidrug resistance transporter MRP1 could be effectively inhibited by some flavonoids. The influence of the two pairs of isoflavones: formononetin/daidzein and biochanin A/genistein on the efflux of fluorescent substrate of MRP1-like protein from erythrocytes and biophysical properties of lipid membranes has been compared. Compounds in each pair differ by the substituent in position 4' of B ring of isoflavone molecule. In the process of O-demethylation, CH(3) group (present in formonetin and biochanin A) is replaced by hydrogen (daidzein, genistein). Inhibition of MRP1-like protein transport activity by methylated and demethylated isoflavones was very similar. Their influence on lipid thermotropic properties and fluidity of lipid bilayer was not also significantly different.     

Foliar flavonoids of Annonaceae from Brazil: taxonomic significance

Foliar flavonoids of 31 species of the Annonaceae native to Brazil, amounting to 76 compounds, were isolated and identified. All phenols found were glycosides of either flavones (apigenin, scutellarein, hispidulin and luteolin) or flavonols (kaempferol, rhamnocitrin, 6-hydroxyrhamnocitrin, quercetin, isorhamnetin and rhamnetin), with the latter predominating. Some members of the tribe Bocageeae are distinctive for accumulating 6-oxygenated flavones and flavonols, in addition to 7-O-methylated flavonols, a feature possibly linked to the assumed advanced condition of the tribe within the family. Members of Duguetia stand out for the apparent absence of quercetin glycosides. Anaxagorea dolichocharpa seemingly lacks flavones and flavonols entirely. A UPGMA analysis based on the distribution of flavonoids does not group the analyzed species according to the available tribal division of the Annonaceae. However, several taxonomically meaningful groupings emerged through the multivariate analysis.     

Isoprenoid flavonoids are new leads in the modulation of chemoresistance

Flavonoid compounds are able to bind to P-glycoprotein (P-gp), a transporter involved in chemoresistance of cancer cells. This interaction involves, at least in part, two overlapping sites in the cytosolic domains of P-gp, the ATP site and a hydrophobic steroid-binding site. We have studied the structure-activity relationships toward binding to P-gp. Modification of the substitution pattern of the flavonoid ring by hydroxylation, methoxylation or introduction of nitrogen-containing substituents had little effect. On the contrary, the presence of a 3-hydroxyl (flavonols), and especially of a C-isoprenoid chain increased the affinity of flavonoids towards P-gp. More detailed examination of the interaction with the ATP site was conducted through inhibition by flavonoids of the photolabeling by radioactive 8-azido-TNP-ATP. Only simple flavonols were demonstrated to bind to the ATP site. When position 3 was free (flavones) or when a hydrophobic C-prenyl substituent was present, interaction was rather directed to the hydrophobic site. A number of flavonoid compounds were tested for their ability to modulate multidrug resistance in resistant leukemic K562/R7 cells. Again, prenyl flavonoids were potent modulators. Simple flavonoids were ineffective in this model. The beneficial effect of prenylation was lower in polyhydroxylated compounds, suggesting a crucial role of hydrophobicity in P-gp modulation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Subcellular localization and activity of multidrug resistance proteins

The multidrug resistance (MDR) phenotype is associated with the overexpression of members of the ATP-binding cassette family of proteins. These MDR transporters are expressed at the plasma membrane, where they are thought to reduce the cellular accumulation of toxins over time. Our data demonstrate that members of this family are also expressed in subcellular compartments where they actively sequester drugs away from their cellular targets. The multidrug resistance protein 1 (MRP1), P-glycoprotein, and the breast cancer resistance protein are each present in a perinuclear region positive for lysosomal markers. Fluorescence-activated cell sorting analysis suggests that these three drug transporters do little to reduce the cellular accumulation of the anthracycline doxorubicin. However, whereas doxorubicin enters cells expressing MDR transporters, this drug is sequestered away from the nucleus, its subcellular target, in vesicles expressing each of the three drug resistance proteins. Using a cell-impermeable inhibitor of MRP1 activity, we demonstrate that MRP1 activity on intracellular vesicles is sufficient to confer a drug resistance phenotype, whereas disruption of lysosomal pH is not. Intracellular localization and activity for MRP1 and other members of the MDR transporter family may suggest different strategies for chemotherapeutic regimens in a clinical setting.     

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.     

ABC drug transporter expression and functional activity in trophoblast-like cell lines and differentiating primary trophoblast

ATP-binding cassette (ABC) efflux transporters are expressed in the human placenta where they are thought to help protect the fetus from xenobiotics. To evaluate models for analysis of ABC transporter function and regulation in the placenta, we have characterized the expression and activity of multidrug resistance (MDR) 1/P glycoprotein (Pgp), MDR3/Pgp, breast cancer resistance protein (BCRP), and multidrug resistance proteins 1 and 2 (MRPs 1, 2) in differentiating primary trophoblast cells and BeWo and Jar cell lines. Real-time PCR and immunoblotting were used for analysis of mRNA and protein expression, respectively. Functional activity was measured using selective inhibitors of efflux of fluorescent substrates, calcein-AM (Pgp and MRPs) and Hoechst 33342 (BCRP). The levels of MDR1 mRNA and protein expression were much higher in trophoblast than in Jar and especially BeWo cells. Expression of MDR3 protein was also lower in BeWo cells. Levels of MDR3 expression were markedly higher than MDR1 levels in all tested cell types. Levels of both MDR1 and MDR3 expression decreased during trophoblast differentiation/syncytialization. BCRP was highly expressed in all cell types and increased with trophoblast differentiation. MRP1 expression was much lower in trophoblasts compared with both cell lines. In contrast to its abundant mRNA expression, MRP2 protein was practically undetectable in BeWo and Jar cells and was present only at very low levels in trophoblast. Functional studies confirmed the presence of active Pgp and BCRP in all studied cell types, whereas MRP functional activity was detected only in BeWo and Jar cells. Both cell lines may be useful models for studying various aspects of placental ABC transporter expression and function, but also have significant limitations. With respect to their ABC protein expression profile, Jar cells are more similar to nondifferentiated cytotrophoblast, whereas BeWo appear to more closely reflect differentiated syncytiotrophoblast.     

Characterization of 3-methoxy flavones for their interaction with ABCG2 as suggested by ATPase activity

Breast Cancer Resistance Protein (BCRP/ABCG2) belongs to the superfamily of ATP binding cassette (ABC) transporters. Characteristic of some of these transporter proteins is the transport of a variety of structurally unrelated substances against a concentration gradient by using the energy of ATP hydrolysis. ABCG2 has been found to confer multidrug resistance (MDR) in cancer cells. Several anticancer drugs have been identified as ABCG2 substrates including mitoxantrone, etoposide and topotecan. As inhibition of the transporter is one of the strategies to overcome MDR, we have synthesized and tested several 3-methoxy flavones and investigated them for their ABCG2 inhibition. Among these, pentamethyl quercetin (compound 4) and pentamethyl morin (compound 5) were found to be fluorescent and hence screened for their possible transport by ABCG2 using confocal microscopy. This study showed that pentamethyl quercetin was far less accumulated in ABCG2 overexpressing MDCK BCRP cells as compared to MDCK sensitive cells, suggesting possible efflux of this compound by ABCG2. Pentamethyl morin showed no visible difference in both cell lines. Based on this observation, we studied several other fluorescent 3-methoxy flavones for their accumulation in ABCG2 overexpressing cells. To confirm the substrate or inhibitor nature of the tested compounds, these compounds were further investigated by ATPase assay. If stimulation of the transporter ATPase activity is detected, one can conclude that the compound is probably a transported substrate. All compounds except pentamethyl morin (compound 5) and tetramethyl quercetin (compound 6) were found to stimulate ATPase activity pointing to possible substrates despite being potent inhibitors of ABCG2.     

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.     

Nuclear Multidrug-Resistance Related Protein 1 Contributes to Multidrug-Resistance of Mucoepidermoid Carcinoma Mainly via Regulating Multidrug-Resistance Protein 1: A Human Mucoepidermoid Carcinoma Cells Model and Spearman's Rank Correlation Analysis

Background

Multidrug resistance-related protein 1 (MRP1/ABCC1) and multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1) are both membrane-bound drug transporters. In contrast to MDR1, MRP1 also transports glutathione (GSH) and drugs conjugated to GSH. Due to its extraordinary transport properties, MRP1/ABCC1 contributes to several physiological functions and pathophysiological incidents. We previously found that nuclear translocation of MRP1 contributes to multidrug-resistance (MDR) of mucoepidermoid carcinoma (MEC). The present study investigated how MRP1 contributes to MDR in the nuclei of MEC cells.

Methods

Western blot and RT-PCR was carried out to investigate the change of multidrug-resistance protein 1 (MDR1) in MC3/5FU cells after MRP1 was downregulated through RNA interference (RNAi). Immunohistochemistry (IHC) staining of 127 cases of MEC tissues was scored with the expression index (EI). The EI of MDR1 and MRP1 (or nuclear MRP1) was analyzed with Spearman''s rank correlation analysis. Using multiple tumor tissue assays, the location of MRP1 in other tissues was checked by HIC. Luciferase reporter assays of MDR1 promoter was carried out to check the connection between MRP1 and MDR1 promoter.

Results

MRP1 downregulation led to a decreased MDR1 expression in MC3/5FU cells which was caused by decreased activity of MDR1 promoter. IHC study of 127 cases of MEC tissues demonstrated a strong positive correlation between nuclear MRP1 expression and MDR1 expression. Furthermore, IHC study of multiple tumor tissue array sections showed that although nuclear MRP1 widely existed in MEC tissues, it was not found in normal tissues or other tumor tissues.

Conclusions

Our findings indicate that nuclear MRP1 contributes to MDR mainly through regulating MDR1 expression in MEC. And the unique location of MRP1 made it an available target in identifying MEC from other tumors.     

Talazoparib nanoparticles for overcoming multidrug resistance in triple-negative breast cancer

Herein, we investigated efflux pumps-mediated talazoparib-resistance in the treatment of triple-negative breast cancer (TNBC). Furthermore, we produced a novel talazoparib-solid lipid nanoparticles (SLNs) and then explored in vitro therapeutic efficacy of talazoparib-SLNs to overcome talazoparib-resistance in TNBC cells. Talazoparib-SLNs formulation was produced and then characterized. Calcein and Rho-123 were used to analyze the functional activity of drug efflux pumps in these cells. Additionally, RT-PCR, western blot and immunofluorescence analysis were used to detect the messenger RNA, and protein expression level, and cellular localization of the multidrug resistance (MDR1), breast cancer resistance protein (BCRP), and MRP1. We found that talazoparib efflux was mediated by BCRP and MRP1 pumps in TNBC cells. Talazoparib-SLNs could significantly enhance therapeutic efficacy of talazoparib. Furthermore, talazoparib-SLNs were more effective in the suppression of MDR1, BCRP, and MRP1 gene and protein expression levels than talazoparib. Consequently, this study suggests that talazoparib-SLNs formulation represents a promising therapeutic carrier to reverse MDR-mediated resistance in TNBC.