Multiple Drug Resistance Transporters Research Articles

Tyrosol induces multiple drug resistance in yeast Saccharomyces cerevisiae.

In yeast, multiple (pleiotropic) drug resistance (MDR) transporters efflux xenobiotics from the cytoplasm to the environment. Additionally, upon the accumulation of xenobiotics in the cells, MDR genes are induced. At the same time, fungal cells can produce secondary metabolites with physico-chemical properties similar to MDR transporter substrates. Nitrogen limitation in yeast Saccharomyces cerevisiae leads to the accumulation of phenylethanol, tryptophol, and tyrosol, which are products of aromatic amino acid catabolism. In this study, we investigated whether these compounds could induce or inhibit MDR in yeast. Double deletion of PDR1 and PDR3 genes, which are transcription factors that upregulate the expression of PDR genes, reduced yeast resistance to high concentrations of tyrosol (4-6 g/L) but not to the other two tested aromatic alcohols. PDR5 gene, but not other tested MDR transporter genes (SNQ2, YOR1, PDR10, PDR15) contributed to yeast resistance to tyrosol. Tyrosol inhibited the efflux of rhodamine 6G (R6G), a substrate for MDR transporters. However, preincubating yeast cells with tyrosol induced MDR, as evidenced by increased Pdr5-GFP levels and reduced yeast ability to accumulate Nile red, another fluorescent MDR-transporter substrate. Moreover, tyrosol inhibited the cytostatic effect of clotrimazole, the azole antifungal. Our results demonstrate that a natural secondary metabolite can modulate yeast MDR. We speculate that intermediates of aromatic amino acid metabolites coordinate cell metabolism and defense mechanisms against xenobiotics.

Successful Prediction of Human Steady-State Unbound Brain-to-Plasma Concentration Ratio of P-gp Substrates Using the Proteomics-Informed Relative Expression Factor Approach.

In order to optimize central nervous system (CNS) drug development, accurate prediction of the drug’s human steady‐state unbound brain interstitial fluid‐to‐plasma concentration ratio (Kp,uu,brain) is critical, especially for drugs that are effluxed by the multiple drug resistance transporters (e.g., P‐glycoprotein, P‐gp). Due to lack of good in vitro human blood‐brain barrier models, we and others have advocated the use of a proteomics‐informed relative expressive factor (REF) approach to predict Kp,uu,brain. Therefore, we tested the success of this approach in humans, with a focus on P‐gp substrates, using brain positron emission tomography imaging data for verification. To do so, the efflux ratio (ER) of verapamil, N‐desmethyl loperamide, and metoclopramide was determined in human P‐gp‐transfected MDCKII cells using the Transwell assay. Then, using the ER estimate, Kp,uu,brain of the drug was predicted using REF (ER approach). Alternatively, in vitro passive and P‐gp–mediated intrinsic clearances (CLs) of these drugs, estimated using a five‐compartmental model, were extrapolated to in vivo using REF (active CL) and brain microvascular endothelial cells protein content (passive CL). The ER approach successfully predicted Kp,uu,brain of all three drugs within twofold of observed data and within 95% confidence interval of the observed data for verapamil and N‐desmethyl loperamide. Using the in vitro–to–in vivo extrapolated clearance approach, Kp,uu,brain was reasonably well predicted but not the brain unbound interstitial fluid drug concentration‐time profile. Therefore, we propose that the ER approach be used to predict Kp,uu,brain of CNS candidate drugs to enhance their success in development.

Do Multiple Drug Resistance Transporters Interfere with Cell Functioning under Normal Conditions?

Eukaryotic cells rely on multiple mechanisms to protect themselves from exogenous toxic compounds. For instance, cells can limit penetration of toxic molecules through the plasma membrane or sequester them within the specialized compartments. Plasma membrane transporters with broad substrate specificity confer multiple drug resistance (MDR) to cells. These transporters efflux toxic compounds at the cost of ATP hydrolysis (ABC-transporters) or proton influx (MFS-transporters). In our review, we discuss the possible costs of having an active drug-efflux system using yeast cells as an example. The pleiotropic drug resistance (PDR) subfamily ABC-transporters are known to constitutively hydrolyze ATP even without any substrate stimulation or transport across the membrane. Besides, some MDR-transporters have flippase activity allowing transport of lipids from inner to outer lipid layer of the plasma membrane. Thus, excessive activity of MDR-transporters can adversely affect plasma membrane properties. Moreover, broad substrate specificity of ABC-transporters also suggests the possibility of unintentional efflux of some natural metabolic intermediates from the cells. Furthermore, in some microorganisms, transport of quorum-sensing factors is mediated by MDR transporters; thus, overexpression of the transporters can also disturb cell-to-cell communications. As a result, under normal conditions, cells keep MDR-transporter genes repressed and activate them only upon exposure to stresses. We speculate that exploiting limitations of the drug-efflux system is a promising strategy to counteract MDR in pathogenic fungi.

Different Efflux Transporter Affinity and Metabolism of 99mTc-2-Methoxyisobutylisonitrile and 99mTc-Tetrofosmin for Multidrug Resistance Monitoring in Cancer.

Little is known about the affinity and stability of 99mTc-labeled 2-methoxyisobutylisonitrile (99mTc-MIBI) and tetrofosmin (99mTc-TF) for imaging of multiple drug resistance transporters in cancer. We examined the affinity of 99mTc-labeled compounds for these transporters and their stability. 99mTc-MIBI and 99mTc-TF were incubated in vesicles expressing P-glycoprotein (MDR1), multidrug resistance-associated protein (MRP)1-4, or breast cancer resistance protein with and without verapamil (MDR1 inhibitor) or MK-571 (MRP inhibitor). Time activity curves of 99mTc-labeled compounds were established using SK-N-SH neuroblastoma, SK-MEL-28 melanoma, and PC-3 prostate adenocarcinoma cell lines, and transporter expression of multiple drug resistance was measured in these cells. The stability was evaluated. In vesicles, 99mTc-labeled compounds had affinity for MDR1 and MRP1. 99mTc-TF had additional affinity for MRP2 and MRP3. In SK-N-SH cells expressing MDR1 and MRP1, MK-571 produced the highest uptake of both 99mTc-labeled compounds. 99mTc-MIBI uptake with inhibitors was higher than 99mTc-TF uptake with inhibitors. 99mTc-TF was taken up more in SK-MEL-28 cells expressing MRP1 and MRP2 than PC-3 cells expressing MRP1 and MRP3. 99mTc-MIBI was metabolized, whereas 99mTc-TF had high stability. 99mTc-MIBI is exported via MDR1 and MRP1 (MRP1 > MDR1) at greater levels and more quickly compared to 99mTc-TF, which is exported via MDR1 and MRP1-3 (MRP1 > MDR1; MRP1, 2 > MRP3). Because 99mTc-MIBI is metabolized, clinical imaging for monitoring MDR and shorter examination times may be possible with an earlier scanning time on late phase imaging. 99mTc-TF has high stability and accurately reflects the function of MDR1 and MRP1-3.

Co-operativity in multiple drug resistance transporter 1 (Mdr1, pGP, ABCB1)

Co-operativity in multiple drug resistance transporter 1 (Mdr1, pGP, ABCB1)

Effect of Korean Red Ginseng extracts on drug-drug interactions

BackgroundGinseng has been the subject of many experimental and clinical studies to uncover the diverse biological activities of its constituent compounds. It is a traditional medicine that has been used for its immunostimulatory, antithrombotic, antioxidative, anti-inflammatory, and anticancer effects. Ginseng may interact with concomitant medications and alter metabolism and/or drug transport, which may alter the known efficacy and safety of a drug; thus, the role of ginseng may be controversial when taken with other medications. MethodsWe extensively assessed the effects of Korean Red Ginseng (KRG) in rats on the expression of enzymes responsible for drug metabolism [cytochrome p450 (CYP)] and transporters [multiple drug resistance (MDR) and organic anion transporter (OAT)] in vitro and on the pharmacokinetics of two probe drugs, midazolam and fexofenadine, after a 2-wk repeated administration of KRG at different doses. ResultsThe results showed that 30 mg/kg KRG significantly increased the expression level of CYP3A11 protein in the liver and 100 mg/kg KRG increased both the mRNA and protein expression of OAT1 in the kidney. Additionally, KRG significantly increased the mRNA and protein expression of OAT1, OAT3, and MDR1 in the liver. Although there were no significant changes in the metabolism of midazolam to its major metabolite, 1′-hydroxymidazolam, KRG significantly decreased the systemic exposure of fexofenadine in a dose-dependent manner. ConclusionBecause KRG is used as a health supplement, there is a risk of KRG overdose; thus, a clinical trial of high doses would be useful. The use of KRG in combination with P-glycoprotein substrate drugs should also be carefully monitored.

A Biophysical Approach Towards Understanding the Transport Mechanism of an ABC L-Methionine Importer

ABC transporters are prevalent in many kingdoms of life. In E. coli, one third of the transporters are ABC transporters. Their proper functions are vital to cell survival. Multiple drug resistance (MDR) transporters are a type of ABC transporters. They pump the drug out of the cell before the drug can exert its effects. Multidrug resistance is a major challenge in cancer and infectious diseases. Such resistance interferes with treatments and leads to longer recovery times and higher mortality rates. In addition, ABC transporters are important in chloride conductance, cholesterol transport, and surface-antigen presentation. ABC transporters in general also play a role in the regulation of movements of small molecules, ions, and macromolecules across membranes. MetNI was discovered by Kadner in the 1970s to efficiently uptake L-methionine in E. coli. In the MetNI system, the interplay between transporter and its binding protein, MetQ delivers L-methionine to the cell. Using a biophysical method, SPR, the kinetics of MetQ binding MetNI have been characterized and rate constants are extrapolated. Using a thermophoresis technique, MST, dissociation constants of this protein-protein interaction with different mutations, substrates, and nucleotide have been quantified. A transport mechanism, which consists of rate and equilibrium constants is being developed. By synthesizing the knowledge on the ATP hydrolysis, binding, transport, and conformational change, we will gain a better understanding of mechanisms of this intricate and important process. Importantly, this model will be able to help predict activities and stability of other members of the type I ABC transporters.

Novel insights into P-glycoprotein in cancer progression

The acquisition of multiple drug resistance (MDR) phenotype is associated with the overexpression of multidrug resistance-associated genes, such as MDR1, MRP1, and BCRP. P-glycoprotein (P-gp), encoded by MDR1, is one of the most extensively characterized MDR transporters in cancer. P-gp mainly functions as a drug pump that excretes chemotherapeutic drugs from cancer cells. However, P-gp participates in cancer progression-related processes, such as cancer cell proliferation, growth, apoptosis, migration, and invasion. Several functions are independent of drug transporter activities. These data suggest that novel mechanisms are employed by P-gp to promote cancer progression. Thus, novel functions of P-gp should be understood and mechanisms by which P-gp promotes cancer aggravation should be determined to improve cancer diagnosis and treatment. In this review, recent research progress on novel contributions of P-gp to cancer progression is summarized.

Proteomics investigation reveals cell death-associated proteins of basidiomycete fungus Trametes versicolor treated with Ferruginol.

Ferruginol has antifungal activity against wood-rot fungi (basidiomycetes). However, specific research on the antifungal mechanisms of ferruginol is scarce. Two-dimensional gel electrophoresis and fluorescent image analysis were employed to evaluate the differential protein expression of wood-rot fungus Trametes versicolor treated with or without ferruginol. Results from protein identification of tryptic peptides via liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) analyses revealed 17 protein assignments with differential expression. Downregulation of cytoskeleton β-tubulin 3 indicates that ferruginol has potential to be used as a microtubule-disrupting agent. Downregulation of major facilitator superfamily (MFS)–multiple drug resistance (MDR) transporter and peroxiredoxin TSA1 were observed, suggesting reduction in self-defensive capabilities of T. versicolor. In addition, the proteins involved in polypeptide sorting and DNA repair were also downregulated, while heat shock proteins and autophagy-related protein 7 were upregulated. These observations reveal that such cellular dysfunction and damage caused by ferruginol lead to growth inhibition and autophagic cell death of fungi.

234 Kinetic analysis of dynamic 11C-verapamil PET study: Compartmental v adaptive mixture models comparison

Background: Temporal aspects of dynamic positron emission tomography (PET) generally rely on compartmental models (CM), with questionable assumptions to summarise functional kinetics of injected radio-tracers in living tissues. Material and Methods: The tracer signal time-course is a combination of vascular delivery and tissue retention effects which do not always satisfy the assumptions of tissue homogeneity and instant mixing within compartments required by the CM. This tissue activity time-course can typically be expressed as a convolution between the signal of the tracer in the arterial supply and the tissue residue function. The residue represents the amount of tracer remaining in the tissue and provides a description of the tracer kinetics measurable by the PET scan. Thus, in statistical terms, the residue can be thought of as a survival function for the residence of the tracer in the tissue which does not require the physiological constraints intrinsic in the exponential-like residue needed for CM. Accordingly more flexible novel approaches − nonparametric and later adaptive mixture models − to estimate the residue based on a piecewise linear form have been developed. The new approaches have been successfully probed on two of the most well-established PET radiotracers, O-H2O and F-fluorodeoxyglucose, used for perfusion and glucose metabolism respectively. This study shows the extension of the new approaches to the multiple-drug resistance transporter P-glycoprotein (P-gp) highly present at the blood–brain barrier (BBB) using C-Verapamil (Vp) in healthy humans before and after inhibition of the P-gp by Cyclosporine (CsA) infusion. Previous PET studies on measuring BBB activity by CsA infusion using Vp have revealed difficulties for the CM to recover metabolic information as they require a full understanding of the tracers metabolisation in the tissue, which is still not certain for Vp. This problem is especially latent when the inhibition of the P-gp allows the tracer through the BBB where it is retained once at the brain tissue level. Results: This work examines and evaluate adaptive mixture model as an alternative to conventional compartment techniques in recovering metabolic information from dynamic PET studies with less biological restrictions. Key bioparameters such as flux, flow, blood volume and volume of distribution from the two different approaches are compared. Cross-validation is used to make regional comparisons and evaluate the fit of the two different estimated residue functions to the tissue activity curves. Conclusions: The accuracy of the standard models for Vp PET studies can be questionable, likely because behaviour of Vp metabolites in tissue is still unclear. Significant statistical evidence in favour of the new adaptivemixture models has been found. Supported by Science Foundation Ireland under SFI-PI 11/27 and by the National Institute of Health (NCI) under PO1-CA-42045.

Beyond cellular detoxification: a plethora of physiological roles for MDR transporter homologs in plants.

Higher plants possess a multitude of Multiple Drug Resistance (MDR) transporter homologs that group into three distinct and ubiquitous families—the ATP-Binding Cassette (ABC) superfamily, the Major Facilitator Superfamily (MFS), and the Multidrug And Toxic compound Extrusion (MATE) family. As in other organisms, such as fungi, mammals, and bacteria, MDR transporters make a primary contribution to cellular detoxification processes in plants, mainly through the extrusion of toxic compounds from the cell or their sequestration in the central vacuole. This review aims at summarizing the currently available information on the in vivo roles of MDR transporters in plant systems. Taken together, these data clearly indicate that the biological functions of ABC, MFS, and MATE carriers are not restricted to xenobiotic and metal detoxification. Importantly, the activity of plant MDR transporters also mediates biotic stress resistance and is instrumental in numerous physiological processes essential for optimal plant growth and development, including the regulation of ion homeostasis and polar transport of the phytohormone auxin.

MicroRNAs and drug resistance in prostate cancers.

Prostate cancer is the second leading cause of cancer related death in American men. Androgen deprivation therapy (ADT) is used to treat patients with aggressive prostate cancers. After androgen deprivation therapy, prostate cancers slowly progress to an androgen-independent status. Taxanes (e.g., docetaxel) are used as standard treatments for androgen-independent prostate cancers. However, these chemotherapeutic agents will eventually become ineffective due to the development of drug resistance. A microRNA (miRNA) is a small noncoding RNA molecule, which can regulate gene expression at the post-transcription level. miRNAs elicit their effects by binding to the 3'-untranslated region (3'-UTR) of their target mRNAs, leading to the inhibition of translation or the degradation of the mRNAs. miRNAs have received increasing attention as targets for cancer therapy, as they can target multiple signaling pathways related to tumor progression, metastasis, invasion, and chemoresistance. Emerging evidence suggests that aberrant expression of miRNAs can lead to the development of resistant prostate cancers. Here, we discuss the roles of miRNAs in the development of resistant prostate cancers and their involvement in various drug resistant mechanisms including androgen signaling, apoptosis avoidance, multiple drug resistance (MDR) transporters, epithelialmesenchymal transition (EMT), and cancer stem cells (CSCs). In addition, we also discuss strategies for treating resistant prostate cancers by targeting specific miRNAs. Different delivery strategies are also discussed with focus on those that have been successfully used in human clinical trials.

Cellular uptake and antiproliferative effects of 11-oxo-eicosatetraenoic acid

Cyclooxygenases (COX) metabolize arachidonic acid (AA) to hydroxyeicosatetraenoic acids (HETE), which can then be oxidized by dehydrogenases, such as 15-hydroxyprostaglandin dehydrogenase (15-PGDH), to oxo-eicosatetraenoic acids (ETE). We have previously established that 11-oxo-eicosatetraenoic acid (oxo-ETE) and 15-oxo-ETE are COX-2/15-PGDH-derived metabolites. Stable isotope dilution (SID) chiral liquid chromatography coupled with electron capture atmospheric pressure chemical ionization (ECAPCI) single reaction monitoring (SRM) MS has been used to quantify uptake of 11-oxo-ETE and 15-oxo-ETE in both LoVo cells and human umbilical vein endothelial cells (HUVEC). Intracellular 11-oxo- and 15-oxo-ETE concentrations reached maximum levels within 1 h and declined rapidly, with significant quantitative differences in uptake between the LoVo cells and the HUVECs. Maximal intracellular concentrations of 11-oxo-ETE were 0.02 ng/4 × 105 cells in the LoVo cells and 0.58 ng/4 × 105 cells in the HUVECs. Conversely, maximal levels of 15-oxo-ETE were 0.21 ng/4 × 105 in the LoVo cells and 0.01 ng/4 × 105 in the HUVECs. The methyl esters of both 11-oxo- and 15-oxo-ETE increased the intracellular concentrations of the corresponding free oxo-ETEs by 3- to 8-fold. 11-oxo-ETE, 15-oxo-ETE, and their methyl esters inhibited proliferation in both HUVECs and LoVo cells at concentrations of 2–10 μM, with 11-oxo-ETE methyl ester being the most potent inhibitor. Cotreatment with probenecid, an inhibitor of multiple drug resistance transporters (MRP)1 and 4, increased the antiproliferative effect of 11-oxo-ETE methyl ester in LoVo cells and increased the intracellular concentration of 11-oxo-ETE from 0.05 ng/4 × 105 cells to 0.18 ng/4 × 105 cells. Therefore, this study has established that the COX-2/15-PGDH-derived eicosanoids 11-oxo- and 15-oxo-ETE enter target cells, that they inhibit cellular proliferation, and that their inhibitory effects are modulated by MRP exporters.

5′‐ectonucleotidase mediates multiple‐drug resistance in glioblastoma multiforme cells

Glioblastoma multiforme (GBM) cells are characterised by their extreme chemoresistance. The activity of multiple-drug resistance (MDR) transporters that extrude antitumor drugs from cells plays the most important role in this phenomenon. To date, the mechanism controlling the expression and activity of MDR transporters is poorly understood. Activity of the enzyme ecto-5'-nucleotidase (CD73) in tumor cells, which hydrolyses AMP to adenosine, has been linked to immunosuppression and prometastatic effects in breast cancer and to the proliferation of glioma cells. In this study, we identify a high expression of CD73 in surgically resected samples of human GBM. In primary cultures of GBM, inhibition of CD73 activity or knocking down its expression by siRNA reversed the MDR phenotype and cell viability was decreased up to 60% on exposure to the antitumoral drug vincristine. This GBM chemosensitization was caused by a decrease in the expression and activity of the multiple drug associated protein 1 (Mrp1), the most important transporter conferring multiple drug resistance in these cells. Using pharmacological modulators, we have recognized the adenosine A(3) receptor subtype in mediation of the chemoresistant phenotype in these cells. In conclusion, we have determined that the activity of CD73 to trigger adenosine signaling sustains chemoresistant phenotype in GBM cells.

The Androgen Metabolite, 5α-androstane-3β,17β-diol, Decreases Cytokine-Induced Cyclooxygenase-2, Vascular Cell Adhesion Molecule-1 Expression, and P-Glycoprotein Expression in Male Human Brain Microvascular Endothelial Cells

P-glycoprotein (Pgp), a multiple drug resistance transporter expressed by vascular endothelial cells, is a key component of the blood-brain barrier and has been shown to increase after inflammation. The nonaromatizable androgen, dihydrotestosterone (DHT), decreases inflammatory markers in vascular smooth muscle cells, independent of androgen receptor (AR) stimulation. The principal metabolite of DHT, 5α-androstane-3β,17β-diol (3β-diol), activates estrogen receptor (ER)β and similarly decreases inflammatory markers in vascular cells. Therefore, we tested the hypothesis that either DHT or 3β-diol decrease cytokine-induced proinflammatory mediators, vascular cell adhesion molecule-1 (VCAM-1) and cyclooxygenase-2 (COX-2), to regulate Pgp expression in male primary human brain microvascular endothelial cells (HBMECs). Using RT-qPCR, the mRNAs for AR, ERα, and ERβ and steroid metabolizing enzymes necessary for DHT conversion to 3β-diol were detected in male HBMECs demonstrating that the enzymes and receptors for production of and responsiveness to 3β-diol are present. Western analysis showed that 3β-diol reduced COX-2 and Pgp expression; the effect on Pgp was inhibited by the ER antagonist, ICI-182,780. IL-1β-caused an increase in COX-2 and VCAM-1 that was reduced by either DHT or 3β-diol. 3β-diol also decreased cytokine-induced Pgp expression. ICI-182,780 blocked the effect of 3β-diol on COX-2 and VCAM-1, but not Pgp expression. Therefore, in cytokine-stimulated male HBMECs, the effect of 3β-diol on proinflammatory mediator expression is ER dependent, whereas its effect on Pgp expression is ER independent. These studies suggest a novel role of 3β-diol in regulating blood-brain barrier function and support the concept that 3β-diol can be protective against proinflammatory mediator stimulation.

Study of resistance to chemotherapy mediated by ABC transporters in biopsies of glioblastoma multiforme

Mortality rate is dramatically high in high grade brain tumors. The presence of multiple drug resistance transporters in glioblastoma multiforme, has contributed largely to the poor efficacy of targeted therapy against cancer in the central nervous system. To analyze the percentage of survival and mortality of patients with glioblastoma multiforme in a cohort of patients in Chile and to co-rrelate the chemo-resistance of these cells with the expression level of multiple drug resistance transporters. Eighteen biopsies of glioblastoma multiforme were obtained from patients at the Institute of Neurosurgery Dr. Asenjo (INCA). The tumor cells were obtained from primary cultures and the expression and activity of multiple drug resistance transporters was assessed by RT-PCR and immunohistochemistry. Population-based study was performed using the databases of the Department of Neurosurgery of INCA. The number of patients with glioblastoma multiforme increased between 2007 and 2009, from 3.5% to 7.9% of total brain tumors. Mortality of these tumors is 90 % at three years. A high expression and activity of the multiple drugs resistance associated protein 1 (Mrp1) transporter was observed in primary cultures of biopsies. We propose that Mrp1 activity is responsible for the chemo-resistance of the glioblastoma multiforme and inhibition of this transporter could represent a plausible strategy for the treatment.

Glioblastoma multiforme y estudio de la resistencia a la quimioterapia mediada por transportadores ABC

Mortality rate is dramatically high in high grade brain tumors. The presence of multiple drug resistance transporters in glioblastoma multiforme, has contributed largely to the poor efficacy of targeted therapy against cancer in the central nervous system.To analyze the percentage of survival and mortality of patients with glioblastoma multiforme in a cohort of patients in Chile and to co-rrelate the chemo-resistance of these cells with the expression level of multiple drug resistance transporters.Eighteen biopsies of glioblastoma multiforme were obtained from patients at the Institute of Neurosurgery Dr. Asenjo (INCA). The tumor cells were obtained from primary cultures and the expression and activity of multiple drug resistance transporters was assessed by RT-PCR and immunohistochemistry. Population-based study was performed using the databases of the Department of Neurosurgery of INCA.The number of patients with glioblastoma multiforme increased between 2007 and 2009, from 3.5% to 7.9% of total brain tumors. Mortality of these tumors is 90 % at three years. A high expression and activity of the multiple drugs resistance associated protein 1 (Mrp1) transporter was observed in primary cultures of biopsies.We propose that Mrp1 activity is responsible for the chemo-resistance of the glioblastoma multiforme and inhibition of this transporter could represent a plausible strategy for the treatment.

Multifunctional Nanoparticles Delivering Small Interfering RNA and Doxorubicin Overcome Drug Resistance in Cancer

Drug resistance is a major challenge to the effective treatment of cancer. We have developed two nanoparticle formulations, cationic liposome-polycation-DNA (LPD) and anionic liposome-polycation-DNA (LPD-II), for systemic co-delivery of doxorubicin (Dox) and a therapeutic small interfering RNA (siRNA) to multiple drug resistance (MDR) tumors. In this study, we have provided four strategies to overcome drug resistance. First, we formed the LPD nanoparticles with a guanidinium-containing cationic lipid, i.e. N,N-distearyl-N-methyl-N-2-(N'-arginyl) aminoethyl ammonium chloride, which can induce reactive oxygen species, down-regulate MDR transporter expression, and increase Dox uptake. Second, to block angiogenesis and increase drug penetration, we have further formulated LPD nanoparticles to co-deliver vascular endothelial growth factor siRNA and Dox. An enhanced Dox uptake and a therapeutic effect were observed when combined with vascular endothelial growth factor siRNA in the nanoparticles. Third, to avoid P-glycoprotein-mediated drug efflux, we further designed another delivery vehicle, LPD-II, which showed much higher entrapment efficiency of Dox than LPD. Finally, we delivered a therapeutic siRNA to inhibit MDR transporter. We demonstrated the first evidence of c-Myc siRNA delivered by the LPD-II nanoparticles down-regulating MDR expression and increasing Dox uptake in vivo. Three daily intravenous injections of therapeutic siRNA and Dox (1.2 mg/kg) co-formulated in either LPD or LPD-II nanoparticles showed a significant improvement in tumor growth inhibition. This study highlights a potential clinical use for the multifunctional nanoparticles with an effective delivery property and a function to overcome drug resistance in cancer. The activity and the toxicity of LPD- and LPD-II-mediated therapy are compared.

Abstract LB-290: Tranilast inhibits breast cancer stem cells

Abstract Cancer stem cells (CSCs) have increased resistance to anti-cancer drugs, and may be responsible for chemotherapeutic failure. CSCs can be enriched from cancer cell lines by growth with anti-cancer drugs (e.g., doxorubicin, mitoxantrone), and form mammospheres in low-adherence, serum-free cultures. We are studying tranilast, a non-toxic orally active drug in clinical use for allergic diseases in Japan, but that we found also targets cancer cells. Previously, we reported that tranilast exerts multiple anti-cancer effects and protects against breast cancer metastasis. Our recent studies show that tranilast strongly inhibits breast CSCs in several assays, at pharmacologically relevant concentrations. We enriched CSCs from breast cancer cell lines by cell sorting for ALDH-1 positive cells (ALDEFLUOR+), or by growth in mitoxantrone-containing medium to select drug-surviving CSCs. In both cases, CSCs readily formed mammospheres in vitro. Tranilast inhibited mammosphere formation,’ and dissociated formed mammospheres. It was more effective than paclitaxel or etoposide. We recently identified molecular targets of this drug. We found it has aryl hydrocarbon receptor (AHR) agonist activity and this might explain its anti-cancer effects. The AHR is a receptor for toxins such as dioxin and polycyclic aromatic hydrocarbons. It is a transcription factor that exerts a number of ligand-dependent effects, such as cell-cycle arrest, reduced RB phosphorylation, and decreased cytokine and growth factor production. Unlike many AHR ligands, tranilast has very low toxicity (LD50 > 1 gm/kg in rats), and is well tolerated by patients. In accord with a role for the AHR, breast CSCs expressed higher levels of this receptor than the main population of cancer cells, and the effects of tranilast were markedly diminished by an AHR antagonist (alpha-naphthoflavone). Moreover, we found tranilast inhibits some ATP-binding cassette (ABC) multiple drug resistance transporters, and it enhances the effects of other anti-cancer drugs. Our studies show that tranilast is a drug of low toxicity that has potential as an anti-CSC drug. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-290.

Mutant TNF-α negatively regulates human breast cancer stem cells from MCF7 in vitro

Understanding the biology of breast cancer stem cells and trying new ways to obliterate these cells would be a key step in developing cures for breast cancer. The objective of this study was to investigate the effect of mutant TNF-α on human breast cancer stem cells derived from MCF7 cell line under the characterization of biologic features of these cells in vitro. By FACS analysis and sorting, we got MCF7 side population (SP) cells and showed that MCF7 SP cells possess cancer stem cell characteristics using the accepted breast cancer stem cell markers, but do not express multiple drug resistance transporters. Furthermore, by RT-PCR, these stem cells were found to constitutively express TNFR-p55 and TNFR-p75. After being treated with Mt rh471 TNF-a, SP cells displayed a decreased self-renewal ability and an increased apoptosis by three different methods. When monocolony antibody against TNFR-p55 was added into the culture medium, the inhibitory effect of Mt rh471 TNF-α on self-renewal was blocked completely, but this was not the case for that of Wt rhTNF-α. The possible reasons might be that the increased binding of Mt rh471 TNF-α mainly to TNFR-p55 results in induction of apoptosis of SP cells, while Wt rhTNF-α could bind to both TNFR-p55 and TNFR-p75 which would lead to NF-κB activity, resulting in a discounted apoptotic effect. These data suggest that Mt rh471 TNF-a might be a negative regulator of the breast cancer stem cell-like cells and have the potential to treat breast cancer in clinic.