MDR1 Gene Expression Research Articles

Expression of Multi-drug Resistance-Related Genes MDR3 and MRP as Prognostic Factors in Clinical Liver Cancer Patients

To investigate the expression of multi-drug resistance-related genes, MDR3 and MRP, in clinical specimens of primary liver cancer and their potential as prognostic factors in liver cancer patients. A total of 26 patients with primary liver cancer were enrolled. The expression of MDR3 and MRP genes was measured by real-time PCR and the association between gene expression and the prognosis of patients was analyzed by the Kaplan-Meier method and COX regression model. This study showed that increases in MDR3 gene expression were identified in cholangiocellular carcinoma, cirrhosis and HBsAg-positive patients, while MRP expression increased in hepatocellular carcinoma, non-cirrhosis and HBsAg-negative patients. Moreover, conjugated bilirubin and total bile acid in the serum were significantly reduced in patients with high MRP expression compared to patients with low expression. The overall survival tended to be longer in patients with high MDR3 and MRP expression compared to the control group. MRP might be an independent prognostic factor in patients with liver cancer by COX regression analysis. MDR3 and MRP may play important roles in liver cancer patients as prognostic factors and their underlying mechanisms in liver cancer are worthy of further investigation.

Perifosine downregulates MDR1 gene expression and reverses multidrug-resistant phenotype by inhibiting PI3K/Akt/NF-κB signaling pathway in a human breast cancer cell line

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is the major clinical impediment to chemotherapy of breast cancers. Down-regulation of PI3K/Akt pathway has been described as related to reversal of MDR in cancer cells. Here, we investigated the reversal effect on MDR phenotype of perifosine, a new Akt inhibitor, in breast cancer cell lines. In this study, MCF-7/ADM cells and MCF-7 cells were treated with different concentrations of perifosine. Our results suggested that perifosine reversed MDR partially by downregulation of P-gp expression and inhibition of PI3K/Akt/NF-κB pathway in the MCF-7/ADM cell line. The novel Akt inhibitor perifosine may be a promising new drug due to its ability to reverse MDR in human breast cancer cells.

The Mevalonate Pathway As a Metabolic Target to Circumvent Multidrug-Resistance in Chronic Lymphocytic Leukemia Cells

Abstract 735The mutational status of the tumor immunoglobulin heavy chain variable region (IGHV) is a very reliable prognosticator in chronic lymphocytic leukemia (CLL): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. We have recently shown that UM CLL cells are more dependent on microenvironment-mediated signals for survival (Coscia M. et al, Leukemia, 2011), but it is currently unknown whether this also translates into an enhanced chemotherapy resistance. Multi drug resistance (MDR) is mediated by the over-expression of membrane transporters, like P-glycoprotein (Pgp), which actively extrudes several anticancer drugs. The Pgp protein is the product of the mdr1 gene whose regulation is under the positive control of the mevalonate (Mev) metabolic pathway, the downstream Ras/RhoA dependent signaling pathways and the transcription factor Hypoxia-Inducible-Factor-1-alfa (HIF-1α). The aim of this study was to investigate the MDR phenotype and signaling cascade in M and UM CLL cells under basal conditions and after exposure to prosurvival microenvironment-mediated signals.Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation from untreated CLL patients. More than 90% pure CLL cells were co-cultured with the murine stromal cell line M2-10B4. Culture conditions included: medium alone, Zoledronic acid (ZA) (1 μmol/L), ERK1/2 kinase inhibitor PD98059 (10 μmol/L) and the HIF-1α inhibitor YC-1 (10 μmol/L). The level of activity of the Mev pathway was evaluated by measuring the intermediate metabolite farnesyl pyrophosphate (FPP) and the final product cholesterol (Chol) as previously described (Coscia M. et al, J Cell Mol Med, 2010). The Ras and RhoA activity, was evaluated by measuring the GTP binding. Rho kinase activity was measured using the CycLex Rho Kinase Assay Kit (CycLex Co., Nagano). The following antibodies were used for Western Blot (WB) analyses: anti phospho-(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2; anti-ERK 1/2; anti-HIF-1α; anti-Pgp; anti-GAPDH. The activity of HIF-1 in the nuclear extracts was assessed by the TransAM™ HIF-1 Transcription Factor Assay Kit (Active Motif). Mdr1 gene expression was evaluated by RT-PCR. Intracellular Doxorubicine (Doxo) accumulation was evaluated by cytofluorimetric analyses.We have found that the Mev pathway is significantly accelerated in freshly isolated UM vs M CLL cells. This hypermetabolic activity translates into a higher activation of the Ras/ERK-1/2 and RhoA/Rho kinase signaling pathways, and to a significantly more active HIF-1α in UM compared to M CLL cells. HIF-1α activation positively regulates mdr1 gene expression in UM CLL cells leading to Pgp expression and more effective Doxo extrusion in these cells. The co-culture of CLL cells with the murine stromal cell line M2-10B4 further accelerated the Mev pathway and activated the HIF-1α/mdr1/PgP axis leading to even lower intracellular Doxo retention. This stroma-mediated effect was particularly evident in UM CLL cells. Targeting of the Mev pathway with ZA and statins, and targeting of ERK-1/2 and HIF-1α with specific inhibitors (PD85 and YC1-10, respectively) significantly reduced basal and stroma-induced activity of HIF-1α/mdr1/PgP axis, and significantly increased the intracellular concentrations of Doxo in UM CLL cells. These data indicate that the pharmacological inhibition of the Mev pathway and of the downstream HIF-1α/mdr1/PgP axis can be regarded as a novel potential strategy to circumvent basal and environment-mediated chemoresistance of CLL cells. Disclosures:Massaia:Novartis Farma S.p.A: Honoraria, Research Funding, scientific adviser board.

Forced expression of Hsp27 Reverses P-Glycoprotein (ABCB1) Mediated Drug Efflux and MDR1 Gene Expression in Adriamycin Resistant Human Breast Cancer Cells

Forced expression of Hsp27 Reverses P-Glycoprotein (ABCB1) Mediated Drug Efflux and MDR1 Gene Expression in Adriamycin Resistant Human Breast Cancer Cells

Anticancer Effects of 15d-Prostaglandin-J2 in Wild-Type and Doxorubicin-Resistant Ovarian Cancer Cells: Novel Actions on SIRT1 and HDAC

15-deoxy-delta-12,14-prostaglandin-J2 (15d-PGJ2), an arachidonic metabolite and a natural PPARγ agonist, is known to induce apoptosis in tumor cells. In this study, we investigated new therapeutic potentials of 15d-PGJ2 by determining its anticancer effects in wild-type and doxorubicin-resistant ovarian carcinoma cells. Despite high expression of resistance-inducing genes like MDR1, Bcl2 and Bcl-xl, 15d-PGJ2 strongly induced apoptosis in doxorubicin-resistant (A2780/AD) cells similar to the wild-type (A2780). This was found to be related to caspase-3/7- and NF-κB pathways but not to its PPARγ agonistic activity. 15d-PGJ2 also was able to reduce the doxorubicin resistance of A2780/AD cells at low doses as confirmed by the inhibition of gene expression of MDR1 (p-glycoprotein) and SIRT1 (a drug senescence gene). We also investigated effects of 15d-PGJ2 on cell migration and transformation using a wound-healing assay and morphological analyses, respectively. We found that 15d-PGJ2 inhibited migration most likely due to NF-κB inhibition and induced transformation of the round-shape A2780/AD cells into elongated epithelial cells due to HDAC1 inhibition. Using a 15d-PGJ2 analog, we found the mechanism of action of these new activities of 15d-PGJ2 on SIRT1 and HDAC1 gene expressions and enzyme activities. In conclusion, the present study demonstrates that 15d-PGJ2 has a high therapeutic potential to kill drug-resistant tumor cells and, the newly described inhibitory effects of this cyclo-oxygenase product on SIRT1 and HDAC will provide new opportunities for cancer therapeutics.

Forced Expression of Heat Shock Protein 27 (Hsp27) Reverses P-Glycoprotein (ABCB1)-mediated Drug Efflux and MDR1 Gene Expression in Adriamycin-resistant Human Breast Cancer Cells

Mutant p53 accumulation has been shown to induce the multidrug resistance gene (MDR1) and ATP binding cassette (ABC)-based drug efflux in human breast cancer cells. In the present work, we have found that transcriptional activation of the oxidative stress-responsive heat shock factor 1 (HSF-1) and expression of heat shock proteins, including Hsp27, which is normally known to augment proteasomal p53 degradation, are inhibited in Adriamycin (doxorubicin)-resistant MCF-7 cells (MCF-7/adr). Such an endogenous inhibition of HSF-1 and Hsp27 in turn results in p53 mutation with gain of function in its transcriptional activity and accumulation in MCF-7/adr. Also, lack of HSF-1 enhances nuclear factor κB (NF-κB) DNA binding activity together with mutant p53 and induces MDR1 gene and P-glycoprotein (P-gp, ABCB1), resulting in a multidrug-resistant phenotype. Ectopic expression of Hsp27, however, significantly depleted both mutant p53 and NF-κB (p65), reversed the drug resistance by inhibiting MDR1/P-gp expression in MCF-7/adr cells, and induced cell death by increased G(2)/M population and apoptosis. We conclude from these results that HSF-1 inhibition and depletion of Hsp27 is a trigger, at least in part, for the accumulation of transcriptionally active mutant p53, which can either directly or NF-κB-dependently induce an MDR1/P-gp phenotype in MCF-7 cells. Upon Hsp27 overexpression, this pathway is abrogated, and the acquired multidrug resistance is significantly abolished so that MCF-7/adr cells are sensitized to Dox. Thus, clinical alteration in Hsp27 or NF-κB level will be a potential approach to circumvent drug resistance in breast cancer.

Long-Chain Polyunsaturated Fatty Acids Promote Paclitaxel Cytotoxicity via Inhibition of the MDR1 Gene in the Human Colon Cancer Caco-2 Cell Line

Objective: Accumulating evidence in both humans and animal models indicates that dietary intake of long-chain polyunsaturated fatty acids (PUFAs) can improve response to chemotherapy. The intent of this study was to determine the mechanisms by which PUFAs affect the response to anticancer chemotherapy.Methods: Human colorectal cancer cell line Caco-2 was used as a model system in this study. Caco-2 cells were treated with different concentrations of three PUFAs: eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (AA). Real-time polymerase chain reaction was used to determine mdr1 gene (codes for P-glycoprotein [P-gp]) expression. Western blotting and calcein-acetoxymethylester efflux assay were used for P-gp expression and functional evaluation, respectively. Furthermore, apoptosis assay was conducted by adding PUFAs with paclitaxel to confirm the synergetic effect. Finally, gene expression of nuclear receptors CAR and PXR were estimated to evaluate the possible mechanisms.Results: Both classes of PUFAs, omega-3 (ω-3) and omega-6 (ω-6), can cause a modest but very reproducible reduction of gene expression, protein production, and pump activity of MDR1. Incubation of cells with PUFAs greatly enhanced the cytotoxicity of the anticancer drug paclitaxel, manifested mainly through enhanced paclitaxel-induced apoptosis. Furthermore, PUFAs increased the messenger RNA (mRNA) levels of the nuclear receptors CAR and PXR, thus implicating these two transcription factors as cellular targets of PUFAs in cells but not directly affecting MDR1 regulation.Conclusions: Our results suggest that inhibition of the multidrug resistance MDR1/P-gp is one mechanism through which dietary polyunsaturated fatty acids exert a synergetic effect on the response of tumor cells to anticancer drugs.

The cross-talk of extracellular signal-regulated protein kinase (ERK) and pregnane X receptor (PXR) and its effect on expression of CYP3A4 and MDR1 genes in primary human hepatocytes and hepatoma cell lines

The cross-talk of extracellular signal-regulated protein kinase (ERK) and pregnane X receptor (PXR) and its effect on expression of CYP3A4 and MDR1 genes in primary human hepatocytes and hepatoma cell lines

Adenovirus-mediated p53 gene therapy reverses resistance of breast cancer cells to adriamycin

The aim of this study was to determine whether adenovirus-mediated p53 gene (Ad-p53) transfection can enhance adriamycin cytotoxicity and reverse adriamycin resistance in human breast cancer cells and explore its effect on the expression of MDR1 gene and permeability-glycoprotein (P-gp). Human breast cancer cell lines, MCF-7 and MCF-7/ADR, were used in in-vitro studies. After infection with Ad-p53, the cytotoxicity of adriamycin was evaluated using the Cell Counting Kit-8 assay. The expression of MDR1 mRNA was detected by quantitative real-time PCR. The expression of P-gp was analyzed using western blotting. In in-vivo studies, MCF-7/ADR tumor cells were inoculated subcutaneously in athymic nude mice. After 14 days of inoculation, tumor size was measured. Apoptosis and expression of P-gp in the tumor tissue were analyzed by fluorescence activated cell sorting and western blotting. After transfection with a multiplicity of infection of 50 for Ad-p53, chemosensitivity of MCF-7/ADR cells increased by 18.1 times (P=0.001), and 50% inhibitory concentration (IC50) of adriamycin decreased from 4.54 ± 0.91 to 0.26 ± 0.11 mg/l. Real-time PCR showed that MDR1 mRNA decreased from 1.32 to 0.85 (P=0.001). Western blotting analysis showed that P-gp also decreased. In in-vivo studies, Ad-p53 combined with adriamycin dramatically inhibited the growth of subcutaneous xenograft of MCF-7/ADR. The fluorescence activated cell sorting assay showed that there were more apoptotic cells in tumor tissues treated with Ad-p53 and adriamycin. The expression of P-gp was significantly decreased in tumor tissues. This study suggests that Ad-p53 can reverse MCF-7/MDR cell resistance to adriamycin. The reversal effect was associated with inhibition of P-gp expression and induction of apoptosis.

Identification of an antifungal peptide from Trapa natans fruits with inhibitory effects on Candida tropicalis biofilm formation

Due to recent emergence of fungal pathogens resistant to current antifungal therapies, several studies have been focused on screening of plant peptides to find novel compounds having antifungal activities. Here, a novel antifungal plant peptide, with molecular mass of 1230Da was purified from fruits of Trapa natans by reverse phase high performance liquid chromatography using 300SB-C18 column and named as Tn-AFP1. Determination of complete amino acid sequences of this peptide by tandem mass spectrometry showed to contain following eleven amino acid residues: LMCTHPLDCSN. Purified Tn-AFP1 showed the inhibition of Candida tropicalis growth in vitro and disrupted the biofilm formation in a concentration dependent manner. It also showed downregulation of MDR1 and ERG11 gene expression in real time-PCR analysis. In silico molecular modeling predicted the structure of Tn-AFP1 as a single coil attached by a unique disulfide bond. Characterization of Tn-AFP1 could contribute in designing novel derivative(s) of this peptide for the development of more effective antimycotic compounds.

The role of paclitaxel in the development and treatment of multidrug resistant cancer cell lines

Paclitaxel (PTX) is used for treatment of wide range of solid tumors, but its efficacy is often limited by appearance of multidrug resistance (MDR). We explored the MDR induced by PTX in human colon cancer DLD1 and glioblastoma U87 cell lines. After confirmation of the cross-resistance to other anticancer agents in newly established DLD1-TxR and U87-TxR, we analyzed the mRNA expression of membrane transporters involved in MDR. The cells had increased levels of mdr1 gene expression, while mrp1 was decreased. Flow cytometry analyzes showed that the accumulation of P-glycoprotein (P-gp) substrates (rhodamine 123 and doxorubicin) was significantly lower in DLD1-TxR and U87-TxR compared to DLD1 and U87, respectively. The significant depletion of gst-π gene expression and glutathione (GSH) concentration was observed in U87-TxR. The analysis of cell cycle kinetics revealed extensive cell death in colon cancer cells that were accumulated in subG0 phase after PTX treatment, while glioblastoma cells died during interphase (G1, S or G2). The secretion of vascular endothelial growth factor (VEGF) was inhibited by single PTX treatment of colon cancer and in continuous treatment of glioblastoma cell lines. In conclusion, continuous PTX treatment caused the over-expression of P-gp and acquisition of MDR in colon cancer and glioblastoma cell lines, while some mechanisms of MDR and tumor progression such as GSH detoxification system and VEGF secretion were suppressed. Hence, the present results implicate that PTX is an important clinical tool for colon cancer and glioblastoma treatment even in the presence of MDR.

Deactivation of Signal Transducer and Activator of Transcription 3 Reverses Chemotherapeutics Resistance of Leukemia Cells via Down-Regulating P-gp

Multidrug resistance (MDR) caused by overexpression of p-glycoprotein is a major obstacle in chemotherapy of malignant cancer, which usually is characterized by constitutive activation of signal transducer and activator of transcription 3 (STAT3), but their relation between MDR and STAT3 remains unclear. Here, we showed that STAT3 was overexpressed and highly activated in adriamycin-resistant K562/A02 cells compared with its parental K562 cells. Blockade of activation of STAT3 by STAT3 decoy oligodeoxynucleotide (ODN) promoted the accumulation and increased their sensitivity to adriamycin by down-regulating transcription of mdr1 and expression of P-gp, which were further confirmed by using STAT3-specific inhibitor JSI-124. Inhibition of STAT3 could also decrease mdr1 promoter mediated luciferase expression by using mdr1 promoter luciferase reporter construct. Otherwise, activation of STAT3 by STAT3C improved mdr1 transcription and P-gp expression. The ChIP results demonstrated that STAT3 could bind to the potential promoter region of mdr1, and STAT3 decoy depressed the binding. Further mutation assay show +64∼+72 region could be the STAT3 binding site. Our data demonstrate a role of STAT3 in regulation of mdr1 gene expression in myeloid leukemia and suggest that STAT3 may be a promising therapeutic target for overcoming MDR resistance in myeloid leukemia.

Caffeic Acid Phenethyl Ester (CAPE) derived from propolis, a honeybee product, inhibits growth of breast cancer stem cells

SummaryCancer stem cells (CSC) are chemoresistant and implicated in tumor recurrence, metastasis and high patient mortality; thus substances impairing CSC activity, could be invaluable as novel cancer therapeutics. We previously showed that CAPE (caffeic acid phenethyl ester), a component of propolis, a honeybee product, inhibits growth of MDA-MB-231 (MDA-231) cells, mdr gene expression, NF-κB, EGFR, and VEGF. We hypothesized that CAPE also acts by interfering with CSC-mediated effects. We isolated breast CSC (bCSC) from MDA-231 cells, a model of human triple-negative breast cancer, and mouse xenografts. bCSC grow as mammospheres (MMS) and when dissociated into single cells, form MMS again, a sign of self-renewal. bCSC exhibited the characteristic CD44+/CD24-/low phenotype and generated progenitors in the presence of serum, a CSC trait responsible for regenerating tumor mass. CAPE caused dose-dependent bCSC self-renewal inhibition and progenitor formation. Clonal growth on soft agar was inhibited dose-dependently, but apoptosis was not induced as determined by Annexin-V/PI assay. Instead, bCSC were noted to significantly progress from a quiescent cell cycle state in G0/G1 (82%), S phase (12%) to a cycling state with an increase in S phase (41%) and subsequent decrease in G0/G1 (54%). Treatment of bCSC with CAPE (4.5-days) decreased CD44 levels by 95%, while another cell population containing 10-100-fold lower CD44 content concurrently increased. Results suggest that CAPE causes pronounced changes in bCSC characteristics manifested by inhibition of self renewal, progenitor formation, clonal growth in soft agar, and concurrent significant decrease in CD44 content, all signs of decreased malignancy potential.

Abstract 701: A mechanistic study of mdr1a gene regulation in vivo using bioimaging

Abstract The human P-glycoprotein (Pgp) encoded by the MDR1 gene is as a transmembrane drug transporter that mediates the efflux of drugs from cells. It confers multidrug resistance in cancer cells that overexpress MDR1. In addition, basal expression of MDR1 in organs such as liver, kidney, and colon affects the pharmacokinetics of drug uptake and excretion for agents that are substrates for Pgp transport. Nuclear receptors such as SXR and CAR have been suggested as possible master regulators of xenobiotic- and drug-inducible expression of MDR1 and of other genes involved in drug metabolism in drug processing organs. However, the mechanism of MDR1 regulation in tumors or in normal, non-malignant tissue, particularly in response to xenobiotics, has yet to be demonstrated in vivo. To facilitate the mechanistic study of MDR1 gene regulation in vivo, we previously reported a mouse model that allows non-invasive bioimaging of the mdr1 gene expression in vivo and in real time by inserting a firefly luciferase (fLUC) gene into the murine mdr1a genetic locus by homologous recombination.1 To determine the role of PXR, the murine homologue of SXR, in drug-mediated induction of mdr1a, we crossed mdr1a.fLUC mice with PXR knockout mice. Pregnenolone-16α-carbonitrile (PCN), a known SXR substrate, induced intestinal mdr1a.fLUC expression in the PXR wild type background but not the PXR knockout background. Notably, mdr1a.fLUC was re-inducible over three rounds of drug dosing, which could be measured longitudinally in our real-time assessment of gene expression. TCPOBOP, a known CAR ligand, induced mdr1a.fLUC in both the wild type and knockout background, consistent with the idea that mdr1a is also regulated by the CAR nuclear receptor. The chemotherapeutic agent docetaxel induced mdr1a.fLUC in the PXR wild type background but induction was dramatically reduced in the PXR KO background; paclitaxel gave only low-level induction in both genetic backgrounds. This result is consistent with the reported ligand specificity of mouse PXR. Future studies in a CAR-null background will determine if the observed effect of paclitaxel on mdr1a expression is mediated through CAR. These studies demonstrate the utility of our murine imaging model for the in vivo study of mdr1 gene regulation in real time. Studies with additional chemotherapeutic drugs and genetic backgrounds will establish the variety of agents capable of inducing mdr1a at the transcriptional level and the trans-acting factors that are involved in such induction. 1Gu et al. Proc Natl Acad Sci 106:5394-5399 (2009) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 701. doi:10.1158/1538-7445.AM2011-701

Reversal effects of two new milbemycin compounds on multidrug resistance in MCF-7/adr cells in vitro

Development of agents to overcome multidrug resistance (MDR) is important in cancer chemotherapy, and the overexpression of P-glycoprotein (P-gp) is one of the major mechanisms of MDR. In this paper, we evaluated the effects of two new milbemycin compounds, milbemycin β 14 and secomilbemycin D, isolated from fermentation broth of S. bingchenggensis on reversing MDR of adriamycin-resistant human breast carcinoma (MCF-7/adr) cells. We observed that the both milbemycins (5 μM) showed strong potency to increase adriamycin cytotoxicity toward MCF-7/adr cells with reversal fold (RF) of 13.5 and 10.59, respectively. In addition, the mechanisms of milbemycins on reversing P-gp-mediated MDR demonstrated that they significantly increased the accumulations of adriamycin and Rh123 via inhibiting P-gp efflux in MCF-7/adr cells. Furthermore, the results also revealed that milbemycin β 14 and secomilbemycin D could regulate down the expression of P-gp, but not affect the expression of MDR1 gene. In conclusion, our observations suggest that the two new milbemycin compounds probably represent the promising agents for reversing MDR in cancer therapy.

Correlation between the Type of BCR-ABL Transcripts and Blood Cell Counts in Chronic Myeloid Leukemia—A Possible Influence of MDR1 Gene Expression

The impact of BCR-ABL mRNA type (b3a2 vs. b2a2) on chronic myeloid leukemia (CML) phenotype is still a subject of controversies. We searched for a correlation between the BCR-ABL transcripts type and CML patients' characteristics, including MDR1 gene expression. Ninety-eight untreated chronic phase CML patients were studied. The type of BCR-ABL fusion transcripts and MDR1 gene expression were determined by reverse transcriptase polymerase chain reaction. B3a2 and b2a2 transcripts were found in 53 [54%] and 44 [45%] patients, respectively. One patient co-expressed b3a2/b2a2 and was excluded from analysis. The only difference in the clinical characteristics between the two groups was the platelets count, that was higher in b3a2(+) patients [791.3±441.3×109/L vs. 440.4±283.4×109/L in b2a2(+); P=0.007]. MDR1 over-expression [MDR1(+)] was observed in 48 patients (49.5%), more frequently in older patients >60 years [71% (24/34) vs. 38% (24/63) in younger; P=0.008], and was associated with a lower white blood cells (WBC) count [105.5±79.8× 109/L vs. 143.6±96.5×109/L in MDR1(−) cases; P=0.047]. On performing the analysis only within the MDR1(+) group, the b3a2(+) cases were characterized with a significantly higher platelets count [908.7±470.1×109/L vs. 472.9±356.1×109/L; P=0.006] and a lower WBC count [85.4±61.2×109/L vs. 130±93.9×109/L; P=0.004) compared to b2a2(+) patients. No similar differences were found between b3a2(+) and b2a2(+) groups with normal MDR1 levels. These results indicate that the type of BCR-ABL transcripts correlates with the hematological parameters of CML, however only in the subgroup of patients characterized by MDR1 over-expression.

Expression and functional activity of P-glycoprotein in passaged primary human nasal epithelial cell monolayers cultured by the air–liquid interface method for nasal drug transport study

P-glycoprotein (P-gp) is an efflux transporter encoded by the multidrug resistance gene (MDR1), which is also known as the human ABCB1 gene (ATP-binding cassette, subfamily-B). The objectives of this study were to investigate the expression of P-gp in passaged primary human nasal epithelial (HNE) cell monolayer, cultured by the air-liquid interface (ALI) method, and to evaluate its feasibility as an in-vitro model for cellular uptake and transport studies of P-gp substrates. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to verify the expression of the MDR1 gene. Transport and cellular uptake studies with P-gp substrate (rhodamine123) and P-gp inhibitors (verapamil and cyclosporin A) were conducted to assess the functional activity of P-gp in HNE cell monolayers cultured by the ALI method. MDR1 gene expression in primary HNE cell monolayers cultured by ALI method was confirmed by RT-PCR. The apparent permeability coefficient (P(app) ) of the P-gp substrate (rhodamine123) in the basolateral to apical (B to A) direction was 6.9 times higher than that in the apical to basolateral (A to B) direction. B to A transport was saturated at high rhodamine123 concentration, and the treatment of P-gp inhibitors increased cellular uptake of rhodamine123 in a time- and concentration-dependent manner. These results support the MDR1 gene expression and the functional activity of P-gp in primary HNE cell monolayers cultured by the ALI method.

Upregulation of mdr1 gene is related to activation of the MAPK/ERK signal transduction pathway and YB-1 nuclear translocation in B-cell lymphoma

Multidrug resistance (MDR) in human B-cell lymphoma constitutes a major obstacle to the effectiveness of chemotherapy. The aim of this study was to investigate the molecular mechanism of MDR in B-cell lymphoma. The B-cell lymphoma MDR sublines were developed by exposing the parental Daudi cells to stepwise increasing concentrations of doxorubicin. Interaction of Y-box binding protein-1 (YB-1) with the Y-box motif of the mdr1 gene promoters was studied by electrophoretic mobility shift assay. The effects of YB-1 on mdr1 promoter activity were examined by luciferase assay. After silencing of YB-1 gene by shRNA, the role of YB-1 nuclear translocation in the formation of induced MDR was examined. Expression of mdr1 and YB-1 was examined further after Daudi cells were pretreated with mitogen-activated protein kinase (MAPK) inhibitor PD98059 for 1 hour. Doxorubicin-resistant sublines was generated from the Daudi cell line by stepwise selection in doxorubicin. We found that acquisition of MDR is associated with enhanced YB-1 nuclear translocation and MAPK/extracellular signal-regulated kinase (ERK) activity. Electrophoretic mobility shift assay revealed that doxorubicin increased binding of YB-1 to the Y-box of mdr1 promoter. Luciferase reporter assays demonstrated that the Y-box region is essential for YB-1 regulation of mdr1 expression. The introduction of exogenous YB-1 shRNA into Daudi cells resulted in decreased levels of the expression of mdr1 gene and P-glycoprotein induced by doxorubicin. When Daudi cells were pretreated with MAPK inhibitor PD98059, the phosphorylation of ERK was effectively inhibited as well as the nuclear translocation of YB-1 and the expression of mdr1 gene. Doxorubicin can increase expression of mdr1/P-glycoprotein through activating MAPK/ERK transduction pathway, then increasing expression of YB-1, inducing YB-1 nuclear translocation, and enhancing DNA-binding activity of YB-1.

Effect of D, L‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol and tetrandrine on the reversion of multidrug resistance in K562/A02 cells

In this study, we applied D, L‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP) hydrochloride as a chemical inhibitor for glucosylceramide synthase (GCS) and tetrandrine (Tet) for P‐glycoprotein (P‐gp) to reverse daunorubicin (DNR) resistance of human leukemia cell line K562/A02. Cytotoxicity assays showed that either PDMP or Tet enhanced cytotoxic effect of DNR on K562/A02 cells, while cotreatment of these two drugs had a more significant effect on chemosensitization. Using flow cytometric analysis, we confirmed that the enhancement effect was accompanied by elevated cellular DNR accumulation and DNR‐induced apoptosis. According to reverse transcription‐polymerase chain reaction and western blot, the reversal effect of that composite might owe to the significant downregulation of mdr1 and GCS gene expressions. Importantly, PDMP diminished mdr1 gene expression and Tet also downregulated GCS gene expression. Moreover, a positive correlation was observed between GCS and P‐gp. Thus, our results suggest that a potential clinical application of PDMP in combination with Tet may enhance chemosensitivity in leukemia.

Reversal of multidrug resistance by small interfering RNA (siRNA) in doxorubicin-resistant MCF-7 breast cancer cells

Purpose Resistance to anticancer drugs is a serious obstacle to cancer chemotherapy. A common form of multidrug resistance (MDR) is caused by the overexpression of transmembrane transporter proteins P-glycoprotein (P-gp) and multidrug resistance-associated protein-1 (MRP1), encoded by MDR1 and MRP1 genes, respectively. These proteins lead to reduced intracellular drug concentration and decreased cytotoxicity by means of their ability to pump the drugs out of the cells. Breast cancer tumor resistance is mainly associated with overexpression of P-gp/MDR1. Although some chemical MDR modulators aim to overcome MDR by interfering functioning of P-gp, their toxicities limit their usage in clinics. Consequently, RNA interference mediated sequence specific inhibition of the expression of P-gp/MDR1 mRNA may be an efficient tool to reverse MDR phenotype and increase the success of chemotherapy. Aim of this study was resensitizing doxorubicin-resistant breast cancer cells to anticancer agent doxorubicin by selective downregulation of P-gp/MDR1 mRNA. Methods The effect of the selected MDR1 siRNA, and MRP1 expression after MDR1 silencing was determined by qPCR analysis. Intracellular drug accumulation and localization was investigated by confocal laser scanning microscopy after treatment with MDR1 siRNA. XTT cell proliferation assay was performed to determine the effect of MDR1 silencing on doxorubicin sensitivity. Results The results demonstrated that approximately 90% gene silencing occurred by the selected siRNA targeting MDR1 mRNA. However, the level of MRP1 mRNA did not change after MDR1 downregulation. Silencing of P-gp encoding MDR1 gene resulted in almost complete restoration of the intracellular doxorubicin accumulation and relocalization of the drug in the nuclei. Introduction of siRNA resulted in about 70% resensitization to doxorubicin. Conclusions Selected siRNA duplex was shown to effectively inhibit MDR1 gene expression, restore doxorubicin accumulation and localization, and enhance chemosensitivity of resistant cells, which makes it a suitable candidate for therapeutic applications.