Topotecan is a substrate for multidrug resistance associated protein 4

Topotecan (TPT) is a semisynthetic water-soluble derivative of camptothecin (CPT) used as second-line therapy in patients with metastatic ovarian carcinoma, small cell lung cancer, and other malignancies. However, both dose-limiting toxicity and tumor resistance hinder the clinical use of TPT. The mechanisms for resistance to TPT are not fully defined, but increased efflux of the drug by multiple drug transporters including P-glycoprotein (PgP), multidrug resistance associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) from tumor cells has been highly implicated. This study aimed to investigate whether overexpression of human MRP4 rendered resistance to TPT by examining the cytotoxicity profiles using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay and cellular accumulation of TPT in HepG2 cells stably overexpressing MRP4. Two kinds of cell lines, HepG2 with insertion of an empty vector plasmid (V/HepG2), HepG2 cells stably expressing MRP4 (MRP4/HepG2), were exposed to TPT for 4 or 48 hr in the absence or presence of various MRP4 inhibitors including DL-buthionine-(S,R)-sulphoximine (BSO), diclofenac, celecoxib, or MK-571. The intracellular accumulation of TPT and paclitaxel (a PgP substrate) by V/HepG2 and MRP4/HepG2 cells was determined by incubation of TPT with the cells and the amounts of the drug in cells were determined by validated HPLC methods. The study demonstrated that MRP4 conferred a 12.03- and 6.86-fold resistance to TPT in the 4- and 48-hr drug-exposure MTT assay, respectively. BSO, MK-571, celecoxib, or diclofenac sensitised MRP4/HepG2 cells to TPT cytotoxicity and partially reversed MRP4-mediated resistance to TPT. In addition, the accumulation of TPT was significantly reduced in MRP4/HepG2 cells compared to V/HepG2 cells, and one-binding site model was found the best fit for the MRP4-mediated efflux of TPT, with an estimated K(m) of 1.66 microM and V(max) of 0.341 ng/min/106 cells. Preincubation of MRP4/HepG2 cells with BSO (200 microM) for 24 hr, celecoxib (50 microM), or MK-571 (100 microM) for 2 hr significantly increased the accumulation of TPT over 10 min in MRP4/HepG2 cells by 28.0%, 37.3% and 32.5% (P < 0.05), respectively. By contrast, there was no significant difference in intracellular accumulation of paclitaxel in V/HepG2 and MRP4/HepG2 cells over 120 min. MRP4 also rendered resistance to adefovir dipivoxil (bis-POM-PMEA) and methotrexate, two reported MRP4 substrates. MRP4 did not exhibit any significant resistance to other model drugs including vinblastine, vincristine, etoposide, carboplatin, cyclosporine and paclitaxel in both long (48 hr) and short (4 hr) drug-exposure MTT assays. These findings indicate that MRP4 confers resistance to TPT and TPT is the substrate for MRP4. Further studies are needed to explore the role of MRP4 in resistance to, toxicity and pharmacokinetics of TPT in cancer patients.     

ABCC2基因过表达细胞株的建立及鉴定

目的 构建携带ABCC2基因的慢病毒载体,转染HEK293细胞,筛选出稳定过表达ABCC2基因的细胞株并进行鉴定,为体外实验确定与多药耐药相关蛋白2(MRP2)外排转运相关的底物药物及其转运机制提供细胞模型。方法 根据GenBank提供的ABCC2基因cDNA序列设计引物,PCR扩增该基因并将其连接至慢病毒载体PZE-LV105,包装病毒并感染HEK293细胞。用嘌呤霉素进行筛选得到过表达ABCC2基因的稳转细胞株,通过基因测序、实时荧光定量PCR(RTQ-PCR)和蛋白免疫印迹(Western blot)对稳转细胞进行鉴定。结果 测序结果证明重组慢病毒过表达载体所携带的ABCC2基因序列与GenBank提供的ABCC2序列一致;RTQ-PCR分析结果显示转染了ABCC2基因的HEK293细胞中MRP2 的mRNA相对表达比正常HEK293细胞和空白载体对照HEK293细胞高出约320倍;蛋白免疫印迹试验显示,转染了ABCC2基因的HEK293细胞中MRP2蛋白表达水平比正常HEK293细胞和空白载体对照HEK293细胞高出约150倍。结论 该实验成功构建了稳定过表达ABCC2基因的细胞株,该细胞株可用于初步筛选确定MRP2的特异性外排转运底物及其转运机制。     

Human Multidrug Resistance Associated Protein 4 Confers Resistance to Camptothecins

Purpose The multidrug resistance associated protein (MRP) 4 is a member of the adenosine triphosphate (ATP)-binding cassette transporter family. Camptothecins (CPTs) have shown substantial anticancer activity against a broad spectrum of tumors by inhibiting DNA topoisomerase I, but tumor resistance is one of the major reasons for therapeutic failure. P-glycoprotein, breast cancer resistance protein, MRP1, and MRP2 have been implicated in resistance to various CPTs including CPT-11 (irinotecan), SN-38 (the active metabolite of CPT-11), and topotecan. In this study, we explored the resistance profiles and intracellular accumulation of a panel of CPTs including CPT, CPT-11, SN-38, rubitecan, and 10-hydroxy-CPT (10-OH-CPT) in HepG2 cells with stably overexpressed human MRP4. Other anticancer agents such as paclitaxel, cyclophosphamide, and carboplatin were also included. Methods HepG2 cells were transfected with an empty vehicle plasmid (V/HepG2) or human MRP4 (MRP4/HepG2). The resistance profiles of test drugs in exponentially growing V/HepG2 and MRP4/HepG2 cells were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay with 4 or 48 h exposure time of the test drug in the absence or presence of various MRP4 inhibitors. The accumulation of CPT-11, SN-38, and paclitaxel by V/HepG2 and MRP4/HepG2 cells was determined by validated high-performance liquid chromatography methods. Results Based on the resistance folds from the MTT assay with 48 h exposure time of the test drug, MRP4 conferred resistance to CPTs tested in the order 10-OH-CPT (14.21) > SN-38 carboxylate (9.70) > rubitecan (9.06) > SN-38 lactone (8.91) > CPT lactone (7.33) > CPT-11 lactone (5.64) > CPT carboxylate (4.30) > CPT-11 carboxylate (2.68). Overall, overexpression of MRP4 increased the IC₅₀ values 1.78- to 14.21-fold for various CPTs in lactone or carboxylate form. The resistance of MRP4 to various CPTs tested was significantly reversed in the presence of dl-buthionine-(S,R)-sulfoximine (BSO, a γ-glutamylcysteine synthetase inhibitor), MK571, celecoxib, or diclofenac (all MRP4 inhibitors). In addition, the accumulation of CPT-11 and SN-38 over 120 min in MRP4/HepG2 cells was significantly reduced compared to V/HepG2 cells, whereas the addition of celecoxib, MK571, or BSO significantly increased their accumulation in MRP4/HepG2 cells. There was no significant difference in the intracellular accumulation of paclitaxel in V/HepG2 and MRP4/HepG2 cells, indicating that P-glycoprotein was not involved in the observed resistance to CPTs in this study. MRP4 also conferred resistance to cyclophosphamide and this was partially reversed by BSO. However, MRP4 did not increase resistance to paclitaxel, carboplatin, etoposide (VP-16), 5-fluorouracil, and cyclosporine. Conclusions Human MRP4 rendered significant resistance to cyclophosphamide, CPT, CPT-11, SN-38, rubitecan, and 10-OH-CPT. CPT-11 and SN-38 are substrates for MRP4. Further studies are needed to explore the role of MRP4 in resistance, toxicity, and pharmacokinetics of CPTs and cyclophosphamide.     

Drug resistance and ATP-dependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells

The multidrug resistance protein MRP1 functions as an ATP-dependent conjugate export pump and confers multidrug resistance. We cloned MRP2 (symbol ABCC2), a MRP family member localized to the apical membrane of polarized cells. Stable expression of MRP2 in transfected human embryonic kidney (HEK-293) and Madin-Darby canine kidney (MDCK) cells was enhanced by inhibitors of histone deacetylase. In polarized MDCK cells, both rat and human MRP2 were sorted to the apical plasma membrane. An antibody raised against the amino terminus of rat MRP2 recognized the recombinant protein on the apical surface of nonpermeabilized cells, providing direct evidence for the extracellular localization of the amino terminus of MRP2. ATP-dependent transport by recombinant human and rat MRP2 was measured with membrane vesicles from stably transfected cells. The Km value of human MRP2 was 1.0 +/- 0.1 microM for leukotriene C4 and 7.2 +/- 0.7 microM for 17beta-glucuronosyl estradiol; the Km values of human MRP1 were 0.1 +/- 0.02 microM for leukotriene C4 and 1.5 +/- 0.3 microM for 17beta-glucoronosyl estradiol. Thus, the conjugate-transporting ATPases MRP2 and MRP1 differ not only by their domain-specific localization but also by their kinetic properties. Drug resistance conferred by recombinant MRP2 was studied in MDCK and HEK-293 cells using cell viability assays. Expression of human and rat MRP2 enhanced the resistance of MDCK cells to etoposide 5.0-fold and 3.8-fold and to vincristine 2.3- and 6.0-fold, respectively. Buthionine sulfoximine reduced resistance to these drugs. Human MRP2 overexpressed in HEK-293 cells enhanced the resistance to etoposide (4-fold), cisplatin (10-fold), doxorubicin (7.8-fold), and epirubicin (5-fold). These results demonstrate that MRP2 confers resistance to cytotoxic drugs.     

HEK293 cells exposed to microcystin‐LR show reduced protein phosphatase 2A activity and more stable cytoskeletal structure when overexpressing α4 protein

Microcystin‐LR (MC‐LR) is one of the most toxic members of microcystins released by freshwater cyanobacterial. The major mechanism of MC‐LR toxicity has been attributed to its inhibition of protein phosphatases 1 (PP1) and 2A (PP2A). In our prior research, α4 protein, a regulator of PP2A, was found not only crucial for PP2A regulation but also for the overall response of HEK 293 cells encountering MC‐LR. To explore the role of α4 in MC‐LR toxicity via PP2A regulation, here, HEK 293 cells overexpressing α4 protein were exposed to MC‐LR and PP2A, cytoskeletal organization, and cytoskeleton‐related proteins were investigated. The results showed that PP2A activity decreased and PP2A/C subunit expression and phosphorylation at Tyr307 increased significantly in the group exposed to high MC‐LR. Vimentin IF became concentrated and formed perinuclear bundles. However, the assembly of actin filament and microtubules remained unchanged and the expression and phosphorylation of the cytoskeleton‐related proteins HSP27 and VASP did not increase significantly. Some of these results differ from those of our previous study in which normal HEK293 cells were exposed to MC‐LR. Our results indicate that elevated α4 expression confers some resistance to MC‐LR‐induced cytoskeletal change These new findings provide helpful insights into the mechanism of MC‐LR toxicity and the role of α4 in regulating PP2A function. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 255–264, 2017.     

A novel gene delivery system for stable transfection of thiopurine-S-methyltransferase gene in versatile cell types

A novel gene delivery system termed artificial viral particles (AVPs) containing a plasmid coding for a recombinant fusion protein of enhanced green fluorescent protein (EGFP) with thiopurine-S-methyltransferase (TPMT) was designed for transfection of selected cell lines to establish stable clones which express recombinant EGFP–TPMT protein for further in vitro investigation of toxic effect of thiopurine drugs. Various AVPs based on a complex of the cationic polymer polyethylenimine (PEI) and anionic liposomes were formulated and transfection conditions were adapted in order to transfect the human Jurkat, HepG2 and HEK 293 cell lines. An adequate transfection rate was achieved with AVP containing branched low molecular weight PEI at a PEI:DNA charge ratio of 4.5:1 and liposomes composed of DOPS, DLPE, cholesterol and an activated N-glutaryl-DOPE membrane anchor. Stably transfected clones were successfully established and expression of recombinant EGFP–TPMT in homogeneous cell populations was demonstrated by flow cytometry, fluorescence microscopy and immunoblotting. The level of the expressed protein in stable clones was highest in HEK 293, followed by HepG2 and Jurkat. The enzymatic activity of the TPMT moiety was demonstrated by decreased sensitivity to 6-thioguanine and increased sensitivity to 6-mercaptopurine in HEK 293 cells expressing EGFP–TPMT. Formulation of AVP as transfection vector succeeded in establishing human cell lines stably expressing EGFP–TPMT, thereby proving a successful delivery system and providing an initial step to enable investigation of the role of the clinically important drug metabolizing enzyme TPMT.     

Identification of two C-terminal amino acids,Ser(355) and Thr(357), required for short-term homologous desensitization of mu-opioid receptors

Our recent study suggests that a cluster of Ser/Thr residues (T(354)S(355)S(356)T(357)) at the intracellular carboxyl tail of rat mu-opioid receptor (MOR1) is required for the development of short-term homologous desensitization. To investigate the functional role played by individual serine or threonine residue of this (TSST) cluster in the agonist-induced mu-opioid receptor desensitization, point mutant (T354A), (S355A), (S356A) and (T357A) mu-opioid receptors were prepared and stably expressed in human embryonic kidney 293 cells (HEK 293 cells). Similar to wild-type mu-opioid receptors, mutant (T354A) and (S356A) mu-opioid receptors stably expressed in HEK 293 cells developed homologous desensitization after 30 min pretreatment of DAMGO ([D-Ala(2),N-methyl-Phe(4),Gly-ol(5)]enkephalin), a specific mu-opioid receptor agonist. Substituting Ser(355)or Thr(357) with alanine resulted in a significant attenuation of agonist-induced mu-opioid receptor desensitization. In HEK 293 cells stably expressing double mutant (S355A/T357A) mu-opioid receptors, DAMGO pretreatment failed to significantly affect the efficacy and potency by which DAMGO inhibits forskolin-stimulated adenylyl cyclase activity. Consistent with the general belief that agonist-induced phosphorylation of guanine nucleotide binding protein (G protein)-coupled receptors is involved in homologous desensitization. Treating HEK 293 cells expressing wild-type mu-opioid receptors with 5 microM DAMGO for 30 min induced the receptor phosphorylation. Mutation of Ser(355) and Thr(357) also greatly impaired DAMGO-induced mu-opioid receptor phosphorylation. These results suggest that two C-terminal amino acids, Ser(355) and Thr(357), are required for short-term homologous desensitization and agonist-induced phosphorylation of mu-opioid receptors expressed in HEK 293 cells.     

ABCB1(G1199A)基因稳定转染HEK293细胞株的建立

目的:将ABCB1(G1199A)突变型和野生型基因分别转入HEK293细胞,建立P-糖蛋白稳定表达细胞株。方法:以野生型ABCB1基因的cDNA为模板质粒,采用PCR点突变的方法合成突变的ABCB1(1199A)基因;在慢病毒的介导下,将实验室构建的pLVX-ABCB1-PGK-Puro和pLVX-ABCB1-mut-PGK-Puro重组质粒转染HEK293细胞,经嘌呤霉素筛选稳定表达的细胞;通过流式细胞术检测P-糖蛋白的表达、RT-PCR检测ABCB1基因的转录水平、CCK-8方法测定外源基因对HEK293细胞增殖的影响。结果:流式细胞术证实P-糖蛋白在HEK193细胞中稳定过表达,RT-PCR确定转染细胞中存在ABCB1(G1199A)基因mRNA的过表达,成果获得ABCB1(G1199A)野生型和突变型基因的稳定表达细胞株。结论:成功建立两种ABCB1(G1199A)稳定表达的HEK293细胞株,为该酶的功能研究奠定了基础。     

Up-regulation of transporters of the MRP family by drugs and toxins

Expression of a variety of ABC efflux pumps including certain conjugate transporters of the multidrug resistance protein (MRP) subfamily is inducible in primate and rodent tissues, and in a variety of cell lines and primary cells in culture. In human cell lines (HepG2, MCF-7), we studied the inducibility of MRPs 1–5. Similar to the rat mrp2 gene, human mrp2 is inducible by the chemical carcinogen 2-AAF, the chemotherapeutic drug cisplatin and the barbiturate phenobarbital, as demonstrated in Northern and Western Blots. Furthermore, the antibiotic rifampicin was identified as MRP2 inducer in HepG2 cells. MRP1 and 4 mRNAs being expressed in human liver at a very low level could not be detected in HepG2 cells after treatment with various agents. However, MRP3 and 5 mRNAs were detected in addition to MRP2 and their expression was found to be increased by 2-AAF, cisplatin and rifampicin. MRP1 expression was studied in MCF-7 cells where the chemotherapeutic drug vinblastine and tert-butyl hydroquinone but not the MRP2 inducing agents described above acted as inducers.     

Characterization of renal tubular apical efflux of zonampanel, an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist, in humans

1. Zonampanel, a novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist, is mainly excreted unchanged via renal tubular secretion. The renal apical transport transport of zonampanel was examined in this study using HEK293 cells expressing human organic anion transporter 4 (OAT4/SLC22A11), and membrane vesicles prepared from Sf-9 insect cells expressing human multidrug resistance-associated protein 2 (MRP2/ABCC2), MRP4 (ABCC4), and breast cancer resistance protein (BCRP/ABCG2). 2. Glutaric acid, a model dicarboxylate, trans-stimulated the uptake of [(14)C]zonampanel by OAT4, suggesting that zonampanel was transported by OAT4 via an exchange with dicarboxylate. Considering the endogenous dicarboxylate gradient, OAT4 seems to transport zonampanel in the direction of reabsorption rather than secretion. For MRP2, MRP4, and BCRP, zonampanel selectively inhibited the activity of MRP4 (K(i) = 41.3 microM). Marked transport of [(14)C]zonampanel was observed only for MRP4 (K(m) = 33.7 microM). 3. In conclusion, the data indicate that MRP4 was the apical efflux transporter that contributed to the active renal tubular secretion of zonampanel in humans, in concert with the apical reabsorption transporter OAT4 and basolateral uptake transporters.     

Lapatinib and erlotinib are potent reversal agents for MRP7 (ABCC10)-mediated multidrug resistance

In recent years, a number of TKIs (tyrosine kinase inhibitors) targeting epidermal growth factor receptor (EGFR) family have been synthesized and some have been approved for clinical treatment of cancer by the FDA. We recently reported a new pharmacological action of the 4-anilinoquinazoline derived EGFR TKIs, such as lapatinib (Tykerb^®) and erlotinib (Tarceva^®), which significantly affect the drug resistance patterns in cells expressing the multidrug resistance (MDR) phenotype. Previously, we showed that lapatinib and erlotinib could inhibit the drug efflux function of P-glycoprotein (P-gp, ABCB1) and ABCG2 transporters. In this study, we determined if these TKIs have the potential to reverse MDR due to the presence of the multidrug resistance protein 7 (MRP7, ABCC10). Our results showed that lapatinib and erlotinib dose-dependently enhanced the sensitivity of MRP7-transfected HEK293 cells to several established MRP7 substrates, specifically docetaxel, paclitaxel, vinblastine and vinorelbine, whereas there was no or a less effect on the control vector transfected HEK293 cells. [³H]-paclitaxel accumulation and efflux studies demonstrated that lapatinib and erlotinib increased the intracellular accumulation of [³H]-paclitaxel and inhibited the efflux of [³H]-paclitaxel from MRP7-transfected cells but not in the control cell line. Lapatinib is a more potent inhibitor of MRP7 than erlotinib. In addition, the Western blot analysis revealed that both lapatinib and erlotinib did not significantly affect MRP7 expression. We conclude that the EGFR TKIs, lapatinib and erlotinib reverse MRP7-mediated MDR through inhibition of the drug efflux function, suggesting that an EGFR TKI based combinational therapy may be applicable for chemotherapeutic practice clinically.     

Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

RNA interference (RNAi) is useful for selective gene silencing. Cytochrome P450 3A4 (CYP3A4), which metabolizes approximately 50% of drugs in clinical use, plays an important role in drug metabolism. In this study, we aimed to develop a short hairpin RNA (shRNA) to modulate CYP3A4 expression. Three new shRNAs (S1, S2 and S3) were designed to target the coding sequence (CDS) of CYP3A4, cloned into a shRNA expression vector, and tested in different cells. The mixture of three shRNAs produced optimal reduction (55%) in CYP3A4 CDS-luciferase activity in both CHL and HEK293 cells. Endogenous CYP3A4 expression in HepG2 cells was decreased about 50% at both mRNA and protein level after transfection of the mixture of three shRNAs. In contrast, CYP3A5 gene expression was not altered by the shRNAs, supporting the selectivity of CYP3A4 shRNAs. In addition, HepG2 cells transfected with CYP3A4 shRNAs were less sensitive to Ginkgolic acids, whose toxic metabolites are produced by CYP3A4. These results demonstrate that vector-based shRNAs could modulate CYP3A4 expression in cells through their actions on CYP3A4 CDS, and CYP3A4 shRNAs may be utilized to define the role of CYP3A4 in drug metabolism and toxicity.KEY WORDS: RNAi, Cytochrome P450, CYP3A4, shRNA, Chemosensitivity     

Carrier-mediated transport of quercetin conjugates: involvement of organic anion transporters and organic anion transporting polypeptides

Flavonoids modulate cell signaling and inhibit oxidative enzymes. After oral consumption, they circulate in human plasma as amphiphilic glucuronide or sulfate conjugates, but it is unknown how these physiological metabolites permeate into cells. We examined the mechanisms of uptake of these conjugates into hepatocellular carcinoma (HepG2) cells, and found that uptake of quercetin-3'-O-sulfate was saturable and temperature-dependent, indicating the involvement of carrier-mediated transport. Quercetin-3-O-glucuronide was taken up predominantly via passive diffusion in these cells. Quantitative real-time PCR analysis showed high expression of OATP4C1, followed by OAT2, OAT4 and low expression of OATP1B1 in HepG2 cells, and addition of inhibitors of OATs and OATPs resulted in a significant reduction in quercetin-3'-O-sulfate uptake. The accumulation of quercetin-3'-O-sulfate was further evaluated in HEK293 cells expressing OAT2, OAT4 and OATP4C1. Uptake of quercetin-3'-O-sulfate was 2.3- and 1.4-fold higher in cells expressing OAT4 and OATP4C1 at pH 6.0, respectively, than in control HEK293 cells. siRNA knockdown of OATP4C1 expression in HepG2 cells reduced uptake of quercetin-3'-O-sulfate by ～40%. This study highlights a role for OATs and OATPs in the cellular uptake of biologically active flavonoid conjugates.     

Inducible expression and pharmacology of the human excitatory amino acid transporter 2 subtype of L-glutamate transporter

1. In this study we have examined the use of the ecdysone-inducible mammalian expression system (Invitrogen) for the regulation of expression of the predominant L-glutamate transporter EAAT2 (Excitatory Amino Acid Transporter) in HEK 293 cells. 2. HEK 293 cells which were stably transformed with the regulatory vector pVgRXR (EcR 293 cells) were used for transfection of the human EAAT2 cDNA using the inducible vector pIND and a clone designated HEK/EAAT2 was selected for further characterization. 3. Na+-dependent L-glutamate uptake activity (3.2 pmol min-1 mg-1) was observed in EcR 293 cells and this was increased approximately 2 fold in the uninduced HEK/EAAT2 cells, indicating a low level of basal EAAT2 activity in the absence of exogenous inducing agent. Exposure of HEK/EAAT2 cells to the ecdysone analogue Ponasterone A (10 microM for 24 h) resulted in a > or = 10 fold increase in the Na+-dependent activity. 4. L-glutamate uptake into induced HEK/EAAT2 cells followed first-order Michaelis-Menten kinetics and Eadie-Hofstee transformation of the saturable uptake data produced estimates of kinetic parameters as follows; Km 52.7+/-7.5 microM, Vmax 3.8+/-0.9 nmol min-1 mg-1 protein. 5. The pharmacological profile of the EAAT2 subtype was characterized using a series of L-glutamate transport inhibitors and the rank order of inhibitory potency was similar to that described previously for the rat homologue GLT-1 and in synaptosomal preparations from rat cortex. 6. Addition of the EAAT2 modulator arachidonic acid resulted in an enhancement (155+/-5% control in the presence of 30 microM) of the L-glutamate transport capacity in the induced HEK/EAAT2 cells. 7. This study demonstrates that the expression of EAAT2 can be regulated in a mammalian cell line using the ecdysone-inducible mammalian expression system.     

ABCC2 (1249G > A) polymorphism implicates altered transport activity for sorafenib

1.?Multidrug resistance-associated protein 2 (MRP2), encoded by the ABCC2 gene, is an efflux transporter of several endogenous substrates and xenobiotics. Here, we investigated whether the 1249G?>?A (rs2273697) polymorphism in ABCC2 affects the ability of MRP2 to pump the multi-tumor drug sorafenib out of cells.

2.?Human embryonic kidney 293 (HEK 293) cell lines transfected with ABCC2-1249G and ABCC2-1249A were used to assess the sensitivity and accumulation to sorafenib. The isolated MRP2 were applied to estimate the ATPase activity.

3.?The HEK293 cell line overexpressing the ABCC2 1249A allele showed a significantly higher 50% inhibitory concentration (IC₅₀) than a cell line overexpressing ABCC2-1249G or a non-overexpressing control cell line. Intracellular accumulation of sorafenib was much lower in ABCC2-1249A cells than in ABCC2-1249G cells expressing comparable levels of MRP2. Isolated ABCC2-1249A protein showed higher ATPase activity than ABCC2-1249G protein.

4.?Our results suggest that the ABCC2 polymorphism 1249G?>?A increases the ATPase activity of MRP2, leading to greater efflux of sorafenib.     

Cepharanthine is a potent reversal agent for MRP7(ABCC10)-mediated multidrug resistance

Multidrug resistance protein 7 (MRP7; ABCC10) is an ABC transporter that confers resistance to anticancer agents such as the taxanes. We previously reported that several inhibitors of P-gp and MRP1 were able to inhibit the in vitro transport of E₂17βG by MRP7 in membrane vesicles transport assays. However, compounds that are able to reverse MRP7-mediated cellular resistance have not been identified. In this study, we examined the effects of cepharanthine (6′,12′-dimethoxy-2,2′-dimethyl-6,7-[methylenebis(oxy)]oxyacanthan), an herbal extract isolated from Stephania cepharantha Hayata, to reverse paclitaxel resistance in MRP7-transfected HEK293 cells. Cepharanthine, at 2 μM, completely reversed paclitaxel resistance in MRP7-transfected cells. In contrast, the effect of cepharanthine on the parental transfected cells was significantly less than that on the MRP7-transfected cells. In addition, cepharanthine significantly increased the accumulation of paclitaxel in MRP7-transfected cells almost to the level of control cells in the absence of cepharanthine. The efflux of paclitaxel from MRP7-transfected cells was also significantly inhibited by cepharanthine. The ability of cepharanthine to inhibit MRP7 was analyzed in membrane vesicle assays using E₂17βG, an established substrate of MRP7, as a probe. E₂17βG transport was competitively inhibited by cepharanthine with a K_i value of 4.86 μM. These findings indicate that cepharanthine reverses MRP7-mediated resistance to paclitaxel in a competitive manner.     

Cloning, expression, and functional analysis of human dopamine D1 receptors

AIM: To construct an HEK293 cell line stably expressing human dopamine D1 receptor (D1R). METHODS: cDNA was amplified by RT-PCR using total RNA from human embryo brain tissue as the template. The PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3.1. The cloned D1R cDNA was sequenced and stably expressed in HEK293 cells. Expression of D1R in HEK293 cells was monitored by the [3H]SCH23390 binding assay. The function of D1R was studied by the cAMP accumulation assay, CRE-SEAP reporter gene activity assay, and intracellular calcium assay. RESULTS: An HEK293 cell line stably expressing human D1R was obtained. A saturation radioligand binding experiment with [3H]SCH23390 demonstrated that the Kd and Bmax values were 1.5+/-0.2 nmol/L and 2.94+/-0.15 nmol/g of protein, respectively. In the [3H]SCH23390 competition assay, D1R agonist SKF38393 displaced [3H]SCH23390 with an IC50 value of 2.0 (1.5-2.8) micromol/L. SKF38393 increased the intracellular cAMP level and CRE-SEAP activity through D1R expressed in HEK293 cells in a concentration-dependent manner with an EC50 value of 0.25 (0.12-0.53) micromol/L and 0.39 (0.27-0.57) micromol/L at 6 h/0.59 (0.22-1.58) micromol/L at 12 h, respectively. SKF38393 also increased the intracellular calcium level in a concentration-dependent manner with EC50 value of 27 (8.6-70) nmol/L. CONCLUSION: An HEK293 cell line stably expressing human D1R was obtained successfully. The study also demonstrated that the CRE-SEAP activity assay could be substituted for the cAMP accumulation assay for measuring increase in cAMP levels. Thus, both intracellular calcium measurements and the CRE-SEAP activity assay are suitable for high-throughput screening in drug research.     

Cell-type specific calcium signaling by corticotropin-releasing factor type 1 (CRF1) and 2a (CRF2(a)) receptors: phospholipase C-mediated responses in human embryonic kidney 293 but not SK-N-MC neuroblastoma cells

The human corticotropin-releasing factor (hCRF) receptors CRF1 and CRF2(a) couple to the Gs protein. It has been postulated that CRF receptors may also signal through phospholipase C (PLC). To test this hypothesis, binding and signaling properties were determined for both receptor subtypes stably expressed in human embryonic kidney 293 (HEK293) and human SK-N-MC neuroblastoma cells. CRF receptors were highly expressed and strongly coupled to Gs in HEK293 and SK-N-MC cells. However, when the calcium mobilization pathway was investigated, marked differences were observed. In SK-N-MC cells, neither CRF receptor stimulated calcium mobilization in the fluorometric imaging plate reader (FLIPR) assay, whereas activation of orexin type 1 and 2 receptors stably expressed in SK-N-MC cells revealed robust calcium responses. In contrast, intracellular calcium was strongly mobilized by agonist stimulation of hCRF1 and hCRF2(a) receptors in HEK293 cells. In HEK293 cells, potency rank orders for calcium and cAMP responses were identical for both receptors, despite a rightward shift of the dose-response curves. Complete inhibition of calcium signaling of both hCRF1 and hCRF2(a) receptors was observed in the presence of the PLC inhibitor U-73,122 whereas ryanodine, an inhibitor of calcium release channels and the protein kinase A inhibitor Rp-cAMPS were ineffective. Finally, CRF agonists produced a small but significant stimulation of inositol 1,4,5-triphosphate (IP3) accumulation in hCRF1-and hCRF2(a)-transfected HEK293 cells. These data clearly show that phospholipase C-mediated signaling of CRF receptors is dependent upon the cellular background and that in HEK293 cells human CRF receptors robustly respond in the FLIPR format.