Theresa May Chin Tan

Role of glutathione in the multidrug resistance protein 4 (MRP4/ABCC4)-mediated efflux of cAMP and resistance to purine analogues.

Multidrug resistance protein 4 (MRP4/ABCC4) is a member of the MRP subfamily, which in turn is a member of the superfamily of ATP-binding-cassette (ABC) transporters. Within the MRP subfamily, ABCC4,ABCC5 (MRP5), ABCC11 (MRP8) and ABCC12 (MRP9) have similar predicted membrane topologies. All lack the additional transmembrane domain, TMD(0), which is present in the other MRPs. Using cells stably overexpressing ABCC4, this study shows that ABCC4 exports GSH. ABCC4 also facilitates the efflux of cAMP. Depletion of intracellular GSH with DL-buthionine-(S,R)-sulphoximine led to decreased export of cAMP and a corresponding increase in intracellular cAMP was observed. ABCC4 also mediates resistance to purine analogues 9-(2-phosphonylmethoxyethyl)-adenine and 6-thioguanine. This resistance can be reversed by the presence of DL-buthionine-(S,R)-sulphoximine. We conclude that as well as nucleotide and nucleoside analogues, ABCC4 can mediate the export of GSH. In addition, GSH plays an important role in the function of ABCC4. Depletion of intracellular GSH adversely affects the export of cAMP by ABCC4. Resistance to nucleoside analogues is also adversely affected by depletion of cellular GSH.

Impact of Curcumin-Induced Changes in P-Glycoprotein and CYP3A Expression on the Pharmacokinetics of Peroral Celiprolol and Midazolam in Rats

The aim of this study was to evaluate whether curcumin could modulate P-glycoprotein (P-gp) and CYP3A expression, and in turn modify the pharmacokinetic profiles of P-gp and CYP3A substrates in male Sprague-Dawley rats. Intragastric gavage of the rats with 60 mg/kg curcumin for 4 consecutive days led to a down-regulation of the intestinal P-gp level. There was a concomitant upregulation of hepatic P-gp level, but the renal P-gp level was unaffected. Curcumin also attenuated the CYP3A level in the small intestine but induced CYP3A expression in the liver and kidney. Regular curcumin consumption also caused the Cmax and area under the concentration-time curve (AUC0–8 and total AUC) of peroral celiprolol (a P-gp substrate with negligible cytochrome P450 metabolism) at 30 mg/kg to increase, but the apparent oral clearance (CLoral) of the drug was reduced. Similarly, rats treated with curcumin for 4 consecutive days showed higher AUC (AUC0–4 and total AUC) and lower CLoral for peroral midazolam (a CYP3A substrate that does not interact with the P-gp) at 20 mg/kg in comparison with vehicle-treated rats. In contrast, curcumin administered 30 min before the respective drug treatments did not significantly modify the pharmacokinetic parameters of the drugs. Analysis of the data suggests that the changes in the pharmacokinetic profiles of peroral celiprolol and midazolam in the rat model were contributed mainly by the curcumin-mediated down-regulation of intestinal P-gp and CYP3A protein levels, respectively.

In vitro and in vivo evaluation of the effects of piperine on P-gp function and expression

Piperine, a major component of black pepper, is used as spice and nutrient enhancer. The purpose of the present study was to evaluate the effects of acute and prolonged piperine exposure on cellular P-gp expression and function in vitro and in vivo. Piperine at concentrations ranging from 10 to 100 microM, determined by MTT assay to be non-cytotoxic, was observed to inhibit P-gp mediated efflux transport of [(3)H]-digoxin across L-MDR1 and Caco-2 cell monolayers. The acute inhibitory effect was dependent on piperine concentration, with abolishment of [(3)H]-digoxin polarized transport attained at 50 microM of piperine. In contrast, prolonged (48 and 72 h) co-incubation of Caco-2 cell monolayers with piperine (50 and 100 microM) increased P-gp activity through an up-regulation of cellular P-gp protein and MDR1 mRNA levels. The up-regulated protein was functionally active, as demonstrated by a higher degree of [(3)H]-digoxin efflux across the cell monolayers, but the induction was readily reversed by the removal of the spice from the culture medium. Peroral administration of piperine at the dose of 112 microg/kg body weight/day to male Wistar rats for 14 consecutive days also led to increased intestinal P-gp levels. However, there was a concomitant reduction in the rodent liver P-gp although the kidney P-gp level was unaffected. Our data suggest that caution should be exercised when piperine is to be co-administered with drugs that are P-gp substrates, particularly for patients whose diet relies heavily on pepper.

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.

Genetic determinants of tetracycline resistance in Vibrio harveyi

ABSTRACT Isolates of Vibrio harveyi, a prawn pathogen, have demonstrated multiple antibiotic resistance to commonly used antimicrobial agents, such as oxytetracycline. In this paper, we describe the cloning and characterization of two tetracycline resistance determinants from V. harveyi strain M3.4L. The first resistance determinant, cloned as a 4,590-bp fragment, was identical to tetA and flanking sequences encoded on transposon Tn10 from Shigella flexneri. The second determinant, cloned as a 3,358-bp fragment in pATJ1, contains two open reading frames, designated tet35 and txr. tet35 encodes a 369-amino-acid protein that was predicted to have nine transmembrane regions. It is a novel protein which has no homology to any other drug resistance protein but has low levels of homology (28%) to Na+/H+ antiporters. Transposon mutagenesis showed that tet35 and txr were required for tetracycline resistance in a heterologous Escherichia coli host. Tetracycline accumulation studies indicate that E. coli carrying tet35 and txr can function as an energy-dependent tetracycline efflux pump but is less efficient than TetA.

Gene profiling reveals hydrogen sulphide recruits death signaling via the N-methyl-D-aspartate receptor identifying commonalities with excitotoxicity.

Recently the role of hydrogen sulphide (H2S) as a gasotransmitter stimulated wide interest owing to its involvement in Alzheimers disease and ischemic stroke. Previously we demonstrated the importance of functional ionotropic glutamate receptors (GluRs) by neurons is critical for H2S‐mediated dose‐ and time‐dependent injury. Moreover N‐methyl‐D‐aspartate receptor (NMDAR) antagonists abolished the consequences of H2S‐induced neuronal death. This study focuses on deciphering the downstream effects activation of NMDAR on H2S‐mediated neuronal injury by analyzing the time‐course of global gene profiling (5, 15, and 24 h) to provide a comprehensive description of the recruitment of NMDAR‐mediated signaling. Microarray analyses were performed on RNA from cultured mouse primary cortical neurons treated with 200 µM sodium hydrosulphide (NaHS) or NMDA over a time‐course of 5–24 h. Data were validated via real‐time PCR, western blotting, and global proteomic analysis. A substantial overlap of 1649 genes, accounting for over 80% of NMDA global gene profile present in that of H2S and over 50% vice versa, was observed. Within these commonly occurring genes, the percentage of transcriptional consistency at each time‐point ranged from 81 to 97%. Gene families involved included those related to cell death, endoplasmic reticulum stress, calcium homeostasis, cell cycle, heat shock proteins, and chaperones. Examination of genes exclusive to H2S‐mediated injury (43%) revealed extensive dysfunction of the ubiquitin‐proteasome system. These data form a foundation for the development of screening platforms and define targets for intervention in H2S neuropathologies where NMDAR‐activated signaling cascades played a substantial role. J. Cell. Physiol. 226: 1308–1322, 2011.

Direct PCR amplification and sequence analysis of extrachromosomal Plasmodium DNA from dried blood spots.

The Plasmodium parasite possesses two extrachromosomal genomes; the mitochondrial genetic element and the extrachromosomal plastid-like DNA. The latter has only been fully described for one culture strain of P. falciparum. In this study, a rapid procedure for amplifying plastid DNA from dried blood spots of blood infected with different malaria species was developed. PCR amplification of a 595 bp fragment within the plastid-like large subunit ribosomal-RNA (LSU-rRNA) gene was achieved using primers derived from the P. falciparum sequence. The PCR product was observed in all Plasmodium species examined. Sequence analysis of amplified products homologous to an LSU-rRNA fragment of the plastid-like extrachromosomal circle revealed extensive conservation between Plasmodium species including P. falciparum, P. vivax, P. malariae and P. berghei.

Lack of Association of Single-Nucleotide Polymorphisms in Pregnane X Receptor, Hepatic Nuclear Factor 4α, and Constitutive Androstane Receptor with Docetaxel Pharmacokinetics

Purpose: This study aims to describe a population pharmacokinetic model for docetaxel in Asian breast cancer patients and to evaluate the effects of single-nucleotide polymorphisms (SNP) in the cytochrome P450 3A (CYP3A) gene expression regulators, constitutive androstane receptor (CAR), pregnane X receptor (PXR), and hepatic nuclear factor 4α (HNF4α), on the pharmacokinetics of docetaxel. Experimental Design: Docetaxel was given as an i.v. infusion of 75 mg/m2 over 1 h to 101 female breast cancer patients. CAR, PXR, and HNF4α were comprehensively sequenced. Docetaxel concentrations were measured using a liquid chromatography/tandem mass spectrometry method and its population pharmacokinetic variables, and the covariate effects of clearance predictors were estimated using a nonlinear mixed effects model. Results: Final estimates for docetaxel clearance was 47.1 L/h/70 kg/1.75 m. Between subject variability in docetaxel clearance was 22.5%. Covariates that showed significant association with docetaxel clearance included body size, α1 acid glycoprotein and liver function. SNPs identified in the coding regions of CAR and HNF4α and 5′ untranslated region of PXR in this Asian breast cancer cohort did not seem to improve predictability of docetaxel clearance. Conclusions: SNPs identified in CYP3A gene expression regulators CAR, HNF4α, and PXR in the Asian female breast cancer population do not seem to have any significant effect on the clearance of docetaxel, a CYP3A substrate.

Mechanism of translation of the bicistronic mRNA encoding human papillomavirus type 16 E6-E7 genes

The transforming genes E6 and E7 of human papillomavirus (HPV) type 16 and other HPV types are expressed from a bicistronic mRNA with a characteristic spacing of 3 to 6 bp between the termination codon of E6 and the initiation codon of E7. Plasmid pSP64E6E7 which contains the reading frames of both E6 and E7 was constructed in order to study the expression of both proteins in a coupled transcription/rabbit reticulocyte translation system. Both E6 and E7 proteins were expressed simultaneously. This translation could be interfered with by antisense oligonucleotides corresponding to various regions of the transcript. Antisense oligonucleotides targeted at sequences flanking either side of the translation initiation codon of the E6 open reading frame were effective in inhibiting the synthesis of both proteins, whereas oligonucleotides complementary to the coding regions downstream of the first start codon showed either a considerably reduced effect or none at all. In particular, there was limited inhibition of E7 translation by antisense oligonucleotides flanking the translation start region of the E7 gene. In the presence of RNase H, it was possible to selectively inhibit the synthesis of either E6 or E7 by several gene-internal antisense oligonucleotides. We conclude that HPV16 E6-E7 bicistronic mRNA is fully functional and that both proteins are translated with equal efficiency via the scanning mechanisms with reinitiation at the second open reading frame. In addition, both AE6 and AE7 may have therapeutical potential as they are capable of inhibiting the proliferation of CaSki cells which contain the HPV16 genome.

NRAMP1 and hGPX1 Gene Polymorphism and Response to Bacillus Calmette-Guérin Therapy for Bladder Cancer

BACKGROUND The natural resistance-associated macrophage protein 1 (NRAMP1) gene is associated with susceptibility to Mycobacterium tuberculosis in humans and to bacillus Calmette-Guérin (BCG) in mice. The detoxification enzyme, human glutathione peroxidase 1 (hGPX1), is associated with recurrence of bladder cancer (BCa). OBJECTIVE To determine whether NRAMP1 and hGPX1 gene polymorphisms correlate with response to BCG immunotherapy for non-muscle-invasive BCa (NMIBC). DESIGN, SETTING, AND PARTICIPANTS DNA was obtained from the peripheral blood of 99 NMIBC patients who were prospectively randomized to receive postresection intravesical BCG (81 mg [n=50] or 27 mg [n=19]) or BCG (27 mg) with interferon alpha (IFN-α; n=30). The median follow-up time was 60 mo. INTERVENTION Intravesical BCG or BCG-IFN-α. MEASUREMENTS Restriction fragment length polymorphism (RFLP) analysis was performed to identify polymorphisms in the NRAMP1 promoter region (GT repeat number) and at position 543 (aspartate [D] and/or asparagine [N] expression) within the NRAMP1 protein (D543N) and position 198 (proline and/or leucine expression) within the hGPX1 protein (Pro198Leu). Data were analyzed using χ(2) analysis, multivariate analysis, and Kaplan-Meier curves. RESULTS AND LIMITATIONS On univariate analysis, the NRAMP1 D543N G:G genotype had decreased cancer-specific survival (CSS; p=0.036). The hGPX1 CT genotype (Pro-Leu) had decreased recurrence time (p=0.03) after BCG therapy. On multivariate analysis, patients with the NRAMP1 D543N G:G genotype and allele 3 (GT)n polymorphism had decreased recurrence time (p=0.014 and p=0.03) after BCG therapy. The limitation of this study was its small sample size. CONCLUSIONS Polymorphisms of the NRAMP1 and hGPX1 genes may be associated with recurrence of BCa after BCG immunotherapy.