ABCB1 G1199A polymorphism and ovarian cancer response to paclitaxel

P-glycoprotein (P-gp), encoded by the ABCB1 gene, confers multi-drug resistance to a variety of antineoplastic agents, for example, paclitaxel. Recently, the G1199T/A polymorphism in the ABCB1 gene was shown to be important for the function of P-gp as well as for the resistance to several chemotherapeutic agents in vitro. We analyzed the allelic distribution of the G1199T/A and other polymorphisms in exons 11 and 12 of the ABCB1 gene in ovarian cancer patients treated with paclitaxel and carboplatin in order to evaluate their predictive value in vivo. The SNPs C1236T, G1199T/A, and A1308G were determined using Pyrosequencing in 51 patients with advanced ovarian cancer and correlated to the progression free survival. The G1199T/A SNP was found to affect the progression free survival. Although only two heterozygous (G/A) patients were found their mean progression free survival was only 2 months as compared to 19 months for the wild-type patients. This is in accordance with the higher resistance for the 1199A genetic variant found in vitro. Genotyping of the ABCB1 gene may be important for determining the tumor resistance to paclitaxel and provide useful information for individualized therapy.     

Inhibition of P-Glycoprotein by D-α-Tocopheryl Polyethylene Glycol 1000 Succinate (TPGS)

Purpose. To investigate whether d--tocopheryl polyethylene glycol 1000 succinate (TPGS) functions as an inhibitor of P-glycoprotein (P-gp), the multidrug resistance transporter. Methods. Two assays were used to measure the function of TPGS on P-gp function. First, we examined the ability of TPGS to modulate the cytotoxicity of established, cytotoxic, P-glycoprotein substrates. Parental NIH 3T3 cells and NIH 3T3 cells transfected with the human MDR1 cDNA (G185) were exposed to doxorubicin, paclitaxel, colchicine, vinblastine and 5-fluorouracil (5FU) in the presence or absence of TPGS. Cytotoxicity was assessed with the MTT assay. Second, polarized transport of the P-gp substrates rhodamine 123 (R123), paclitaxel and vinblastine was measured using the human intestinal HCT-8 and Caco-2 cell lines grown in Transwell dishes. Drug flux was measured by liquid scintillation counting or fluorescence spectroscopy of the media. Results. G185 cells were 27–135 fold more resistant to the cytotoxic drugs doxorubicin, vinblastine, colchicine and paclitaxel than the parental NIH 3T3 cells. In contrast 5FU, which is not a P-gp substrate, is equally cytotoxic to parental and G185 cells. Co-administration of TPGS enhanced the cytotoxicity of doxorubicin, vinblastine, paclitaxel, and colchicine in the G185 cells to levels comparable to the parental cells. TPGS did not increase the cytotoxicity of 5FU in the G185 cells. Using a polarized epithelial cell transport assay, TPGS blocked P-gp mediated transport of Rl 23 and paclitaxel in a dose responsive manner. Conclusions. These data demonstrate that TPGS acts as a reversal agent for P-glycoprotein mediated multidrug resistance and inhibits P-gp mediated drug transport. These results suggest that enhanced oral bioavailability of drugs co-administered with TPGS may, in part, be due to inhibition of P-glycoprotein in the intestine.     

ABCB1(1199G>A)基因多态性对多西他赛转运影响的分子机制

目的：在体外研究ABCB1（1199G>A）基因多态性对多西他赛转运影响的分子机制。方法：将ABCB1（1199G>A）野生型和突变型基因分别导入HEK293细胞,研究2种重组细胞株对多西他赛的摄取以及跨膜转运的差异。结果：在细胞毒性分析中,ABCB1_1199A/mut细胞对多西他赛表现出更强的耐药性。多西他赛在2种重组细胞中的含量均显著性的低于对照组细胞,证实了多西他赛是由P-糖蛋白介导转运的,并且在ABCB1_1199A/mut细胞中跨膜转运更高。ABCB1_1199A/mut细胞介导转运多西他赛的P-糖蛋白的活性较ABCB1_1199G/wt细胞的更强。结论：ABCB1（1199G>A）基因多态性能够显著性影响P-糖蛋白转运多西他赛的能力,由ABCB1突变型基因编码的P-糖蛋白能够更有效的转运多西他赛。因此ABCB1（1199G>A）基因多态性可能会影响P-糖蛋白的活性,并对药物的分布和消除产生影响,从而影响药物的治疗作用。     

A Novel MDR1 GT1292-3TG (Cys431Leu) Genetic Variation and Its Effect on P-glycoprotein Biologic Functions

P-glycoprotein (P-gp) is a membrane-bound transporter protein that is encoded by the human multidrug resistance gene MDR1 (ABCB1). P-gp recognizes a wide range of xenobiotics, is pivotal in mediating cancer drug resistance, and plays an important role in limiting drug penetration across the blood–brain barrier. MDR1 genetic variation can lead to changes in P-gp function and may have implications on drug pharmacokinetics. We have identified a novel MDR1_GT1292-3TG (Cys431Leu) genetic variation through systematic profiling of subjects with leukemia. The cellular and transport function of this variation was investigated with recombinant human embryonic kidney cells expressing MDR1. Compared with the wild type, MDR1_GT1292-3TG recombinant cells exhibited a lower drug resistance phenotype for a panel of chemotherapeutic agents. When compared with wild type, MDR1_GT1292-3TG recombinant cells exposed exhibited a 75% decrease in IC₅₀ for doxorubicin (162.6 ± 17.4 to 37.9 ± 2.6 nM) and a 50% decrease in IC₅₀ for paclitaxel (155.7 ± 27.5 to 87.7 ± 9.2 nM), vinblastine (128.0 ± 15.9 to 65.9 ± 5.1 nM), and vincristine (593.7 ± 61.8 to 307.3 ± 17.0 nM). The effects of the Cys431Leu variation, due to MDR1_GT1292-3TG nucleotide transition, on P-gp-dependent intracellular substrate accumulation appeared to be substrate dependent where doxorubicin, vinblastine, and paclitaxel exhibit an increased accumulation (p < 0.05), while verapamil and Hoechst33342 exhibit a decreased intracellular concentration compared with wild type (p < 0.05). Collectively, these data suggest MDR1_GT1292-3TG variation of P-gp may reduce drug resistance and that subjects with this genotype undergoing chemotherapy with drugs that are transported by P-gp could potentially be more responsive to therapy than those with MDR1 wild-type genotype.Key words: ABC transporter, drug resistance, genetic variation, MDR1, P-glycoprotein, polymorphism, transporter     

不同性激素对P-糖蛋白表达水平及甲磺酸伊马替尼转运的影响

目的：探讨在ABCB1（1199G/A）重组细胞模型中不同性激素对P-糖蛋白（P-gp）表达水平及甲磺酸伊马替尼转运的影响。方法：将构建的ABCB1_1199G/wt和ABCB1_1199A/mut重组质粒稳定转染入HEK293细胞中,采用RT-PCR和Western Blot分别检测不同性激素对P-gp mRNA和蛋白的表达水平;采用HPLC检测性激素对甲磺酸伊马替尼累积量的影响。结果：浓度梯度的性激素（雌酮、雌三醇雌二酮、黄体酮和睾酮）刺激ABCB1_1199G/wt 和ABCB1_1199A/mut重组细胞后,雌酮和雌三醇能刺激P-gp mRNA和蛋白表达水平,且雌三醇上调ABCB1_1199A/mut mRNA的表达水平显著高于ABCB1_1199G/wt;雌酮和雌三醇可降低甲磺酸伊马替尼在细胞内的累积量,产生对伊马替尼的耐药性。结论：雌酮和雌三醇增加了细胞内P-gp的表达,并促进P-gp介导甲磺酸伊马替尼的转运能力。     

Effects of photoactivated 5-aminolevulinic acid hexyl ester on MDR1 over-expressing human uterine sarcoma cells

The role of multi-drug resistance (MDR1) and its product, P-glycoprotein (P-gp) on 5-aminolevulinic acid hexyl ester (Hexyl-ALA) mediated phototoxicity was determined with human uterine sarcoma cells, MES-SA control and MDR1 expressing MES-SA-Dx5. MDR1 expression reduced intracellular levels of the Hexyl-ALA metabolite, protoporphyrin IX (PpIX) to a limited degree and could be reversed with a P-gp inhibitor, verapamil. P-gp expression also reduced Hexyl-ALA photosensitivity. More importantly, photoactivated Hexyl-ALA reduced at the mRNA and protein levels without altering housekeeping GAPDH mRNA. These findings suggest that Hexyl-ALA could be used to selectively reduce P-gp expression in overcoming resistance to chemotherapy agents such as doxorubicin and paclitaxel.     

Impacts of ABCB1 (G1199A) polymorphism on resistance,uptake, and efflux to steroid drugs

1.?P-glycoprotein (P-gp) substrates, including steroid drugs, involve in the inter-individual differences in resistant phenotype. This study was performed to evaluate whether G1199A polymorphism in ABCB1 gene can alter the sensitivity, accumulation, and transepithelial efflux to steroids in LLC-PK1 cells.

2.?The stable recombinant LLC-PK1 cell lines transfected with ABCB1 1199G and ABCB1 1199A were used to assess the sensitivity, accumulation, and transepithelial permeability to steroids.

3.?The cells transfected with 1199A allele displayed stronger resistance to aldosterone, dexamethasone, and cortisol (2.5-, 2.0-, and 1.6-fold, respectively) than cells overexpressing 1199G allele, while the two types of recombinant cells showed a similar resistance to corticosterone. The accumulation of aldosterone, dexamethasone, and cortisol in recombinant 1199A cells were significantly decreased when compared to 1199G cells (2.9-, 4.4-, and 3.9-fold, respectively). The net efflux ratios of P-gp-mediated aldosterone, dexamethasone, and cortisol in cells expressing 1199A allele were apparently greater than cells transfected with 1199G allele (3.3-, 3.5-, and 4.0-fold, respectively).

4.?The impacts of ABCB1 (G1199A) single nucleotide polymorphism on the efflux of P-gp substrates presented as drug-specific. Overall, the transport ability of P-gp-dependent steroid drugs in recombinant model overexpressing variant 1199A allele is stronger in comparison to cells overexpressing wild-type 1199G allele. Therefore, the ABCB1 (G1199A) polymorphism may affect effective steroids concentration in target cells by regulating the drug transport and distribution.     

Flavonoid dimers as bivalent modulators for P-glycoprotein-based multidrug resistance: synthetic apigenin homodimers linked with defined-length poly(ethylene glycol) spacers increase drug retention and enhance chemosensitivity in resistant cancer cells

Much effort has been spent on searching for better P-glycoprotein- (P-gp-) based multidrug resistance (MDR) modulators. Our approach was to target the binding sites of P-gp using dimers of dietary flavonoids. A series of apigenin-based flavonoid dimers, linked by poly(ethylene glycol) chains of various lengths, have been synthesized. These flavonoid dimers modulate drug chemosensitivity and retention in breast and leukemic MDR cells with the optimal number of ethylene glycol units equal to 2-4. Compound 9d bearing four ethylene glycol units increased drug accumulation in drug-resistant cells and enhanced cytotoxicity of paclitaxel, doxorubicin, daunomycin, vincristine, and vinblastine in drug-resistant breast cancer and leukemia cells in vitro, resulting in reduction of IC50 by 5-50 times. This compound also stimulated P-gp's ATPase activity by 3.3-fold. Its modulating activity was presumably by binding to the substrate binding sites of P-gp and disrupting drug efflux.     

Reversal of P-glycoprotein and multidrug-resistance protein-mediated drug resistance in KB cells by 5-O-benzoylated taxinine K

A newly synthesized taxoid originally from the Japanese yew Taxus cuspidata, 5-O-benzoylated taxinine K (BTK) was examined for its ability to reverse P-glycoprotein (P-gp) and multidrug resistance protein (MRP)-mediated multidrug resistance. BTK reversed the resistance to paclitaxel, doxorubicin (ADM), and vincristine (VCR) of KB-8-5 and KB-C2 cells that overexpress P-gp by directly interacting with P-gp. BTK also moderately reversed the resistance to ADM of KB/MRP cells that overexpress MRP. However, BTK neither inhibited the transporting activity of MRP nor reduced intracellular glutathione levels in KB/MRP cells. BTK shifted the distribution of ADM in KB/MRP cells from punctate cytoplasmic compartments to the nucleoplasm and cytoplasm by inhibiting acidification of cytoplasmic organelles. These two functions of BTK make it able to reverse both P-gp- and MRP-mediated MDR. BTK in combination with ADM should be useful for treating patients with tumors that overexpress both P-gp and MRP.     

ABCB1（1199G/A）基因多态性对甲磺酸伊马替尼转运的分子机制

摘 要 目的：探讨P 糖蛋白（P-gp）活性和所介导的甲磺酸伊马替尼胞内累积量及药物跨膜渗透性的影响。 方法: 将构建的ABCB1 1199G/wt和1199A/mut重组质粒分别转染HEK293细胞,利用RT-PCR法考察细胞中P-gp的mRNA表达水平。CCK-8法检测药物对细胞的毒性,高效液相色谱法（HPLC）检测细胞中药物浓度和胞内累积量,跨膜电阻实验考察药物跨膜渗透率,评价P-gp活性对药物转运的作用。结果: 细胞毒性实验表明,转染细胞内的药物浓度均低于对照组,证明P-gp具有介导药物转出胞外的作用。HPLC和跨膜电阻实验表明,与野生型ABCB1(1199G)细胞相比,突变型ABCB1(1199A)细胞抗甲磺酸伊马替尼的作用更强,P-gp对介导甲磺酸伊马替尼外排转运的作用较强且药物的跨膜渗透性也相应较强。结论:实验表明ABCB1（1199G/A）位点突变导致其编码蛋白P-gp活性改变,该位点多态性会导致甲磺酸伊马替尼清除率增加,抑制了药物在靶细胞中有效药物浓度,因此临床上应对ABCB1基因进行分型,指导甲磺酸伊马替尼的个体化用药。     

Effects of MDR1 1236C > T-2677G > T-3435C > T polymorphisms on the intracellular accumulation of tacrolimus,cyclosporine A,sirolimus and everolimus

Abstract

1. Overexpression of P-glycoprotein (P-gp, encoded by MDR1) mediates resistance to multiple immunosuppressors. Several common MDR1 variants (1236C?>?T, 2677G?>?T, 3435C?>?T) impact the efflux activity of P-gp-mediated substrates. We assessed the effect of these polymorphisms on the sensitivity, intracellular accumulation, and efflux of tacrolimus, cyclosporine A, sirolimus and everolimus in transfected LLC-PK1 cells.

2. LLC-PK1 cell lines were transfected with empty vector (pcDNA3.1) and recombinant MDR1_T-T-T, MDR1_C-T-T, MDR1_C-G-T and MDR1_C-G-C vectors, respectively and further screened in the presence of puromycin. The IC₅₀ values, intracellular accumulation, and apparent permeability ratios of tacrolimus, cyclosporine A, sirolimus and everolimus were evaluated.

3. MDR1 overexpression increased the resistance of LLC-PK1 cells to tacrolimus, cyclosporine A, sirolimus and everolimus. The resistance of cells expressing MDR1_C-G-C wild-type haplotypes to tacrolimus were increased compared to MDR1_T-T-T, MDR1_C-T-T, MDR1_C-G-T variant haplotypes. The efflux ability of P-gp-mediated tacrolimus in cells transfected with MDR1_C-G-C was higher than cells overexpressing MDR1_T-T-T, MDR1_C-T-T, MDR1_C-G-T variant haplotypes. In addition, the resistance of cells expressing MDR1_C-G-C wild-type haplotypes to sirolimus were increased compared to MDR1_C-T-T, MDR1_C-G-T variant haplotypes. The efflux ability of P-gp-mediated sirolimus in cells overexpressing MDR1_C-G-C was higher than cells transfected with MDR1_C-T-T, MDR1_C-G-T variant haplotypes.

4. These findings indicate that wild-type MDR1 exports tacrolimus and sirolimus more efficiency than the MDR1_T-T-T, MDR1_C-T-T, MDR1_C-G-T variant protein. This observation indicates that 1236C?>?T, 2677G?>?T, 3435C?>?T variant haplotypes drastically decrease the efflux ability of P-gp-mediated tacrolimus and sirolimus in a substrate-specific manner.     

Effect of MDR1 gene polymorphism on progression of end-stage renal disease

AIM: P-glycoprotein is localized at the apical brush-border membrane of the proximal renal tubule and functions as extruding toxins and xenobiotics out of cells. The difference of P-glycoproteinos function resulted from single nucleotide polymorphisms in MDR1 (multidrug resistance gene encoding for P-gp) and may be the cause of interindividual differences in susceptibility to end-stage renal disease (ESRD). The purpose of this study is to compare the genotype frequency of C3435T and G1199A polymorphisms in MDR1 between ESRD patients and healthy controls in the Chinese population to determine whether the alteration of the P-gp function is associated with ESRD. METHODS: Two hundred and eighty-four healthy Chinese controls and 244 Chinese patients with ESRD were involved in this study. Allele specific PCR and polymerase chain reaction-restriction fragment length polymorphism assay were used to determine the genotype MDR1 G1199A and C3435T, respectively. RESULTS: The genotype distribution of 3435CC, 3435CT, and 3435TT were 0.35, 0.50, and 0.15, respectively, in the control group and 0.38, 0.47, and 0.15 in the group with the ESRD patients. No variant allele 1199G>A was found in any of the patients. The value of serum creatinine for genotypes 3435CC, 3435CT, and 3435TT in the ESRD patients were 753.8+/-276.0 mumol/L, 849.6+/-342.2 micromol/L, and 987.0+/-512.0 micromol/L, respectively. The difference between 3435TT and 3435CC reached statistical significance (P<0.05). CONCLUSION: The low expression of P-glycoprotein was not the etiological factor for the kidney disease, but it may contribute to the progression of ESRD and affect the severity. Chinese people do not carry the 1199G>A variant allele. More studies are needed to clarify the cause and interindividual differences in the susceptibility for the risk of ESRD.     

Development and characterization of P-glycoprotein 1 (Pgp1, ABCB1)-mediated doxorubicin-resistant PLHC-1 hepatoma fish cell line

The development of the multidrug resistance (MDR) phenotype in mammals is often mediated by the overexpression of the P-glycoprotein1 (Pgp, ABCB1) or multidrug resistance-associated protein (MRP)-like ABC transport proteins. A similar phenomenon has also been observed and considered as an important part of the multixenobiotic resistance (MXR) defence system in aquatic organisms. We have recently demonstrated the presence of ABC transporters in the widely used in vitro fish model, the PLHC-1 hepatoma cell line. In the present study we were able to select a highly resistant PLHC-1 sub-clone (PLHC-1/dox) by culturing the wild-type cells in the presence of 1 microM doxorubicin. Using quantitative PCR a 42-fold higher expression of ABCB1 gene was determined in the PLHC-1/dox cells compared to non-selected wild-type cells (PLHC-1/wt). The efflux rates of model fluorescent Pgp1 substrates rhodamine 123 and calcein-AM were 3- to 4-fold higher in the PLHC-1/dox in comparison to the PLHC-1/wt cells. PLHC-1/dox were 45-fold more resistant to doxorubicin cytotoxicity than PLHC-1/wt. Similarly to mammalian cell lines, typical cross-resistance to cytotoxicity of other chemotherapeutics such as daunorubicin, vincristine, vinblastine, etoposide and colchicine, occurred. Furthermore, cyclosporine A, verapamil and PSC833, specific inhibitors of Pgp1 transport activity, completely reversed resistance of PLHC-1/dox cells to all tested drugs, resulting in EC50 values similar to the EC50 values found for PLHC-1/wt. In contrast, MK571, a specific inhibitor of MRP type of efflux transporters, sensitized PLHC-1/dox cells, neither to doxorubicin, nor to any other of the chemotherapeutics used in the study. These data demonstrate for the first time that a specific Pgp1-mediated doxorubicin resistance mechanism is present in the PLHC-1 fish hepatoma cell line. In addition, the fact that low micromolar concentrations of specific inhibitors may completely reverse a highly expressed doxorubicin resistance points to the fragility of Pgp1-mediated MXR defence mechanism in fish.     

P-Glycoprotein mediated resistance to 5′-nor-anhydro-vinblastine (Navelbine®)

Summary Navelbine^? (NVB, vinorelbine tartrate) is a semisyntheticVinca alkaloid in which the catharanthine moiety contains an eight-membered ring in place of the nine-membered ring that is present in all naturally occurring members of the vinblastine group. This modification selectively reduces interaction with anoxalvs mititotic microtubules and may account for the lower neurotoxicity with improved antitumor activity that has been observed in clinical trials with breast, lung and ovarian cancer. We were interested in whether the structural modification in NVB would also alter the drug resistance profile. Specifically, our aim was to determine whether NVB, like vinblastine (VBL), participates in P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). NVB-resistant, murine P388 cells (P388/NVB), were derivedin vivo and used in conjunction with a battery of drug-resistant P388 cell linesin vivo and murine and human tumor cell linesin vitro to develop a resistance profile for NVB. P388/NVB bells were cross-resistant to drugs involved in MDR (doxorubicin, etoposide, amsacrine, vinblastine, vincristine and actinomycin D), but not to the alkylating agents, cyclophosphamide, carmustine, and cisplatin, or to the antimetabolites, 5-fluorouracil and methotrexate. P388/NVB cellular resistance to NVB was stable without drug pressure during continuous passagein vivo for more than ten weeks andin vitro for at least five weeks. These cells exhibited increased expression of P-gp, and a 30-fold level of resistance of NVBin vitro, which was completely reversable with verapamil. The MDR phenotype was confirmed in other tumor models. P388 tumors resistant to vinblastine, vincristine, doxorubicin, and etoposide were cross-resistant to NVBin vivo. Likewise, human KB carcinoma cells resistant to colchicine, MDA-A1^R breast carcinoma cells resistant to doxorubicin, and murine B16/F10 melanoma cells transfected with the humanmdr1 gene were cross-resistant to NVBin vitro. Finally, P388 cells resistantin vivo to melphalan, a drug that does not participate in MDR, were cross-resistant to NVB. Therefore, cellular resistance to NVB, like otherVinca alkaloids, may arise by a P-glycoprotein-mediated MDR mechanism, but may also involve non-MDR mechanisms in cells that do not overexpress P-gp.     

Sensitization of ABCB1 overexpressing cells to chemotherapeutic agents by FG020326 via binding to ABCB1 and inhibiting its function

The effectiveness of chemotherapeutic treatment is usually limited by the overexpression of adenosine triphosphate binding cassette (ABC) transporters, which mediate multidrug resistance (MDR) by acting as efflux pumps to remove chemotherapeutic agents from MDR cancer cells. Thus, the inhibition of ABC transporters may represent a promising strategy to reverse MDR. This study was to characterize the actions of FG020326, a newly synthesized triaryl-substituted imidazole derivative, to reverse MDR in vitro and in vivo. FG020326 significantly potentiated the cytotoxicity of paclitaxel, doxorubicin, and vincristine in the ABCB1 (P-glycoprotein, P-gp) overexpressing cells KBv200 and MCF-7/adr, but not in the ABCB1 negative parental cell lines KB and MCF-7. However, FG020326 did not alter the cytotoxicity of the aforementioned drugs in ABCC1 (MRP1), ABCC4 (MRP4), ABCG2 (BCRP) and LRP overexpressing cell lines, KB-CV60, NIH3T3/MRP4-2, S1-M1-80 and SW1573/2R120, respectively. FG020326, following p.o. administration, was present in concentrations sufficient for reversal of MDR in mice. The co-administration of FG020326 with paclitaxel or vincristine significantly enhanced the antitumor activity of these drugs without significantly increasing toxicity in the mice bearing the KBv200 cell xenografts. In addition, FG020326, at concentrations that reversed MDR, did not significantly affect the activity of CYP3A4 or alter the pharmacokinetic profile of paclitaxel after co-administration with paclitaxel. FG020326 produced a significant concentration-dependent displacement of [³H]azidopine and inhibition of efflux of drug from cells. Furthermore, FG020326 was co-localized with ABCB1 in cell membranes. Hence, FG020326 is characterized as a third generation MDR modulator that holds great promise for the treatment of cancer patients with ABCB1-mediated MDR.     

Screening of Multidrug-Resistance Sensitive Drugs by In Situ Brain Perfusion in P-Glycoprotein-Deficient Mice

Purpose. This study was conducted to assess the influence of P-glycoprotein (P-gp) on brain uptake of multidrug resistance sensitive drugs using an in situbrain perfusion technique in P-gp-deficient (mdr1a[–/–]) and wild-type mice. Methods. The blood-brain transport of radiolabeled vinblastine, vincristine, doxorubicin, colchicine, and morphine was evaluated in mdr1a(–/–) and wild-type CF-1 mice with the in situ brain perfusion technique. Brain uptake of drugs after intravenous pretreatment with P-gp reversal agents, (PSC 833, GF 120918, or (±)-verapamil), or vehicle also was studied in wild-type mice. In all experiments, cerebral vascular volume was determined by co-perfusion of sucrose. Results. Cerebral vascular volume was preserved during perfusion, indicating maintenance of blood-brain barrier integrity in both types of mice within the concentration range of substrates in the perfusate. The apparent brain transport of colchicine, vinblastine, doxorubicin, and morphine was increased 3.0, 2.7, 1.5, and 1.4-fold, respectively, in mdr1a(–/–) mice compared with the wild-type; the brain uptake of vincristine was not affected by P-gp. Preadministration of PSC 833 or GF 120918 in wild-type mice led to a 3-fold increase in the brain transport of colchicine and vinblastine, but no effect was observed for the other compounds. Intravenous verapamil enhanced colchicine brain transport (1.8-fold), but failed to increase the brain uptake of vinblastine and morphine. Conclusion. The in situ brain perfusion technique appears to be a sensitive and powerful tool for medium throughput screening of the brain uptake of multidrug resistance sensitive drugs. The effect of P-gp is characterized more efficiently with mdr1a(–/–) mice than by using modulators of P-gp in wild-type mice.     

Synthesis and evaluation of (2-(4-methoxyphenyl)-4-quinolinyl)(2-piperidinyl)methanol (NSC23925) isomers to reverse multidrug resistance in cancer

Development of multidrug resistance (MDR) during chemotherapy is a fundamental obstacle associated with cancer care. Prior studies have identified (2-(4-methoxyphenyl)-4-quinolinyl)(2-piperidinyl)methanol (5) (NSC23925) to be a small molecule agent that reverses MDR in cancer cells. We synthesized all four isomers of 5 and analyzed them by liquid chromatography-mass spectrometry (LCMS). Structure-activity relationships for reversing MDR were evaluated. Isomer 11 demonstrated the most potent activity. 11 reversed MDR in several drug-resistant cell lines expressing Pgp, including ovarian, breast, colon, uterine, and sarcoma cancer. 11 resensitized these cell lines to paclitaxel, doxorubicin, mitoxantrone, vincristine, and trabectedin with no effect on cell sensitivity to cisplatin, topotecan, and methotrexate. 11 significantly enhanced in vivo antitumor activity of paclitaxel in MDR xenograft models, without increasing the level of paclitaxel toxicity. In conclusion, 11 and derivatives of this compound may hold therapeutic value in the treatment of MDR-dependent cancers.     

Modulation of human placental P-glycoprotein expression and activity by MDR1 gene polymorphisms

The ABC transporter P-glycoprotein is a product of the MDR1 gene and its function in human placenta is to extrude xenobiotics from the tissue thus decreasing fetal exposure. The goal of this investigation was to examine the effect of three polymorphisms in the MDR1 gene on the expression and activity of placental P-gp. In 199 term placentas examined, the C1236T variant was associated with 11% lower P-gp protein expression than wild-type, while the C3435T and G2677T/A variants each were associated with a 16% reduction (p < 0.05). Homozygotes for the C1236T and C3435T variant allele (TT) were associated with 42% and 47% increase in placental P-gp transport activity, respectively (p = 0.04 and p = 0.02) of the prototypic substrate, [³H]-paclitaxel. These findings indicate that the C3435T and G2677T/A SNPs in MDR1 are significantly associated with decreased placental P-gp protein expression, while the C1236T and C3245T homozygous variants are significantly associated with an increase in its efflux activity.     

Collateral sensitivity of multidrug-resistant cells to the orphan drug tiopronin

A major challenge in the treatment of cancer is multidrug resistance (MDR) that develops during chemotherapy. Here we demonstrate that tiopronin (1), a thiol-substituted N-propanoylglycine derivative, was selectively toxic to a series of cell lines expressing the drug efflux pump P-glycoprotein (P-gp, ABCB1) and MRP1 (ABCC1). Treatment of MDR cells with 1 led to instability of the ABCB1 mRNA and consequently a reduction in P-gp protein, despite functional assays demonstrating that tiopronin does not interact with P-gp. Long-term exposure of P-gp-expressing cells to 1 sensitized them to doxorubicin and paclitaxel, both P-gp substrates. Treatment of MRP1-overexpressing cells with tiopronin led to a significant reduction in MRP1 protein. Synthesis and screening of analogues of tiopronin demonstrated that the thiol functional group was essential for collateral sensitivity while substitution of the amino acid backbone altered but did not destroy specificity, pointing to future development of targeted analogues.     

Human MDR1 polymorphism: G2677T/A and C3435T have no effect on MDR1 transport activities

The two most frequently observed single nucleotide polymorphisms (SNPs) of the human multidrug resistance 1 (MDR1) gene are 2677G/T/A (893Ala/Ser/Thr) and 3435C/T (no amino acid substitution). In this study, six forms of MDR1 cDNAs with the SNPs were expressed in LLC-PK1 cells and their transport activities were determined. Nearly identical amounts of the recombinant MDR1 proteins were expressed in the established cell lines using the Flp recombinase, which integrates a gene of interest at a specific genomic location. Four structurally diverse compounds: verapamil, digoxin, vinblastine and cyclosporin A, were examined for transcellular transport activities and intracellular accumulation. No significant differences were observed between cells expressing five polymorphic types of the MDR1 cDNAs (2677G/3435T, 2677A/3435C, 2677A/3435T, 2677T/3435C, 2677T/3435T) and cells expressing the wild-type (2677G/3435C). These results suggested that the two frequently observed MDR1 SNPs had no effect on the transport activities of MDR1 proteins expressed in LLC-PK1 cells in vitro, and other genetic or environmental factors might control the expression of MDR1 and the in vivo activity of MDR1.