Overexpression of ATP-binding cassette (ABC) transporters is one of the most important mechanisms responsible for the development of multidrug resistance (MDR). Selonsertib, a serine/threonine kinase ...inhibitor, targets apoptosis signal-regulating kinase 1 (ASK1) and is now in phase III clinical trial for the treatment of non-alcoholic steatohepatitis (NASH). In this study, we investigated whether selonsertib could reverse MDR-mediated by ABC transporters, including ABCB1, ABCG2, ABCC1 and ABCC10. The results showed that selonsertib significantly reversed ABCB1- and ABCG2-mediated MDR, but not MDR-mediated by ABCC1 or ABCC10. Mechanism studies indicated that the reversal effect of selonsertib was related to the attenuation of the efflux activity of ABCB1 and ABCG2 transporters, without the protein level decrease or change in the subcellular localization of ABCB1 or ABCG2. Selonsertib stimulated the ATPase activity of ABCB1 and ABCG2 in a concentration-dependent manner, and in silico docking study showed selonsertib could interact with the substrate-binding sites of both ABCB1 and ABCG2. This study provides a clue into a novel treatment strategy, which includes a combination of selonsertib with antineoplastic drugs to attenuate MDR-mediated by ABCB1 or ABCG2 in cancer cells overexpressing these transporters.
•Selonsertib, a selective ASK1 inhibitor, significantly overcome MDR in cancer.•Selonsertib stimulates the ATPase activity of ABCB1 and ABCG2.•Selonsertib interacts with the substrate-binding sites of both ABCB1 and ABCG2.•The combination of selonsertib with anticancer drugs could be a novel treatment strategy to evade MDR in cancer.
P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs. Understanding ...the structural basis of P-gp, substrate polyspecificity has been hampered by its intrinsic flexibility, which is facilitated by a 75-residue linker that connects the two halves of P-gp. Here we constructed a mutant murine P-gp with a shortened linker to facilitate structural determination. Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shortened mutant P-gp possesses basal ATPase activity and binds ATP only in its N-terminal nucleotide-binding domain. Nine independently determined structures of wild type, the linker mutant, and a methylated P-gp at up to 3.3 Å resolution display significant movements of individual transmembrane domain helices, which correlated with the opening and closing motion of the two halves of P-gp. The open-and-close motion alters the surface topology of P-gp within the drug-binding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions.
Interindividual variability in protein expression of organic anion-transporting polypeptides (OATPs) OATP1B1, OATP1B3, OATP2B1, and multidrug resistance-linked P-glycoprotein (P-gp) or ABCB1 was ...quantified in frozen human livers (n = 64) and cryopreserved human hepatocytes (n = 12) by a validated liquid chromatography tandem mass spectroscopy (LC-MS/MS) method. Membrane isolation, sample workup, and LC-MS/MS analyses were as described before by our laboratory. Briefly, total native membrane proteins, isolated from the liver tissue and cryopreserved hepatocytes, were trypsin digested and quantified by LC-MS/MS using signature peptide(s) unique to each transporter. The mean ± S.D. (maximum/minimum range in parentheses) protein expression (fmol/µg of membrane protein) in human liver tissue was OATP1B1- 2.0 ± 0.9 (7), OATP1B3- 1.1 ± 0.5 (8), OATP2B1- 1 1.7 ± 0.6 (5), and P-gp- 0.4 ± 0.2 (8). Transporter expression in the liver tissue was comparable to that in the cryopreserved hepatocytes. Most important is that livers with SLCO1B1 (encoding OATP1B1) haplotypes *14/*14 and *14/*1a i.e., representing single nucleotide polymorphisms (SNPs), c.388A > G, and c.463C > A had significantly higher (P < 0.0001) protein expression than the reference haplotype (*1a/*1a). Based on these genotype-dependent protein expression data, we predicted (using Simcyp) an up to ∼40% decrease in the mean area under the curve of rosuvastatin or repaglinide in subjects harboring these variant alleles compared with those harboring the reference alleles. SLCO1B3 (encoding OATP1B3) SNPs did not significantly affect protein expression. Age and sex were not associated with transporter protein expression. These data will facilitate the prediction of population-based human transporter-mediated drug disposition, drug-drug interactions, and interindividual variability through physiologically based pharmacokinetic modeling.
Sunitinib malate (Sutent, SU11248) is a small-molecule receptor tyrosine kinase inhibitor that inhibits cellular signaling of multiple targets such as the platelet-derived growth factor receptors and ...the vascular endothelial growth factor receptors and is used in the treatment of renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumors. Because tyrosine kinase inhibitors are known to increase the p.o. bioavailability and brain penetration of chemotherapy drugs in animal models, we sought to examine the effect of sunitinib on the ATP-binding cassette (ABC) drug transporters P-glycoprotein (P-gp, ABCB1), the multidrug resistance-associated protein 1 (ABCC1), and ABCG2, which are known to transport a wide variety of anticancer drugs. In this study, we show that sunitinib inhibits P-gp- and ABCG2-mediated efflux of fluorescent substrates in cells overexpressing these transporters. In 4-day cytotoxicity assays, at a nontoxic concentration (2 microM) sunitinib was able to partially reverse drug resistance mediated by P-gp and completely reverse resistance mediated by ABCG2. We further show a direct interaction of sunitinib with the substrate binding pocket of these transporters as it inhibited binding of the photoaffinity substrate (125)Iiodoarylazidoprazosin to P-gp (IC(50) = 14.2 microM) and ABCG2 (IC(50) = 1.33 microM). Sunitinib stimulated the ATP hydrolysis by both transporters in a concentration-dependent manner. Conformation-sensitive antibody binding assays with the P-gp- and ABCG2-specific antibodies, UIC2 and 5D3, respectively, also confirmed the interaction of sunitinib with these transporters. Taken together, this is the first report showing that sunitinib inhibits transport mediated by ABC drug transporters, which may affect the bioavailability of drugs coadministered with sunitinib.
Human P-glycoprotein (P-gp) is an ATP-binding cassette transporter that has been implicated in altering the pharmacokinetics of anticancer drugs in normal tissues and development of multidrug ...resistance in tumor cells via drug efflux. There is still no definitive explanation of the mechanism by which P-gp effluxes drugs. One of the challenges of large-scale purification of membrane transporters is the selection of a suitable detergent for its optimal extraction from cell membranes. In addition, further steps of purification can often lead to inactivation and aggregation, decreasing the yield of purified protein. Here we report the large-scale purification of human P-gp expressed in High-Five insect cells using recombinant baculovirus. The purification strategies we present yield homogeneous functionally active wild type P-gp and its E556Q/E1201Q mutant, which is defective in carrying out ATP hydrolysis. Three detergents (1,2-diheptanoyol-sn-glycero-3-phosphocholine, dodecyl maltoside and n-octyl-β-d-glucopyranoside) were used to solubilize and purify P-gp from insect cell membranes. P-gp purification was performed first using immobilized metal affinity chromatography, then followed by a second step of either anion exchange chromatography or size exclusion chromatography to yield protein in concentrations of 2–12 mg/mL. Size exclusion chromatography was the preferred method, as it allows separation of monomeric transporters from aggregates. We show that the purified protein, when reconstituted in proteoliposomes and nanodiscs, exhibits both basal and substrate or inhibitor-modulated ATPase activity. This report thus provides a convenient and robust method to obtain large amounts of active homogeneously purified human P-gp that is suitable for biochemical, biophysical and structural characterization.
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•Human P-glycoprotein is expressed at high levels in High-Five insect cells.•Active P-glycoprotein can be purified and solubilized using different detergents.•Functional P-glycoprotein can be purified in large quantities.
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.
Sildenafil is a potent and selective inhibitor of the type 5 cGMP (cyclic guanosine 3',5'-monophosphate)-specific phosphodiesterase that is used clinically to treat erectile dysfunction and pulmonary ...arterial hypertension. Here, we report that sildenafil has differential effects on cell surface ABC transporters such as ABCB1, ABCC1, and ABCG2 that modulate intracompartmental and intracellular concentrations of chemotherapeutic drugs. In ABCB1-overexpressing cells, nontoxic doses of sildenafil inhibited resistance and increased the effective intracellular concentration of ABCB1 substrate drugs such as paclitaxel. Similarly, in ABCG2-overexpressing cells, sildenafil inhibited resistance to ABCG2 substrate anticancer drugs, for example, increasing the effective intracellular concentration of mitoxantrone or the fluorescent compound BODIPY-prazosin. Sildenafil also moderately inhibited the transport of E(2)17βG and methotrexate by the ABCG2 transporter. Mechanistic investigations revealed that sildenafil stimulated ABCB1 ATPase activity and inhibited photolabeling of ABCB1 with (125)I-iodoarylazidoprazosin (IAAP), whereas it only slightly stimulated ABCG2 ATPase activity and inhibited photolabeling of ABCG2 with (125)I-IAAP. In contrast, sildenafil did not alter the sensitivity of parental, ABCB1-, or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drugs, nor did sildenafil affect the function of another ABC drug transporter, ABCC1. Homology modeling predicted the binding conformation of sildenafil within the large cavity of the transmembrane region of ABCB1. Overall, we found that sildenafil inhibits the transporter function of ABCB1 and ABCG2, with a stronger effect on ABCB1. Our findings suggest a possible strategy to enhance the distribution and potentially the activity of anticancer drugs by jointly using a clinically approved drug with known side effects and drug-drug interactions.
Purpose To evaluate the in vivo efficacy of curcumin as an inhibitor of the multidrug-resistance-linked ATP Binding Cassette (ABC) drug transporter, ABCG2. Methods Photoaffinity labeling with ...¹²⁵I-iodoarylazidoprazosin was used to characterize the interaction of sulfasalazine, a substrate of the mouse ABCG2, with human ABCG2. In addition, the inhibitory effect of curcumin on ABCG2 was evaluated in brain capillaries from rats. Furthermore, the effect of curcumin on absorption of orally administered sulfasalazine in wild-type and abcg2 ⁻/⁻ mice was also determined. Results Sulfasalazine interacted at the drug-substrate site(s) of human ABCG2. Curcumin inhibited ABCG2 activity at nanomolar concentrations at the rat blood-brain barrier in the ex vivo assay. Based on studies in wild type and abcg2 ⁻/⁻ mice, we observed that oral curcumin increased C max and relative bioavailability of sulfasalazine by selectively inhibiting ABCG2 function. Conclusions This study validates our previous in vitro results with human ABCG2 (Chearwae et al., Mol. Cancer Ther. 5:1995-2006, 2006) and provides the first in vivo evidence for the inhibition by curcumin of ABCG2-mediated efflux of sulfasalazine in mice. Based on these studies, we propose that non-toxic concentrations of curcumin may be used to enhance drug exposure when the rate-limiting step of drug absorption and/or tissue distribution is impacted by ABCG2.
Castration-resistant prostate cancer (CRPC) has greater intratumoral testosterone concentrations than similar tumors from eugonadal men; simple diffusion does not account for this observation. This ...study was undertaken to ascertain the androgen uptake kinetics, functional, and clinical relevance of
expression of the steroid hormone transporter OATP1B3 (
). Experiments testing the cellular uptake of androgens suggest that testosterone is an excellent substrate of OATP1B3 (
= 23.2 μmol/L;
= 321.6 pmol/mg/minute), and cells expressing a doxycycline-inducible
construct had greater uptake of a clinically relevant concentration of 3H-testosterone (50 nmol/L; 1.6-fold,
= 0.0027). When compared with
(-/-) mice,
(-/-)/
knockins had greater hepatic uptake (15% greater AUC,
= 0.0040) and lower plasma exposure to 3H-testosterone (17% lower AUC,
= 0.0030). Of 82 transporters genes,
is the second-most differentially expressed transporter in CRPC cell lines (116-fold vs. androgen-sensitive cells), with a differentially spliced cancer-type ct-
making up the majority of
expression. Overexpression of
in androgen-responsive cells results in 1.5- to 2-fold greater testosterone uptake, whereas siRNA knockdown of
in CRPC cells did not change intracellular testosterone concentration. Primary human prostate tumors express
to a greater extent than
(26% of total
expression vs. 0.08%), suggesting that androgen uptake in these tumor cells also is greater. Non-liver tumors do not differentially express SLCO1B3.
This study suggests that
OATP1B3 expression in prostate cancer drives greater androgen uptake and is consistent with previous observations that greater OATP1B3 activity results in the development of androgen deprivation therapy resistance and shorter overall survival.
.
It has been reported that gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), has the ability to modulate the function of certain ATP-binding cassette (ABC) ...transporters and to reverse ABC subfamily B member 1 (ABCB1; P-glycoprotein)- and ABC subfamily G member 2 (ABCG2; breast cancer resistance protein/mitoxantrone resistance protein)-mediated multidrug resistance (MDR) in cancer cells. However, it is unknown whether other EGFR TKIs have effects similar to that of gefitinib. In the present study, we have investigated the interaction of another EGFR TKI, erlotinib, with selected ABC drug transporters. Our findings show that erlotinib significantly potentiated the sensitivity of established ABCB1 or ABCG2 substrates and increased the accumulation of paclitaxel or mitoxantrone in ABCB1- or ABCG2-overexpressing cells. Furthermore, erlotinib did not significantly alter the sensitivity of non-ABCB1 or non-ABCG2 substrates in all cells and was unable to reverse MRP1-mediated MDR and had no effect on the parental cells. However, erlotinib remarkably inhibited the transport of E(2)17 beta G and methotrexate by ABCG2. In addition, the results of ATPase assays show that erlotinib stimulated the ATPase activity of both ABCB1 and ABCG2. Interestingly, erlotinib slightly inhibited the photolabeling of ABCB1 with (125)Iiodoarylazidoprazosin (IAAP) at high concentration, but it did not inhibit the photolabeling of ABCG2 with IAAP. Overall, we conclude that erlotinib reverses ABCB1- and ABCG2-mediated MDR in cancer cells through direct inhibition of the drug efflux function of ABCB1 and ABCG2. These findings may be useful for cancer combinational therapy with erlotinib in the clinic.
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