Intracellular Doppler Spectroscopy detects altered drug response in SKOV3 tumor spheroids with silenced or inhibited P-glycoprotein.

Intracellular Doppler spectroscopy is a form of low-coherence digital holography based upon Doppler detection of scattered light that measures drug response/resistance in tumor spheroids, xenografts, and clinical biopsies. Multidrug resistance (MDR) is one of the main causes of ineffective cancer treatment. One MDR mechanism is mediated by the MDR1 gene that encodes the drug efflux pump P-glycoprotein (Pgp). Overexpression of Pgp in some cancers is associated with poor chemotherapeutic response. This paper uses intracellular Doppler spectroscopy to detect Pgp-mediated changes to drug response in 3D tissues grown from an ovarian cancer cell line (SKOV3). The SKOV3 cell line was incrementally exposed to cisplatin to create a cell line with increased Pgp expression (SKOV3cis). Subsequently, MDR1 in a subset of these cells was silenced in SKOV3cis using shRNA to create a doxycycline inducible, Pgp-silenced cell line (SKOV3cis-sh). A specific Pgp inhibitor, zosuquidar, was used to study the effects of Pgp inhibition on the Doppler spectra. Increased drug sensitivity was observed with Pgp silencing or inhibition as determined by drug IC50s of paclitaxel-response of silenced Pgp and doxorubicin-response of inhibited Pgp, respectively. These results indicate that intracellular Doppler spectroscopy can detect changes in drug response due to silencing or inhibition of a single protein associated with drug resistance with important consequences for personalized medicine.

Molecular cloning and tissue distribution of a novel marmoset ABC transporter.

Common marmosets (Callithrix jacchus) have been recognized as a useful small non-human primate model in preclinical testing for drug development. In this study, a cDNA of novel ATP-dependent efflux transporter ABCB1 was cloned from marmoset liver tissue. Marmoset ABCB1 cDNA encodes a protein of 1279 amino acid residues (MW = 141.4 kDa) containing characteristic regions of an ATP-binding cassette (ABC) protein, two hydrophobic transmembrane regions and two cytoplasmic nucleotide-binding regions, similar to human ABCB1. The deduced amino acid sequences were more highly identical (95%) to those of human ABCB1 compared with non-primate species such as dogs, pigs and rodents (79-90%). A close evolutionary relationship of ABCB1 among marmosets, cynomolgus and rhesus monkeys and humans was evident from a phylogenetic analysis using ABCB1 amino acid sequences from primates, dogs, pigs and rodents. Tissue distribution analyses by quantitative reverse transcription-polymerase chain reaction indicated that marmoset ABCB1 mRNA was most abundant in kidneys, followed by small intestines and livers, similar to human ABCB1, and marmoset ABCB1 proteins in these tissues were also detected by immunoblotting. These results indicated that the primary structure and tissue distribution of ABCB1 in marmosets were similar to those of humans, suggesting similar molecular characteristics of ABCB1 between marmosets and humans.

Novel biomarkers GEP/ABCB5 regulate response to adjuvant transarterial chemoembolization after curative hepatectomy for hepatocellular carcinoma.

BACKGROUND: Transarterial chemoembolization (TACE) is the most commonly used adjuvant therapy for hepatocellular carcinoma (HCC) after curative resection. Responses to TACE are variable due to tumor and patient heterogeneity. We had previously demonstrated that expression of Granulin-epithelin precursor (GEP) and ATP-dependent binding cassette (ABC)B5 in liver cancer stem cells was associated with chemoresistance. The present study aimed to evaluate the association between GEP/ABCB5 expression and response to adjuvant TACE after curative resection for HCC. METHODS: Patients received adjuvant TACE after curative resection for HCC and patients received curative resection alone were identified from a prospectively collected database. Clinical samples were retrieved for biomarker analysis. Patients were categorized into 3 risk groups according to their GEP/ABCB5 status for survival analysis: low (GEP-/ABCB5-), intermediate (either GEP+/ABCB5- or GEP-/ABCB5+) and high (GEP+/ABCB5+). Early recurrence (recurrence within 2 years after resection) and disease-free survival were analyzed. RESULTS: Clinical samples from 44 patients who had followed-up for more than 2 years were retrieved for further biomarker analysis. Among them, 18 received adjuvant TACE and 26 received surgery alone. Patients with adjuvant TACE in the intermediate risk group was associated with significantly better overall survival and 2-year disease-free survival than those who had surgery alone (P = 0.036 and P = 0.011, respectively). Adjuvant TACE did not offer any significant differences in the early recurrence rate, 2-year disease-free survival and overall survival for patients in low and high risk groups. CONCLUSIONS: Adjuvant TACE can only provide survival benefits for patients in the intermediate risk group (either GEP+/ABCB5- or GEP-/ABCB5+). A larger clinical study is warranted to confirm its role in patient selection for adjuvant TACE.

P-gp/ABCB1 exerts differential impacts on brain and fetal exposure to norbuprenorphine.

Norbuprenorphine is the major active metabolite of buprenorphine which is commonly used to treat opiate addiction during pregnancy. Norbuprenorphine produces marked respiratory depression and was 10 times more potent than buprenorphine. Therefore, it is important to understand the mechanism that controls fetal exposure to norbuprenorphine, as exposure to this compound may pose a significant risk to the developing fetus. P-gp/ABCB1 and BCRP/ABCG2 are two major efflux transporters regulating tissue distribution of drugs. Previous studies have shown that norbuprenorphine, but not buprenorphine, is a P-gp substrate. In this study, we systematically examined and compared the roles of P-gp and BCRP in determining maternal brain and fetal distribution of norbuprenorphine using transporter knockout mouse models. We administered 1mg/kg norbuprenorphine by retro-orbital injection to pregnant FVB wild-type, Abcb1a-/-/1b-/-, and Abcb1a-/-/1b-/-/Abcg2-/- mice on gestation day 15. The fetal AUC of norbuprenorphine was ∼64% of the maternal plasma AUC in wild-type mice, suggesting substantial fetal exposure to norbuprenorphine. The maternal plasma AUCs of norbuprenorphine in Abcb1a-/-/1b-/- and Abcb1a-/-/1b-/-/Abcg2-/- mice were ∼2 times greater than that in wild-type mice. Fetal AUCs in Abcb1a-/-/1b-/- and Abcb1a-/-/1b-/-/Abcg2-/- mice were also increased compared to wild-type mice; however, the fetal-to-maternal plasma AUC ratio remained relatively unchanged by the knockout of Abcb1a/1b or Abcb1a/1b/Abcg2. In contrast, the maternal brain-to-maternal plasma AUC ratio in Abcb1a-/-/1b-/- or Abcb1a-/-/1b-/-/Abcg2-/- mice was increased ∼30-fold compared to wild-type mice. Protein quantification by LC-MS/MS proteomics revealed significantly higher amounts of P-gp protein in the wild-type mice brain than that in the placenta. These results indicate that fetal exposure to norbuprenorphine is substantial and that P-gp has a minor impact on fetal exposure to norbuprenorphine, but plays a significant role in restricting its brain distribution. The differential impacts of P-gp on norbuprenorphine distribution into the brain and fetus are likely, at least in part, due to the differences in amounts of P-gp protein expressed in the blood-brain and blood-placental barriers. BCRP is not as important as P-gp in determining both the systemic and tissue exposure to norbuprenorphine. Finally, fetal AUCs of the metabolite norbuprenorphine-ß-d-glucuronide were 3-7 times greater than maternal plasma AUCs, while the maternal brain AUCs were <50% of maternal plasma AUCs, suggesting that a reversible pool of conjugated metabolite in the fetus may contribute to the high fetal exposure to norbuprenorphine.

P-gp expression levels in the erythrocytes of brown trout: a new tool for aquatic sentinel biomarker development.

CONTEXT: P-glycoprotein (P-gp) is a ubiquitous membrane detoxification pump involved in cellular defence against xenobiotics. Blood is a hub for the trade and transport of physiological molecules and xenobiotics. Our recent studies have highlighted the expression of a 140-kDa P-gp in brown trout erythrocytes in primary cell culture and its dose-dependent response to Benzo[a]pyrene pollutant. OBJECTIVE: The purpose of this study was focused on using P-gp expression in brown trout erythrocytes as a biomarker for detecting the degree of river pollution. METHODS: abcb1 gene and P-gp expression level were analysed by reverse transcriptase-PCR and Western blot, in the erythrocytes of brown trouts. The latter were collected in upstream and downstream of four rivers in which 17 polycyclic aromatic hydrocarbons and 348 varieties of pesticides micro-residues were analysed by liquid chromatography and mass spectrometry. RESULTS: The abcb1 gene and the 140-kDa P-gp were not expressed in trout erythrocytes from uncontaminated river. In contrast, they are clearly expressed in contaminated rivers, in correlation with the river pollution degree and the nature of the pollutants. CONCLUSIONS: This biological tool may offer considerable advantages since it provides an effective response to the increasing need for an early biomarker.

Effects of high-intensity focused ultrasound on cisplatin-resistant human lung adenocarcinoma in vitro and in vivo.

It is widely accepted that high-intensity focused ultrasound (HIFU) is a minimally invasive treatment option for different tumors, but its roles and the corresponding mechanism in cisplatin (DDP) chemoresistance in lung adenocarcinoma (LA) remain unclear. In this study, we investigated the response of DDP-resistant LA cells to HIFU and its underlying molecular mechanisms using molecular biology techniques. It was found that HIFU exposure inhibited the proliferation of DDP-resistant A549 (A549/DDP) cells through arresting cell cycle at the G1/G0 phase via the Cyclin-dependent pathway and promoting apoptosis in a Bcl-2-dependent manner. Furthermore, the results also showed that HIFU exposure could down-regulate the expressions of MDR1, MRP1, and LRP mRNAs, as well as P-gp, MRP1, and LRP proteins related to drug resistance in A549/DDP cells. In vivo experiments also demonstrated that HIFU could reduce the size and mass of subcutaneously transplanted tumors produced by A549/DDP cells through mediating Cyclin-dependent and Bcl-2-dependent pathways. These results suggested that HIFU treatment could inhibit the proliferation of DDP-resistant lung cancer cells and might be a novel therapeutic method for patients with DDP resistance.

The use of simultaneous confidence bands for comparison of single parameter fluorescent intensity data.

Despite the utility of multiparameter flow cytometry for a wide variety of biological applications, comparing single parameter histograms of fluorescence intensity remains a mainstay of flow cytometric analysis. Even comparisons requiring multiparameter gating strategies often end with single parameter histograms as the final readout. When histograms overlap, analysis relies on comparison of mean or median fluorescence intensities, or determination of percent positive based on an arbitrary cutoff. Earlier attempts to address this problem utilized either simple channel-by-channel subtraction without statistical evaluation, or the Kolmogorov-Smirnov (KS) or Chi-square test statistics, both of which proved to be overly sensitive to small and biologically insignificant differences. Here we present a method for the comparison of two single-parameter histograms based on difference curves and their simultaneous confidence bands generated by bootstrapping raw channel data. Bootstrapping is a nonparametric statistical approach that can be used to generate confidence intervals without distributional assumptions about the data. We have constructed simultaneous confidence bands and show them to be superior to KS and Cox methods. The method constructs 95% confidence bands about the difference curves, provides a P value for the comparison and calculates the area under the difference curve (AUC) as an estimate of percent positive and the area under the confidence band (AUCSCB95), providing a lower estimate of the percent positive. To demonstrate the utility of this new approach we have examined single-color fluorescence intensity data taken from a cell surface proteomic survey of a lung cancer cell line (A549) and a published fluorescence intensity data from a rhodamine efflux assay of P-glycoprotein activity, comparing rhodamine 123 loading and efflux in CD4 and CD8 T-cell populations. SAS source code is provided as supplementary material.

Prognostic value of glucosylceramide synthase and P-glycoprotein expression in oral cavity cancer.

BACKGROUND: Glucosylceramide synthase (GCS) and P-glycoprotein (P-gp) overexpression are associated with multidrug resistance in several human cancers. This study investigated the prognostic value of GCS and P-gp in oral cavity squamous cell carcinoma (OSCC). METHODS: The association between GCS and P-gp overexpression and clinical outcomes was assessed in 186 human clinical specimens of primary tumors obtained from curative surgery. Immunohistochemistry staining results were scored as high or low for GCS, and positive or negative for P-gp. Univariate and multivariate analyses using the Cox proportional hazards model were conducted to assess the significance of differences in recurrence or survival outcomes between variables. RESULTS: GCS overexpression was observed in 128 (68.8 %) patients and P-gp overexpression in 43 (23.1 %) patients. High GCS expression was significantly correlated with P-gp immunopositivity (P = 0.005). GCS and P-gp overexpression was significantly correlated with cervical nodal metastasis (P < 0.05). Univariate analyses showed that tumor lymphovascular invasion, positive neck lymph nodes, advanced overall TNM stage, high GCS expression, and P-gp immunopositivity were associated with poor locoregional control (LRC), disease-free survival (DFS), and overall survival (OS) (P < 0.05). Multivariate analyses showed that lymphovascular invasion, nodal positivity, and P-gp overexpression remained independent prognostic variables for LRC, DFS, and OS, and that GCS expression was an independent predictor of LRC and DFS (P < 0.05). CONCLUSION: GCS and P-gp expression is associated with poor prognosis, suggesting suitability as novel biomarkers in OSCC.

Assessment of melanoma-initiating cell markers and conventional parameters in sentinel lymph nodes of malignant melanoma.

Sentinel lymph node (SLN) biopsies have widely been used for the detection of occult LN metastasis of malignant melanoma (MM). In addition to conventional biomarkers, we assessed the diagnostic and prognostic significance of melanoma-initiating cell (MIC) markers in SLNs of MM. We examined the expressions of gp100, MART-1 and tyrosinase mRNA for routine diagnosis and those of ABCB5, CD133, nestin, KDM5B, NGFR and RANK mRNA as MIC markers. The presence of micrometastasis was confirmed immunohistochemically using antibodies to S-100, HMB-45, MART-1, and tyrosinase. Discordance between immunohistochemical and molecular data was observed in 14 of 70 (20.0%) patients, among whom five (7.1%) were positive for only molecular markers;two of these five patients tested positive for micrometastasis by repeated immunohistochemical stainings. The quantitative expression levels of gp100, MART-1, and tyrosinase mRNA were significantly higher in the metastatic LNs;the cut-off values remain to be elucidated. ABCB5 mRNA expression was detected more frequently in the metastatic SLNs (p＜0.05) and in the group of patients with recurrence. To make a definite diagnosis of metastasis, we still need a combination of immunohistochemical and molecular probes. ABCB5 might be a suitable molecular marker for the detection of melanoma-initiating cells in SLNs.

Expression of PKCα, PKCε, and P-gp in epithelial ovarian carcinoma and the clinical significance.

AIM: To inspect the expression of two protein kinase PKC isozyme hypotype PKCα and PKCε in the epithelial ovarian carcinoma tissue, and investigate their relation with multi-drug resistance with P-glycoprotein (P-gp) medium. MATERIALS AND METHODS: Adopted immunohistochemistry SP method to determine expression of PKCα, PKCε, and P-gp in 64 cases of epithelial ovarian carcinoma, 18 cases of epithelial borderline ovarian carcinoma, 15 cases of epithelial ovarian benign tumor, and 15 cases of normal ovarian tissue. RESULTS: The expression of PKCα, PKCε, and P-gp in the epithelial ovarian carcinoma is obviously higher than expression in the normal, benign. and borderline epithelial ovarian carcinoma; the expression of PKCα, PKCε, and P-gp in the recurrent carcinoma tissue is obviously higher than that in the person with initial treatment; the expression of above-mentioned three indicators in epithelial ovarian carcinoma is unrelated with the pathological type, pathological grade, and clinical stage during initial treatment of the carcinoma; there is a close relation among PKCα, PKCε, and P-gp in epithelial ovarian carcinoma (p < 0.01). It is indicated through research that PKCα, PKCε, and P-gp is related with the survival time and poor prognosis of the patient of epithelial ovarian carcinoma, i.e., the positive expression rate of PKCα, PKCε, and P-gp of the person with recurrent carcinoma is higher than that of the person without recurrent car- cinoma (p < 0.05). However, the survival rate of the patients with positive expression of three indicators is remarkably lower than those with negative expression (p < 0.05). CONCLUSION: There is a consistency between expression of PKCα, PKCε, and P-gp in the epithelial ovarian carcinoma, which indicates that the expression of both plays an important role in generation of drug resistance in chemotherapy of ovarian carcinoma with P-gp medium. Joint detection of three indicators has an active guiding role in judgment of the therapeutic effect of clinical chemotherapy and prognosis estimation of the patient.

Apoptosis resistance-related ABCB5 and DNaseX (Apo10) expression in oral carcinogenesis.

BACKGROUND: Apoptosis resistance is a crucial factor for the carcinogenesis of oral squamous cell carcinoma (OSCC). METHODS: Expression of apoptosis resistance-related ATP-binding cassette (ABC) transporter ABCB5 [subfamily B (MDR/TAP) member 5] and DNaseX (Apo10) were analyzed in normal oral mucosa (n = 5), oral precursor lesions (simple hyperplasia, n = 11; squamous intraepithelial neoplasia, SIN I-III, n = 35), and OSCC specimen (n = 42) by immunohistochemistry. RESULTS: Expression of ABCB5 and Apo10 were significantly increased in the carcinogenesis of OSCC compared with normal tissue. Compared with SIN I-III, ABCB5 expression was significantly decreased in OSCC. Apo10 expression did not significantly differ from OSCC compared with SIN I-III. CONCLUSIONS: This study provides the first evidence of the expression of ABCB5 and Apo10 in the multi-step carcinogenesis of OSCC. Overcoming drug resistance of ABCB5+ and Apo10+ cells in precursor lesions and tumors by natural compounds may act as sensitizers for apoptosis or could be useful for chemoprevention.

An efficient nanomaterial-based electrochemical biosensor for sensitive recognition of drug-resistant leukemia cells.

A novel electrochemical cytosensor was developed for the fast and high-sensitivity recognition of drug-resistant leukemia K562/ADM cells based on the P-glycoprotein (P-gp) expression level on a cell membrane. The nanocomposite interface of the gold nanoparticles/polyaniline nanofibers (AuNPs/PANI-NF) was chosen to design the biosensor for electrochemical detection. Au/PANI-NF-based cytosensors coated with anti-P-glycoprotein (anti-P-gp) molecules could provide a biomimetic interface for the immunosensing of cell surface P-glycoprotein, and thus could capture the over-expression P-gp cells. Transmission electron microscopy (TEM) indicated that the gold nanoparticles were uniformly anchored along the structure of the PANI-NF surface, displaying fibrillar morphology with a diameter of ∼70 nm, and atomic force microscopy (AFM) further presented the morphology of the nanocomposite film. Owing to the high affinity of anti-P-gp for leukemia K562/ADM cells of the propounded sensing platform, the proposed biosensor exhibited excellent analytical performance for leukemia K562/ADM cells, ranging from 1.6 × 10(2) to 1.6 × 10(6) cells per mL with a detection limit of 80 cells per mL. Recovery experiments indicated that the sensitivity reported here is suitable for practical application. The cell surface P-gp expression level was analysed by flow cytometric experiments, which confirmed the above recognized result. This strategy is also a cost-effective and convenient operation, implying great promise for the sensitive recognition of cancer cells and cell surface receptors; thus, it is helpful in cancer diagnosis.

The multidrug-resistance transporter ABCB5 is expressed in human placenta.

ATP-binding cassette (ABC) transporters in placenta protectively transport drugs and xenobiotics. ABCB5 [subfamily B (MDR/TAP)] is a novel ABC multidrug-resistance transporter that also mediates cell fusion, stem cell function, and vasculogenic plasticity. Immunohistochemistry and double-labeling immunofluorescence staining for ABCB5 and ABCB5/CD200, respectively, was performed on formalin-fixed, paraffin-embedded placental tissue from 5 first trimester, 5 second trimester, and 5 term pregnancies as well as 5 partial moles, and 5 complete moles. In addition, tumor cells from 5 choriocarcinoma and 5 placental site trophoblastic tumor cases were examined. ABCB5 staining was observed in villous trophoblasts in 100% (5/5) of first trimester placentas (with progressive decrease in term placentas); 100% of partial moles (5/5); and 100% of complete moles (5/5). Notably, reactivity was discretely restricted to the inner trophoblast layer, with no staining of overlying syncytiotrophoblast. Antibody specificity and localization was confirmed further by in situ hybridization. ABCB5 expression was retained in 20% of choriocarcinomas (1/5) and 40% of placental site trophoblastic tumors (2/5). Prior studies have localized expression of multidrug-resistance-1, also known as ABCB1, within the syncytiotrophoblast of early placentas, where it serves a protective function as an efflux transporter. Our results show that ABCB5 is preferentially expressed in the cytotrophoblast layer of placental villi. The expression of this novel biomarker at the maternal-fetal interface raises questions on its role in placental structure and function as well as on its potential contribution to the protective efflux provided by other P-glycoprotein transporters.

Alteration of pharmacokinetics of grepafloxacin in type 2 diabetic rats.

PURPOSE: Patients with type 2 diabetes are generally treated with various pharmacological compounds and are exposed to a high risk of drug-drug interactions. However, alterations of pharmacokinetics in a type 2 diabetes model have been obscure. The present study was undertaken to investigate the effects of type 2 diabetes on the pharmacokinetics of the fluoroquinolone grepafloxacin (GPFX) and the expression level of P-glycoprotein (P-gp), one of the drug efflux transporters. METHODS: We used Goto-Kakizaki (GK) rats, a lean model of type 2 diabetes. Plasma concentration and intestinal, renal, and biliary clearance of GPFX were measured after intravenous and intraintestinal administration in Wistar and GK rats. Real-time PCR and Western blotting were used to assess mRNA and protein expression levels. RESULTS: We found a significant increase in the plasma concentrations of GPFX at 90, 120 and 240 minutes after intraintestinal administration in GK rats compared with the concentrations in Wistar rats but not after intravenous administration. The increase in plasma GPFX concentration was associated with reduction in jejunal clearance of GPFX caused by a decrease in secretory transport of GPFX. However, there was no correlation between the decrease in secretory transport of GPFX and P-gp expression level. CONCLUSION: Type 2 diabetic conditions alter P-gp function as well as expression level and correlate poorly with each other.

Effect of 9-cis retinoic acid and all-trans retinoic acid in combination with verapamil on P-glycoprotein expression in L1210 cells.

The development of the most common multidrug resistance (MDR) phenotype is associated with a massive overexpression of P-glycoprotein (P-gp) in neoplastic cells. In the current study, we used three L1210 cell variants: S cells - parental drug-sensitive cells; R cells - drug-resistant cells with P-gp overexpression due to selection with vincristine; T cells - drug-resistant cells with P-gp overexpression due to stable transfection with the pHaMDRwt plasmid, which encodes human full-length P-gp. Several authors have described the induction of P-gp expression/activity in malignant cell lines after treatment with all-trans retinoic acid (AtRA; ligand of retinoic acid nuclear receptors, RARs). An isomer of AtRA also exists, 9-cis retinoic acid, which is a ligand of both RARs and nuclear retinoid X receptors (RXRs). In a previous work, we described that the combined treatment of R cells with verapamil and AtRA induces the downregulation of P-gp expression/activity. In the current study, we studied the expression of RARs and RXRs in S, R and T cells and the effects of treatment with AtRA, 9cRA and verapamil on P-gp expression, cellular localization and efflux activity in R and T cells. We found that the overexpression of P-gp in L1210 cells is associated with several changes in the specific transcription of both subgroups of nuclear receptors, RARs and RXRs. We also demonstrated that treatment with AtRA, 9cRA and verapamil induces alterations in P-gp expression in R and T cells. Particularly, combined treatment of R cells with verapamil and AtRA induced downregulation of P-gp content/activity. In contrast, similar treatment of T cells induced slight increase of P-gp content without any changes in efflux activity of this protein. These findings indicate that active crosstalk between the RAR and RXR regulatory pathways and P-gp-mediated MDR could take place.

Quantitative assessment of p-glycoprotein expression and function using confocal image analysis.

P-glycoprotein is implicated in clinical drug resistance; thus, rapid quantitative analysis of its expression and activity is of paramout importance to the design and success of novel therapeutics. The scope for the application of quantitative imaging and image analysis tools in this field is reported here at "proof of concept" level. P-glycoprotein expression was utilized as a model for quantitative immunofluorescence and subsequent spatial intensity distribution analysis (SpIDA). Following expression studies, p-glycoprotein inhibition as a function of verapamil concentration was assessed in two cell lines using live cell imaging of intracellular Calcein retention and a routine monolayer fluorescence assay. Intercellular and sub-cellular distributions in the expression of the p-glycoprotein transporter between parent and MDR1-transfected Madin-Derby Canine Kidney cell lines were examined. We have demonstrated that quantitative imaging can provide dose-response parameters while permitting direct microscopic analysis of intracellular fluorophore distributions in live and fixed samples. Analysis with SpIDA offers the ability to detect heterogeniety in the distribution of labeled species, and in conjunction with live cell imaging and immunofluorescence staining may be applied to the determination of pharmacological parameters or analysis of biopsies providing a rapid prognostic tool.

Altered brain uptake of therapeutics in a triple transgenic mouse model of Alzheimer's disease.

PURPOSE: The purpose of this study was to systematically assess the impact of Alzheimer's disease (AD)-associated blood-brain barrier (BBB) alterations on the uptake of therapeutics into the brain. METHODS: The brain uptake of probe compounds was measured in 18-20 month old wild type (WT) and triple transgenic (3×TG) AD mice using an in situ transcardiac perfusion technique. These results were mechanistically correlated with immunohistochemical and molecular studies. RESULTS: The brain uptake of the paracellular marker, [(14)C] sucrose, did not differ between WT and 3×TG mice. The brain uptake of passively diffusing markers, [(3)H] diazepam and [(3)H] propranolol, decreased 54-60% in 3×TG mice, consistent with a 33.5% increase in the thickness of the cerebrovascular basement membrane in 3×TG mice. Despite a 42.4% reduction in P-gp expression in isolated brain microvessels from a sub-population of 3×TG mice (relative to WT mice), the brain uptake of P-gp substrates ([(3)H] digoxin, [(3)H] loperamide and [(3)H] verapamil) was not different between genotypes, likely due to a compensatory thickening in the cerebrovascular basement membrane counteracting any reduced efflux of these lipophilic substrates. CONCLUSION: These studies systematically assessed the impact of AD on BBB drug transport in a relevant animal model, and have demonstrated a reduction in the brain uptake of passively-absorbed molecules in this mouse model of AD.

Potent and selective tariquidar bioisosters as potential PET radiotracers for imaging P-gp.

Compounds 8a-d have been designed as bioisosters of tariquidar for imaging P-gp expression and density by PET. The results displayed that compounds 8b and 8d could be considered potential P-gp/BCRP ligands suitable as (11)C and (18)F radiotracers, respectively.

Single nucleotide polymorphism detection by optical DNA-based sensing coupled with whole genomic amplification.

The work presented here deals with the optimization of a strategy for detection of single nucleotide polymorphisms based on surface plasmon resonance imaging. First, a sandwich-like assay was designed, and oligonucleotide sequences were computationally selected in order to study optimized conditions for the detection of the rs1045642 single nucleotide polymorphism in the gene ABCB1. Then the strategy was optimized on a surface plasmon resonance imaging biosensor using synthetic DNA sequences in order to evaluate the best conditions for the detection of a single mismatching base. Finally, the assay was tested on DNA extracted from human blood which was subsequently amplified using a whole genome amplification kit. The direct detection of the polymorphism was successfully achieved. The biochip was highly regenerable and reusable for up to 20 measurements. Furthermore, coupling these promising results with the multiarray assay, we can foresee applying this biosensor in clinical research extended to concurrent analysis of different polymorphisms.

Modulation of multidrug resistance P-glycoprotein activity by antiemetic compounds in human doxorubicin-resistant sarcoma cells (MES-SA/Dx-5): implications on cancer therapy.

Multidrug resistance (MDR) in cancer cells is often caused by the high expression of the plasma membrane drug transporter P-glycoprotein (Pgp) associated with an elevated intracellular glutathione (GSH) content in various human tumors. Several chemosensitizers reverse MDR but have significant toxicities. Antiemetic medications are often used for controlling chemotherapy-induced nausea and vomiting in cancer patient. In this in vitro study we investigated if the effects of two common antiemetic drugs such as dimenhydrinate (dime) and ondansentron (onda) and a natural compound (6)-gingerol (ginger), the active principle of ginger root, interfere on Pgp activity and intracellular GSH content in order to evaluate their potential use as chemosensitizing agents in anticancer chemotherapy. The human doxorubicin (doxo) resistant uterine sarcoma cells (MES-SA/Dx5) that overexpress Pgp, were treated with each antiemetic alone (1, 10 and 20 microM) or in combination with different doxo concentrations (2, 4, and 8 microM). We measured the intracellular accumulation and cytotoxicity of doxo (MTT assay), the cellular GSH content (GSH assay) and ROS production (DFC-DA assay), in comparison with verapamil (Ver), a specific inhibitor for Pgp, used as reference molecule. We found that exposure at 2, 4 and 8 microM doxo concentrations in the presence of dime, onda and ginger enhanced significantly doxo accumulation and cytotoxicity on resistant MES-SA/Dx5 cells when compared with doxo alone. Moreover, treatment with ginger (20 microM) increased cellular GSH content (greater than 10 percent) in resistant cells, while ROS production remained below the control values for all antiemetic compounds at all concentrations. These findings provide the rationale for innovative clinical trials of antiemetics or their derivatives as a new potential generation of chemosensitizers to improve effectiveness of the anticancer drugs in MDR human tumours.