Involvement of multidrug resistance proteins (MDR) in the modulation of glucocorticoid response

Glucocorticoid resistance is a problem in the treatment of many diseases. One possible factor involved in the modulation of a glucocorticoid response is the export of glucocorticoids out of the cell. It has been shown that multidrug resistance protein 1 (MDR1, ABCB1), a member of the ABC family, is capable of transporting some glucocorticoids. This paper uses a mouse cell line, LMCAT in which the glucocorticoid response can be modulated by inhibitors of multidrug resistance proteins. Glucocorticoids fall into three categories. Firstly, those that are transported by an Abcb1a/Abcb1b transporter and whose transport can be inhibited by inhibitors of ABCB1 activity. Functional Abcb1a/Abcb1b was detected by inhibition of rhodamine efflux by these drugs and mRNA for Abcb1a and Abcb1b were detected in these cells. Secondly, those that are not transported. Finally, those that are transported by an Abcc1a transporter. Calcein transport out of these cells was blocked by treatment with probenecid indicating a functional Abcc1a transporter. Abcc1a mRNA was also detected in these cells. Thus, this paper provides insight into the mechanisms of glucocorticoid transport in cells and demonstrates a diversity of two independent mechanisms of transport of glucocorticoids by Abcb1a/Abcb1b and Abcc1a with individual patterns of steroid specificity.     

Effects of plant sterols on human multidrug transporters ABCB1 and ABCC1

The effects of dietary plant sterols on human drug efflux transporters P-glycoprotein (P-gp, ABCB1) and multidrug resistance protein 1 (MRP1, ABCC1) were investigated using P-gp-overexpressing human carcinoma KB-C2 cells and human MRP1 gene-transfected KB/MRP cells. The effects of natural phytosterols found in foods, herbs, and dietary supplements such as β-sitosterol, campesterol, stigmasterol, fucosterol, and z-guggulsterone were investigated. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-gp, increased in the presence of guggulsterone in KB-C2 cells. The efflux of rhodamine 123 from KB-C2 cells was inhibited by guggulsterone. Guggulsterone also increased the accumulation of calcein, a fluorescent substrate of MRP1, in KB/MRP cells. The ATPase activities of P-gp and MRP1 were stimulated by guggulsterone. These results suggest that guggulsterone, a natural dietary hypolipidemic agent have dual inhibitory effects on P-gp and MRP1 and the potencies to cause food-drug interactions.     

肿瘤多药耐药与自噬、DNA修复和肿瘤干细胞相关的分子机制研究进展

肿瘤多药耐药(multi-drug resistance,MDR)所导致的化疗失败,依旧是肿瘤治疗中存在的难点。虽然针对MDR,已经成功开发了三代靶向三磷酸腺苷结合盒转运体(ATP binding cassette transporter,ABC)的抑制剂,也有MDR调节剂和化学增敏剂,多功能纳米载体和RNA干扰等方法可有效逆转MDR,但是由于肿瘤多药耐药机制的复杂性,MDR依然是肿瘤治疗中面临的难题。将从目前研究比较集中的ABC转运体异常表达; DNA损伤修复的改变和细胞凋亡的异常;自噬诱导与耐药及肿瘤干细胞与耐药等四方面入手,针对目前MDR机制的研究进展做一综述,旨在为MDR的研究提供思路和方法。     

The Mechanism of Action of Multidrug-Resistance-Linked P-Glycoprotein

P-glycoprotein (Pgp), the ATP-binding cassette (ABC) transporter, confers multidrug resistance to cancer cells by extruding cytotoxic natural product amphipathic drugs using the energy of ATP hydrolysis. Our studies are directed toward understanding the mechanism of action of Pgp and recent work deals with the assessment of interaction between substrate and ATP sites and elucidation of the catalytic cycle of ATP hydrolysis. The kinetic analyses of ATP hydrolysis by reconstituted purified Pgp suggest that ADP release is the rate-limiting step in the catalytic cycle and the substrates exert their effect by modulating ADP release. In addition, we provide evidence for two distinct roles for ATP hydrolysis in a single turnover of Pgp, one in the transport of drug and the other in effecting conformational changes so as to reset the transporter for the next catalytic cycle. Detailed kinetic measurements determined that both nucleotide-binding domains behave symmetrically and during individual hydrolysis events the ATP sites are recruited in a random manner. Furthermore, only one nucleotide site hydrolyzes ATP at any given time, causing (in this site) a conformational change that drastically decreases (>30-fold) the affinity of the second site for ATP-binding. Thus, the blocking of ATP-binding to the second site while the first one is in catalytic conformation appears to be the basis for the alternate catalytic cycle of ATP hydrolysis by Pgp, and this may be applicable as well to other ABC transporters linked with the development of multidrug resistance.     

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.     

Functional expression of a multidrug P-glycoprotein transporter of Leishmania

P-glycoprotein (Pgp) transporters play an important role in multidrug resistance in eukaryotic cells and in protozoan parasites such as Leishmania. To search for new reversal agents of the Leishmania tropica Pgp, we developed a screening assay using the Baculovirus-insect cell expression system. We demonstrated a MgATP-dependent, vanadate-sensitive transport of Hoechst 33342 in membrane preparations of Sf9 insect cells expressing Pgp. We have found that dihydro-beta-agarofuran sesquiterpenes from Maytenus cuzcoina inhibited Hoechst 33342 transport that correlates with their reversal effect in a multidrug-resistant L. tropica line overexpressing Pgp. The results suggest that Sf9 cell membrane Hoechst 33342 transport system represents an efficient tool for examining the interactions of Leishmania Pgp with pharmacological agents.     

Tetrandrine and fangchinoline,bisbenzylisoquinoline alkaloids from Stephania tetrandra can reverse multidrug resistance by inhibiting P-glycoprotein activity in multidrug resistant human cancer cells

The overexpression of ABC transporters is a common reason for multidrug resistance (MDR) in cancer cells. In this study, we found that the isoquinoline alkaloids tetrandrine and fangchinoline from Stephania tetrandra showed a significant synergistic cytotoxic effect in MDR Caco-2 and CEM/ADR5000 cancer cells in combination with doxorubicin, a common cancer chemotherapeutic agent. Furthermore, tetrandrine and fangchinoline increased the intracellular accumulation of the fluorescent P-glycoprotein (P-gp) substrate rhodamine 123 (Rho123) and inhibited its efflux in Caco-2 and CEM/ADR5000 cells. In addition, tetrandrine and fangchinoline significantly reduced P-gp expression in a concentration-dependent manner. These results suggest that tetrandrine and fangchinoline can reverse MDR by increasing the intracellular concentration of anticancer drugs, and thus they could serve as a lead for developing new drugs to overcome P-gp mediated drug resistance in clinic cancer therapy.     

MRP Subfamily Transporters and Resistance to Anticancer Agents

The MRP subfamily of ABC transporters from mammals consists of at least seven members, six of which have been implicated in the transport of amphipathic anions. MRP1, MRP2, and MRP3 bear a close structural resemblance, confer resistance to a variety of natural products as well as methotrexate, and have the facility for transporting glutathione and glucuronate conjugates. MRP1 is a ubiquitously expressed efflux pump for the products of phase II of xenobiotic detoxification, while MRP2, whose hereditary deficiency results in Dubin–Johnson syndrome, functions to extrude organic anions into the bile. MRP3 is distinguished by its capacity to transport the monoanionic bile constituent glycocholate, and may function as a basolateral back-up system for the detoxification of hepatocytes when the usual canalicular route is impaired by cholestatic conditions. MRP4 and MRP5 resemble each other more closely than they resemble MRPs 1–3 and confer resistance to purine and nucleotide analogs which are either inherently anionic, as in the case of the anti-AIDS drug PMEA, or are phosphorylated and converted to anionic amphiphiles in the cell, as in the case of 6-MP. Given their capacity for transporting cyclic nucleotides, MRP4 and MRP5 have also been implicated in a broad range of cellular signaling processes. The drug resistance activity and physiological substrates of MRP6 are unknown. However, its hereditary deficiency results in pseudoxanthoma elasticum, a multisystem disorder affecting skin, eyes, and blood vessels. It is hoped that elucidation of the resistance profiles and physiological functions of the different members of the MRP subfamily will provide new insights into the molecular basis of clinical drug resistance and spawn new strategies for combating this phenomenon.     

Availability and applications of <Emphasis Type="Italic">A</Emphasis>TP-binding <Emphasis Type="Italic">c</Emphasis>assette (ABC) transporter blockers

ATP-binding cassette (ABC) transporters encompass membrane transport proteins that couple the energy derived from ATP hydrolysis to the translocation of solutes across biological membranes. The functions of these proteins include ancient and conserved mechanisms related to nutrition and pathogenesis in bacteria, spore formation in fungi, and signal transduction, protein secretion and antigen presentation in eukaryotes. Furthermore, one of the major causes of drug resistance and chemotherapeutic failure in both cancer and anti-infective therapies is the active movement of compounds across membranes carried out by ABC transporters. Thus, the clinical relevance of ABC transporters is enormous, and the membrane transporters related to chemoresistance are among the best-studied members of the ABC transporter superfamily. As ABC transporter blockers can be used in combination with current drugs to increase their efficacy, the (possible) impact of efflux pump inhibitors is of great clinical interest. The present review summarizes the progress made in recent years in the identification, design, availability, and applicability of ABC transporter blockers in experimental scenarios oriented towards improving the treatment of infectious diseases caused by microorganisms including parasites.     

A novel class of highly potent multidrug resistance reversal agents: Disubstituted adamantyl derivatives

Novel disubstituted adamantyl derivatives were synthesized and evaluated in a P-glycoprotein dependent multidrug resistance cancer cell line. The hit to lead optimization provided potent MDR reversal agents. Some potent adamantyl derivatives were more than 10-fold more potent than verapamil without considerable intrinsic cytotoxicity. The 3-trifluorophenyl derivative 14f did not affect the metabolism of CYP450 3A4, whereas most of MDR revertants had a weak inhibitory effect.     

Transport proteins of the ABC family and multidrug resistance of tumor cells

Some new data concerning the role of transport proteins of the ABC family in multidrug resistance (MDR) of human tumor cells, and problems connected with regulation of these proteins are considered. MDR is a complex phenomenon that may be caused simultaneously by several mechanisms functioning in one and the same cell. Among them there may be the alterations of activity of several transport proteins. Activation of these proteins may be associated with alterations of activities of different cell protective systems and of the signal transduction pathways involved in regulation of proliferation, differentiation, and apoptosis. Clinical significance of multifactor MDR is discussed.     

Nitensidine A,a guanidine alkaloid from Pterogyne nitens,is a novel substrate for human ABC transporter ABCB1

The Pterogyne nitens (Fabaceae) tree, native to South America, has been found to produce guanidine alkaloids as well as bioactive flavonols such as kaempferol, quercetin, and rutin. In the present study, we examined the possibility of interaction between human ATP-binding cassette (ABC) transporter ABCB1 and four guanidine alkaloids isolated from P. nitens (i.e., galegine, nitensidine A, pterogynidine, and pterogynine) using human T cell lymphoblast-like leukemia cell line CCRF-CEM and its multi-drug resistant (MDR) counterpart CEM/ADR5000. In XTT assays, CEM/ADR5000 cells were resistant to the four guanidine alkaloids compared to CCRF-CEM cells, although the four guanidine alkaloids exhibited some level of cytotoxicity against both CCRF-CEM and CEM/ADR5000 cells. In ATPase assays, three of the four guanidine alkaloids were found to stimulate the ATPase activity of ABCB1. Notably, nitensidine A was clearly found to stimulate the ATPase activity of ABCB1 as strongly as the control drug, verapamil. Furthermore, the cytotoxic effect of nitensidine A on CEM/ADR5000 cells was synergistically enhanced by verapamil. Nitensidine A inhibited the extrusion of calcein by ABCB1. In the present study, the possibility of interaction between ABCB1 and two synthetic nitensidine A analogs (nitensidine AT and AU) were examined to gain insight into the mechanism by which nitensidine A stimulates the ATPase activity of ABCB1. The ABCB1-dependent ATPase activity stimulated by nitensidine A was greatly reduced by substituting sulfur (S) or oxygen (O) for the imino nitrogen atom (N) in nitensidine A. Molecular docking studies on human ABCB1 showed that, guanidine alkaloids from P. nitens dock to the same binding pocket as verapamil. Nitensidine A and its analogs exhibit similar binding energies to verapamil. Taken together, this research clearly indicates that nitensidine A is a novel substrate for ABCB1. The present results also suggest that the number, binding site, and polymerization degree of the isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their stimulation of ABCB1's ATPase activity.