Levetiracetam plus Oxcarbazepine Combination Treatment Downregulates Serum Multidrug Resistance Protein 1 Levels and Upregulates Neuropeptide Y Levels in Children with Epilepsy

Abstract The aim of the present study was to investigate the usefulness of multidrug resistance protein 1 (MDR1) and neuropeptide Y (NPY) levels in predicting the efficacy of levetiracetam (LEV) plus oxcarbazepine (OXC) treatment administered to children with epilepsy and to determine their prognosis. Overall, 193 children with epilepsy admitted to the hospital were enrolled and randomly divided into two groups according to different treatment methods: group A (n = 106, treated with LEV plus OXC combination) and group B (n = 87, treated with OXC only). After treatment, compared with group B, group A exhibited a remarkably higher total effective rate and a significantly lower total adverse reaction rate. Areas under the curve for MDR1 and NPY for predicting ineffective treatment were 0.867 and 0.834, whereas those for predicting epilepsy recurrence were 0.916 and 0.829, respectively. Electroencephalography abnormalities, intracranial hemorrhage, neonatal convulsion, premature delivery, and MDR1 and NPY levels were independent risk factors for poor prognosis in children with epilepsy. Serum MDR1 and NPY levels exhibited a high predictive value for early epilepsy diagnosis, treatment efficacy assessment, and prognostication in children with epilepsy treated with LEV plus OXC combination.

Egr-1 Enhances Drug Resistance of Breast Cancer Cells by MDR1 Dependence

Abstract Paclitaxel (PTX) is one of the most effective drugs used in the treatment of breast cancer. Nonetheless, the appearance of MDR1 (multidrug resistance 1) in tumor cells has become a significant hindrance for efficacious chemotherapy. In this study, we show that the expression level of Egr-1 (early growth response gene-1) in cancer tissues (from paclitaxel chemotherapy failure patients) and MCF-7/PTX cells (the breast cancer cell line that was resistant to paclitaxel) was increased. Cell proliferation assay and apoptosis assay revealed that Egr-1 could promote cell growth and inhibit apoptosis in MCF-7/PTX. Mechanistic studies indicated that Egr-1 could bind to the proximal MDR1 promoter and enhance MDR1 transcription. These findings indicate that paclitaxel induced Egr-1 accumulation and upregulated the expression of MDR1, thereby inducing the drug resistance in MCF-7/PTX. Our results suggest a novel pathway by which paclitaxel induces MDR1 expression, possibly illuminating a potential target pathway for the prevention of MDR1-mediated drug resistance.

Air pollution exposure increases ABCB1 and ASCT1 transporter levels in mouse cortex.

Membrane transporters are important for maintaining brain homeostasis by regulating the passage of solutes into, out of, and within the brain. Growing evidence suggests neurotoxic effects of air pollution exposure and its contribution to neurodegenerative disorders, including Alzheimer's disease (AD), yet limited knowledge is available on the exact cellular impacts of exposure. This study investigates how exposure to ubiquitous solid components of air pollution, ultrafine particles (UFPs), influence brain homeostasis by affecting protein levels of membrane transporters. Membrane transporters were quantified and compared in brain cortical samples of wild-type and the 5xFAD mouse model of AD in response to subacute exposure to inhaled UFPs. The cortical ASCT1 and ABCB1 transporter levels were elevated in wild-type and 5xFAD mice subjected to a 2-week UFP exposure paradigm, suggesting impairment of brain homeostatic mechanisms. This study provides new insight on the molecular mechanisms underlying adverse effects of air pollution on the brain.

Similarities and differences in the localization, trafficking, and function of P-glycoprotein in MDR1-EGFP-transduced rat versus human brain capillary endothelial cell lines.

BACKGROUND: In vitro models based on brain capillary endothelial cells (BCECs) are among the most versatile tools in blood-brain barrier research for testing drug penetration into the brain and how this is affected by efflux transporters such as P-glycoprotein (Pgp). However, compared to freshly isolated brain capillaries or primary BCECs, the expression of Pgp in immortalized BCEC lines is markedly lower, which prompted us previously to transduce the widely used human BCEC line hCMEC/D3 with a doxycycline-inducible MDR1-EGFP fusion plasmid. The EGFP-labeled Pgp in these cells allows studying the localization and trafficking of the transporter and how these processes are affected by drug exposure. Here we used this strategy for the rat BCEC line RBE4 and performed a face-to-face comparison of RBE4 and hCMEC/D3 wild-type (WT) and MDR1-EGFP transduced cells. METHODS: MDR1-EGFP-transduced variants were derived from WT cells by lentiviral transduction, using an MDR1-linker-EGFP vector. Localization, trafficking, and function of Pgp were compared in WT and MDR1-EGFP transduced cell lines. Primary cultures of rat BCECs and freshly isolated rat brain capillaries were used for comparison. RESULTS: All cells exhibited typical BCEC morphology. However, significant differences were observed in the localization of Pgp in that RBE4-MDR1-EGFP cells expressed Pgp primarily at the plasma membrane, whereas in hCMEC/D3 cells, the Pgp-EGFP fusion protein was visible both at the plasma membrane and in endolysosomal vesicles. Exposure to doxorubicin increased the number of Pgp-EGFP-positive endolysosomes, indicating a lysosomotropic effect. Furthermore, lysosomal trapping of doxorubicin was observed, likely contributing to the protection of the cell nucleus from damage. In cocultures of WT and MDR1-EGFP transduced cells, intercellular Pgp-EGFP trafficking was observed in RBE4 cells as previously reported for hCMEC/D3 cells. Compared to WT cells, the MDR1-EGFP transduced cells exhibited a significantly higher expression and function of Pgp. However, the junctional tightness of WT and MDR1-EGFP transduced RBE4 and hCMEC/D3 cells was markedly lower than that of primary BCECs, excluding the use of the cell lines for studying vectorial drug transport. CONCLUSIONS: The present data indicate that MDR1-EGFP transduced RBE4 cells are an interesting tool to study the biogenesis of lysosomes and Pgp-mediated lysosomal drug trapping in response to chemotherapeutic agents and other compounds at the level of the blood-brain barrier.

NIR-II light triggered nitric oxide release nanoplatform combined chemo-photothermal therapy for overcoming multidrug resistant cancer.

The overexpression of P-glycoprotein (P-gp) in multidrug resistance (MDR) cancer cells increases the efflux of anticancer drugs thereby causing the failure of clinical chemotherapy. To address this obstacle, in this study, we rationally designed a near-infrared (NIR) light-responsive nitric oxide (NO) delivery nanoplatform for targeting the MDR tumors based on core-shell structured nanocomposites. The mesoporous silica shell provided abundant sites for modification of the NO donor, N-diazeniumdiolate, and tumor-targeting molecule, folic acid (FA), and enabled high encapsulation capacity for doxorubicin (DOX) loading. Under NIR light irradiation, the generation of NO gas can efficiently augment chemotherapeutic effects via the inhibition of P-gp expression. Simultaneously, the photothermal conversion agents of the Cu2-xSe core produce a large amount of heat for photothermal therapy (PTT). Finally, this combinational gas/chemo/PTT not only displays a superior and synergistic effect for overcoming MDR cancer, but also provides an efficient strategy to construct a multifunctional nano-drug delivery system with diversified therapeutic modalities.

Drug resistance gene expression and chemotherapy sensitivity detection in Chinese women with different molecular subtypes of breast cancer.

Objective: The aim of the study was to identify specific chemosensitivity drugs for various molecular subtypes of breast tumors in Chinese women, by detecting the expression of drug resistance genes and by using the drug sensitivity test on different molecular subtypes of breast cancers. Methods: The expression of drug resistance genes including Topo II, GST-π, P-gp, LRP, and CD133 were detected with immunohistochemistry in a tissue microarray. Drug sensitivity tests included those for paclitaxel, epirubicin, carboplatin, vinorelbine, and fluorouracil and were conducted on primary cancer tissue cells and cell lines, including the T47D, BT-474, and MDA-MB-231 cells and human breast cancer xenografts in nude mice. Results: The different drug resistant genes Topo II, GST-π, P-gp, and LRP were differentially expressed among different molecular subtypes of breast cancers (P < 0.05). Positive expression of CD133 was highest in basal-like breast cancer (P < 0.05). Kaplan-Meier survival analysis showed that positive expressions of Topo II and CD133 both correlated with shorter disease-free survival (DFS) (P < 0.05) and overall survival (P < 0.05), and positive expression of LRP correlated only with shorter DFS (P < 0.05). BT-474 showed chemosensitivity to paclitaxel and epirubicin, while MDA-MB-231 showed chemosensitivities to paclitaxel, epirubicin, carboplatin, and fluorouracil (T/C ≤ 50%). The basal-like and HER2+ breast cancer primary cells showed chemosensitivities to paclitaxel and epirubicin with significant differences compared with luminal breast cancer primary cells (P < 0.05). Conclusions: The differential expression of drug resistance genes and the differential chemosensitivities of drugs in different molecular subtype of breast cancers suggested that individual treatment should be given for each type of breast cancer.

P-Gp and TOPO II Expression and Their Clinical Significance in Colon Cancer.

OBJECTIVE: Analysis of P-Gp and TOPO II expression levels and their clinical significance in colon cancer. METHODS: A total of 300 cases of paraffin embedded specimens of primary colon cancer were collected from the Affiliated Hospital of Putian University. The levels of P-Gp and TOPO II expression in colon cancer tissues were detected by the two-step En Vison. RESULTS: P-glycoprotein (P-Gp) was mainly localized in the membrane and nucleus of colon cancer cells and the positive expression rate was 62% (186/300). The positive expression of P-Gp correlated with gender, invasion depth and lymph node metastasis and the differences were statistically significant (P<0.05). Topoisomerase II (TOPO II) was mainly expressed in the cell nucleus and the positive rate was 83.3%. A total of 250 out of 300 samples were TOPO II positive and exhibited significant correlation with invasion depth, lymph node metastasis and differentiation degree (P<0.05). Spearman correlation analysis indicated that P-Gp and TOPO II expression levels in colon cancer were positively correlated (r=0.480). CONCLUSION: P-Gp and TOPO II expression levels were associated with colon cancer progression.

Reversal of multidrug resistance in leukemia cells using a transferrin-modified nanomicelle encapsulating both doxorubicin and psoralen.

To ameliorate multidrug resistance (MDR) observed in leukemia cells, nanomicelles modified by transferrin (Tf-M-DOX/PSO), coencapsulating doxorubicin (DOX) and psoralen (PSO), were designed, synthesized and tested in K562 and doxorubicin-resistant K562 (K562/DOX) cells. In vitro drug release kinetics for constructed nanomicelles were measured using high-performance liquid chromatography. Characterization of the produced nanomicelles was completed using transmission electron microscopy and dynamic light scattering. Uptake of the nanomicelles in K562 cells was investigated using both confocal microscopy and flow cytometry. Apoptosis levels as well as the expression of glycoprotein (P-gp) were analyzing by western blotting and flow cytometry. Cellular cytotoxicity resulting from the exposure of nanomicelles was evaluated using MTT assays. The nanomicelles all showed mild release of DOX in PBS solution. In K562/DOX cells, Tf-M-Dox/PSO exhibited higher uptake compared to the other nanomicelles observed. Furthermore, cellular cytotoxicity when exposed to Tf-M-Dox/PSO was 2.8 and 1.6-fold greater than observed in the unmodified DOX and Tf-nanomicelles loaded with DOX alone, respectively. Tf-M-Dox/PSO strongly increased apoptosis of K562/DOX cells. Finally, the reversal of the drug resistance when cells are exposed to Tf-M-DOX/PSO was associated with P-gp expression inhibition. The Tf-M-Dox/PSO nanomicelle showed a reversal of MDR, with enhanced cellular uptake and delivery release.

Expression and Cellular Distribution of P-Glycoprotein and Breast Cancer Resistance Protein in Amyotrophic Lateral Sclerosis Patients.

For amyotrophic lateral sclerosis (ALS), achieving and maintaining effective drug levels in the brain is challenging due to the activity of ATP-binding cassette (ABC) transporters which efflux drugs that affect drug exposure and response in the brain. We investigated the expression and cellular distribution of the ABC transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) using immunohistochemistry in spinal cord (SC), motor cortex, and cerebellum from a large cohort of genetically well characterized ALS patients (n = 25) and controls (n = 14). The ALS group included 17 sporadic (sALS) and 8 familial (fALS) patients. Strong P-gp expression was observed in endothelial cells in both control and ALS specimens. Immunohistochemical analysis showed higher P-gp expression in reactive astroglial cells in both gray (ventral horn) and white matter of the SC, as well as in the motor cortex of all ALS patients, as compared with controls. BCRP expression was higher in glia in the SC and in blood vessels and glia in the motor cortex of ALS patients, as compared with controls. P-gp and BCRP immunoreactivity did not differ between sALS and fALS cases. The upregulation of both ABC transporters in the brain may explain multidrug resistance in ALS patients and has implications for the use of both approved and experimental therapeutics.

Can curcumin along with chemotherapeutic drug and lipid provide an effective treatment of metastatic colon cancer and alter multidrug resistance?

Cancer is one of the most deadly diseases spreading all over the world and a major cause of fear in the society. Colon cancer is the 4th most common cancer causing death in both male and female equally, mainly caused due to the improper diet plans, consumption of the red meat and lack of exercise. Although the design of the chemotherapeutic drugs is well advanced, many of them developed resistance towards the cancer cells. The major reason behind the drug resistance in the colon cancer cells is due to the action exhibited by P-gp, which belongs to a member of ABC transporter family. P-glycoprotein (P-gp) effluxes the drug from its entry into the cancer cells, by treating it as a foreign body and hence decreases the therapeutic concentration of chemotherapeutic drugs inside the cancer cells. For overcoming this scenario, we posit the use of the curcumin (as a flavonoid) along with the lipid and the chemotherapeutic drug to provide an effective therapy and to overcome the possible issues associated with the failure in the therapy. Curcumin possesses dual mode of actions as a chemosensitizing agent and also as a chemotherapeutic drug. It generally acts as a chemosensitizer which can alter or inhibit the efflux pump exhibited by P-gp and provide a pathway for the entry of the chemotherapeutic drug into the cancer cells. Lipids have the potential to overcome the Multidrug resistance (MDR) and related issues; in addition, lipids are used for targeting colon cancer cells and also can act during the metastatic condition of the cancer which is hypothesised to be proven by using various studies. If our hypothesis is proven, the use of curcumin with lipids and the chemotherapeutic drug in a novel combination will reduces the majority of the issues related to the multidrug resistance, the recurrence and the spread of cancer could be overcome in a safe and effective manner.

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.

Proteomic analysis of small intestinal epithelial cells in antibiotic-treated mice: Changes in drug transporters and metabolizing enzymes.

Antibiotics act on bacterial flora originally present in the intestine, and changes in the intestinal flora have various effects on the host. This study investigated changes in the protein levels of drug transporters and metabolizing enzymes in the small intestines of antibiotic-treated mice by proteomic analysis. After the oral administration of non-absorbable antibiotics (vancomycin and polymyxin B) for 5 days, 15 drug transporter or metabolizing enzyme proteins had significantly changed levels among 1780 proteins identified in small intestinal epithelial cells. Of these, the levels of peptide transporter 1 (Pept1), multidrug resistance protein 1a (Mdr1a), and multidrug resistance-associated protein 2 (Mrp2) were increased approximately 2-fold. In addition, the levels of two Cyp4f proteins were decreased and those of Cyp4b1, Ces1d, and three glutathione S-transferase (Gst) proteins were increased. Our results indicate that the oral administration of antibiotics changes the levels of proteins related to the absorption and metabolism of drugs in the small intestine, and suggest that substrate drugs of these proteins have a risk for indirect drug interactions with antibacterial drugs via the intestinal flora.

Analysing the antidepressant and drug efflux competence of Clitoria ternateaL. as P-glycoprotein inhibitor to facilitate blood brain barrier

Clitoria ternateaL. is a vital ayurvedic herbfeatured with a wide spectrum of mental health benefits. The present study investigates the competence of the plant against depression and to inhibit the membrane efflux protein P-glycoprotein (P-gp) that can regulate and restrict drug permeation into the brain. Antidepressant competence of the aqueous plant extract was assessed by animal despair studies on depression induced female mice models. The P-glycoprotein inhibitory potential of active phytocompounds was evaluated by molecular docking assay and substantiated by a cell line study. The in vivostudies exhibited a significant difference in the immobility time thereby establishing antidepressant activity. The histopathological sections from cortex region of treated brain showed decreased degenerative changes. Ten imperative phytocompounds facilitated docking complexes against P-glycoprotein among which Kaempferol 3-O-(2״,6״-di-O-rhamnopyranosyl) glucopyranoside exhibited a finest docking score of -12.569 kcal mol-1. Conversely it was attested by the rhodamine transport assay that demonstrated the inhibitory potential to surpass blood brain barrier. The outcome of the study endorses the efficacy of Clitoria ternateaL. as an idyllic brain drug and facilitates brain permeability.

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.

Development and application of a UPLC-MS/MS method for P-glycoprotein quantification in human tumor cells.

Multidrug resistance (MDR) of tumors occurs when tumor cells exhibit reduced sensitivity to a large number of unrelated drugs. The molecular mechanism of MDR commonly involves overexpression of the plasma membrane drug efflux pump P-glycoprotein (P-gp). Overexpression of P-gp may be induced by the selection and/or adaptation of cells during exposure to chemotherapeutic drugs, referred to as acquired P-gp-mediated MDR. This study aimed to establish a P-gp quantification method by Ultra Performance Liquid Chromatography and Tandem Mass Spectrometry (UPLC-MS/MS) to better understand the regulation of P-gp expression and its relationship with the level of drug resistance. Absolute P-gp expression was determined in the human tumor cells MCF-7, HepG-2, and SMMC-7721 and their corresponding drug-resistant subclones MCF-7/ADMs, MCF-7/MXs, MCF-7/MTXs, HepG-2/ADMs, HepG-2/MXs, HepG-2/MTXs, SMMC-7721/ADMs, SMMC-7721/MXs and SMMC-7721/MTXs. A unique 10-mer tryptic peptide (IATEAIENFR) of P-gp was synthesized for developing the quantitative UPLC-MS/MS method with the stable isotope labeled signature peptide IATEAI (13C6,15N1) ENFR as the internal standard (IS). The detection signal was linear in the range of 0.1-100 ng mL-1. Quality control (QC) data showed that the within-run and between-run precision (%RSD) and accuracy (%RE) conformed to acceptable criteria of ±15% for the calibration standards and QCs (±20% at the LLOQ). The UPLC-MS/MS method was first applied to quantify P-gp in HepG-2 and SMMC-7721 cells and their drug-resistant subclones. The results confirmed that P-gp expression in most drug-resistant subclones increase significantly compared to parental tumor cells but varied among different types of drugs or tumor cells. This outcome was then compared with published reports and discrepancy was observed in HepG2 cell lines mainly due to different sample types and samples sources. Additionally, P-gp mRNA results ascertained that overexpression of P-gp in subclones was not only regulated by MDR1. The linear correlation between RI and logarithm-transformed P-gp expression was moderate or high and statistically significantly different in subclones, except for SMMC-7721/ADMs. The present study is the first to demonstrate the quantitative relationship between RI and P-gp expression by linear regression modeling and expanded the number of efflux transporters related to MDR quantifiable by LC-MS/MS to better understand the biological significance of effluent transporter expression.

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.

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.

Magnetic-Immuno-Loop-Mediated Isothermal Amplification Based on DNA Encapsulating Liposome for the Ultrasensitive Detection of P-glycoprotein.

Determination of proteins, especially low-abundance proteins with high sensitivity and specificity, is essential for characterizing proteomes and studying their biochemical functions. Herein, a novel Magnetic-Immuno-Loop-Mediated Isothermal Amplification (Im-LAMP) based on DNA-encapsulating liposomes (liposome-Im- LAMP), was developed for trace amounts of proteins. To the best of our knowledge, this is our first report about the magnetic Im-LAMP approach based on liposomes encapsulated template DNA as the detection reagent. The DNA template was released from liposomes and then initiated an Im-LAMP reaction, generating the fluorescence signal with high sensitivity and rapidity. This technique was applied for the determination of P-glycoprotein as a model protein. It was demonstrated that the technique exhibited a dynamic response to P-glycoprotein ranging from 1.6*10-2 to 160 pg/ml with a greatly low detection limit of 5*10-3 pg/ml (5 fg/ml) which is substantially better than conventional enzyme-linked immunosorbent assays (ELISA). This ultra sensitivity was attributed to the LAMP reaction initiated by the enormous DNA targets encapsulated in liposomes. This magnetic liposome-Im-LAMP as an alternative approach is attractive for applications in other low-abundance proteins detection in clinical diagnostics.

Strategies of Drug Transporter Quantitation by LC-MS: Importance of Peptide Selection and Digestion Efficiency.

Huge variation of drug transporter abundance was seen in the literature, making PBPK prediction difficult when transporters play a major role. Among multiple factors such as membrane fraction, digestion, and peptide selection that contributed to such variation, peptide selection is the least discussed. Herein, a strategy was established by using a small amount of purified protein standard to select a peptide with near 100% digestion efficiency for quantitation of a transporter protein MDR1. The impact of native membrane protein's tertiary structure on the digestion efficiency of surrogate peptides of MDR1 was investigated. Peptides in more solvent accessible regions are found to be digested much more efficiently than those in large stretches of helical structures. The concentration of peptide EALDESIPPVSFWR(EAL) in the most solvent accessible linker region of MDR1 was found closest to the true protein concentration. When using EAL for MDR1 quantitation, the abundance is over 10 times higher than previously reported, indicating the importance of peptide selection for transporter quantitation. In addition, this study also proposes a screening strategy to select peptides appropriate for relative quantitation for in vitro-in vivo extrapolation in the absence of any protein standard.

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.