Activity Of Drug Efflux Transporters Research Articles

Expressional Study of Permeability Glycoprotein and Multidrug Resistance Protein 1 in Drug-resistant Mesial Temporal Lobe Epilepsy.

About 30% of patients with epilepsy do not respond to anti-epileptic drugs, leading to refractory seizures. The pathogenesis of drug-resistance in mesial temporal lobe epilepsy (MTLE) is not completely understood. Increased activity of drug-efflux transporters might be involved, resulting in subclinical concentrations of the drug at the target site. The major drug-efflux transporters are permeability glycoprotein (P-gp) and multidrug-resistance associated protein-1 (MRP-1). The major drawback so far is the expressional analysis of transporters in equal numbers of drug-resistant epileptic tissue and age-matched non-epileptic tissue. We have studied P-gp and MRP-1 drug-efflux transporters in the sclerotic hippocampal tissues resected from the epilepsy surgery patients (n=15) and compared their expression profile with the tissues resected from non-epileptic autopsy cases (n=15). Statistically significant over expression of both P-gp (P<0.0001) and MRP-1 (P=0.01) at gene and protein levels were found in the MTLE cases. The fold change of P-gp was more pronounced than MRP-1. Immunohistochemistry of the patient group showed increased immunoreactivity of P-gp at blood-brain barrier and increased reactivity of MRP-1 in the parenchyma. The results were confirmed by confocal immunofluorescence microscopy. Our results suggested that P-gp in association with MRP-1 might be responsible for the multi-drug resistance in epilepsy. P-gp and MRP-1 could be important determinants of bio availability and tissue distribution of anti-epileptic drugs in the brain which can pharmacologically inhibited to achieve optimal drug penetration to target site.

Cancer-associated mesothelial cells promote ovarian cancer chemoresistance through paracrine osteopontin signaling.

Ovarian cancer is the leading cause of gynecological malignancy-related deaths, due to its widespread intraperitoneal metastases and acquired chemoresistance. Mesothelial cells are an important cellular component of the ovarian cancer microenvironment that promote metastasis. However, their role in chemoresistance is unclear. Here, we investigated whether cancer-associated mesothelial cells promote ovarian cancer chemoresistance and stemness in vitro and in vivo. We found that osteopontin is a key secreted factor that drives mesothelial-mediated ovarian cancer chemoresistance and stemness. Osteopontin is a secreted glycoprotein that is clinically associated with poor prognosis and chemoresistance in ovarian cancer. Mechanistically, ovarian cancer cells induced osteopontin expression and secretion by mesothelial cells through TGF-β signaling. Osteopontin facilitated ovarian cancer cell chemoresistance via the activation of the CD44 receptor, PI3K/AKT signaling, and ABC drug efflux transporter activity. Importantly, therapeutic inhibition of osteopontin markedly improved the efficacy of cisplatin in both human and mouse ovarian tumor xenografts. Collectively, our results highlight mesothelial cells as a key driver of ovarian cancer chemoresistance and suggest that therapeutic targeting of osteopontin may be an effective strategy for enhancing platinum sensitivity in ovarian cancer.

Targeting Discoidin Domain Receptor 1 (DDR1) Signaling and Its Crosstalk with β1-integrin Emerges as a Key Factor for Breast Cancer Chemosensitization upon Collagen Type 1 Binding.

Collagen type 1 (COL1) is a ubiquitously existing extracellular matrix protein whose high density in breast tissue favors metastasis and chemoresistance. COL1-binding of MDA-MB-231 and MCF-7 breast cancer cells is mainly dependent on β1-integrins (ITGB1). Here, we elucidate the signaling of chemoresistance in both cell lines and their ITGB1-knockdown mutants and elucidated MAPK pathway to be strongly upregulated upon COL1 binding. Notably, Discoidin Domain Receptor 1 (DDR1) was identified as another important COL1-sensor, which is permanently active but takes over the role of COL1-receptor maintaining MAPK activation in ITGB1-knockdown cells. Consequently, inhibition of DDR1 and ERK1/2 act synergistically, and sensitize the cells for cytostatic treatments using mitoxantrone, or doxorubicin, which was associated with an impaired ABCG2 drug efflux transporter activity. These data favor DDR1 as a promising target for cancer cell sensitization, most likely in combination with MAPK pathway inhibitors to circumvent COL1 induced transporter resistance axis. Since ITGB1-knockdown also induces upregulation of pEGFR in MDA-MB-231 cells, inhibitory approaches including EGFR inhibitors, such as gefitinib appear promising for pharmacological interference. These findings provide evidence for the highly dynamic adaptation of breast cancer cells in maintaining matrix binding to circumvent cytotoxicity and highlight DDR1 signaling as a target for sensitization approaches.

Abstract IA07: Genetic determinants of tumor development, therapy response and resistance in mouse models of BRCA-deficient breast cancer

Abstract Heterozygous germline mutations in BRCA1 or BRCA2 strongly predispose to development of breast and ovarian cancer (as well as other cancer types) via loss of the remaining wildtype allele. BRCA1/2-deficient cancers are defective in DNA double-strand break (DSB) repair via homologous recombination (HR) and therefore hypersensitive to DNA-damaging agents, including platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors. However, these treatments do not result in tumor eradication and eventually resistance develops. To maximize therapeutic efficacy of these drugs and achieve durable remissions, it is important to unravel the mechanisms by which these tumors acquire resistance to platinum drugs and PARP inhibitors, in order to develop combination therapies that prevent development of resistance or re-sensitize resistant tumors. To study therapy response and resistance in a realistic in vivo setting, we have established several genetically engineered mouse models (GEMMs) and patient-derived tumor xenograft (PDX) models for BRCA-deficient breast cancer. These mice develop mammary tumors that are characterized by genomic instability and hypersensitivity to DNA-damaging agents, including platinum drugs and PARP inhibitors. Using cross-species oncogenomics and reverse genetics, we have identified several cancer genes including p53, MYC and RB as critical drivers in BRCA1-associated breast cancer. In addition, we have used these mammary tumor models for preclinical evaluation of therapy response and elucidation of mechanisms of acquired drug resistance. Using functional genetic screens, reverse genetics and genomic analysis of therapy-resistant tumors, we found that therapy response and resistance of BRCA1-deficient mammary tumors to cisplatin and the clinical PARP inhibitor olaparib is affected by several factors, including drug efflux transporter activity, type of BRCA1 founder mutation and restoration of HR repair via loss of 53BP1 or REV7. Also BRCA1 re-activation via genetic or epigenetic mechanisms contributes to therapy resistance in PDX models of BRCA1-deficient breast cancer. Importantly, pharmacokinetic or HR-related mechanisms underlie olaparib-resistance in only a fraction of BRCA1/2-deficient mammary tumors, indicating the existence of additional, unknown mechanisms. Citation Format: Jos Jonkers. Genetic determinants of tumor development, therapy response and resistance in mouse models of BRCA-deficient breast cancer [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA07.

Abstract IA08: Studying therapy response and resistance in mouse models of breast cancer

Abstract Women carrying germline mutations in BRCA1 are strongly predisposed to developing triple-negative breast cancers, which frequently contain TP53 mutations. To study the role of BRCA1 loss-of-function in development of triple-negative breast cancer, we have established genetically engineered mouse models (GEMMs) for BRCA1-mutated breast cancer based on cre-mediated tissue-specific deletion of p53 and Brca1. In addition to GEMMs for Brca1-null tumors, we have generated mammary tumor models carrying specific BRCA1 founder mutations (C61G, 185delAG and 5382insC). The mammary tumors that arise in these mouse models show strong similarity to BRCA1-associated breast cancer with respect to expression of basal cell markers, high tumor grade, and lack of expression of hormone receptors and ERBB2. In addition to these GEMMs, we have also established a panel of patient-derived xenograft (PDX) models for BRCA1-proficient and –deficient triple-negative breast cancer. BRCA1 deficiency in these PDX tumors is driven either by genetic mutation or by BRCA1 promoter methylation. The BRCA1-deficient tumors that arise in our various mouse models are in all cases characterized by a high degree of genomic instability and hypersensitivity to DNA-damaging agents due to loss of homology-directed double-strand break (DSB) repair. We have successfully used our GEMMs and PDX models of BRCA1-deficient breast cancer for preclinical evaluation of therapy response and elucidation of mechanisms of acquired drug resistance. BRCA1-deficient mammary tumors are highly sensitive to DNA-damaging agents such as platinum drugs and to PARP inhibitors, which indirectly induce DSBs by inhibiting DNA single-strand break repair. However, none of these drugs are capable of causing tumor eradication and all tumors grow back after drug treatment. By employing in vitro functional genetic screens and in vivo intervention studies, we found that therapy response and resistance is affected by several factors, including type of BRCA1 founder mutation, drug efflux transporter activity, and 53BP1 status. The lack of tumor eradication prompted us to look for additional drugs targeting BRCA-deficient tumors. Using a cell-based screening approach, we found that bifunctional alkylators such as nimustine may cause durable complete remission of BRCA-deficient mouse mammary tumors, suggesting that BRCA-mutated hereditary breast cancers and BRCA-like sporadic tumors may be eradicated by dose-intensive treatment with bifunctional alkylators. In support of this notion, patients with advanced breast cancers that display a BRCA1-like profile of genomic aberrations show a high complete remission rate and long progression-free survival after treatment with high-dose alkylating chemotherapy. Citation Format: Jos Jonkers. Studying therapy response and resistance in mouse models of breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA08.

ES2-2: Mouse Models of Basal-Like Breast Cancer.

Abstract Mouse models of human cancer provide powerful in vivo tools to study the mechanisms underlying drug response and acquired resistance. Once these processes are understood in sufficient detail it may be possible to design (combination) therapies that not only cause complete remissions but also eliminate remnant cells that might elicit recurrent disease. Women carrying germline mutations in BRCA1 are strongly predisposed to developing basal-like breast cancers, which frequently contain TP53 mutations. To study the role of BRCA1 loss-of-function in development of basal-like breast cancer, we and others have established human tumor xenograft models for basal-like-breast cancer as well as genetically engineered mouse models (GEMMs) for BRCA1-mutated basal-like breast cancer based on tissue-specific deletion of p53 and Brca1 [1]. The mammary tumors that arise in these mouse models show strong similarity to basal-like breast cancer with respect to expression of basal cell markers, high tumor grade, and lack of expression of hormone receptors and HER2 [2]. In addition, the BRCA1-mutated basal-like tumors in our mouse models are characterized by a high degree of genomic instability and hypersensitivity to DNA-damaging agents due to loss of homology-directed double-strand break (DSB) repair [3]. We have successfully used the xenograft models and GEMMs of BRCA1-deficient basal-like breast cancer for preclinical evaluation of therapy response and elucidation of mechanisms of acquired drug resistance. BRCA-deficient mammary tumors are highly sensitive to PARP inhibitors and platinum drugs, but none of these drugs is capable of causing tumor eradication and all tumors grow back after drug treatment. Using functional genetic screens and mouse genetics, we found that therapy response and resistance is affected by several factors, including drug efflux transporter activity [3,4], type of BRCA1 founder mutation and 53BP1 status [5]. The lack of tumor eradication prompted us to look for additional drugs targeting BRCA-deficient tumors. Using a cell-based screening approach, we found that bifunctional alkylators such as nimustine may cause durable complete remission of BRCA-deficient mouse mammary tumors [6], suggesting that BRCA-mutated hereditary breast cancers and BRCA-like sporadic tumors may be eradicated by dose-intensive treatment with bifunctional alkylators. In support of this notion, patients with breast cancers that display a BRCA1-like profile of genomic aberrations show a high complete remission rate and long progression-free survival after treatment with high-dose alkylating chemotherapy [7].

Inhibition of P-glycoprotein and multidrug resistance-associated proteins modulates the intracellular concentration of lopinavir in cultured CD4 T cells and primary human lymphocytes

HIV protease inhibitors (HPIs) are an important component of highly active antiretroviral therapy. However, the activity of drug efflux transporters, such as P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRP1/MRP2), may limit intracellular drug accumulation. Drugs that inhibit the activity of drug efflux proteins may, in combination with HPIs, enhance the clinical efficacy of the drugs. The transport of [(14)C]lopinavir was evaluated in peripheral blood mononuclear cells (PBMCs) in the absence or presence of known inhibitors: tariquidar (P-gp), MK571 (MRP), frusemide (MRP1/2), dipyridamole (MRP1/P-gp) and probenecid (MRP2/OATP). The effects of ritonavir, amprenavir and atazanavir on the accumulation of lopinavir were also evaluated in cultured CD4(+) T-lymphoblastoid cells [CEM (parental), CEM(VBL) (P-gp-overexpressing) and CEM(E1000) (MRP1-overexpressing)] and PBMCs. The relative expression of the drug efflux proteins on the PBMCs was assessed by flow cytometric and real-time PCR methods. Tariquidar, MK571, frusemide and dipyridamole all significantly increased the intracellular accumulation of lopinavir in the PBMCs, whereas probenecid decreased it. The cellular accumulation ratio (CAR) of lopinavir was also increased by ritonavir, amprenavir and atazanavir in a concentration-dependent manner in both cell types. The expression of P-gp, MRP1 and MRP2 mRNA were variable and individually did not correlate with CARs of lopinavir. We provide evidence that lopinavir is a substrate of P-gp, MRP1 and MRP2. The intracellular concentration of lopinavir is increased when co-incubated with ritonavir, amprenavir and atazanavir in PBMCs. Manipulation of drug efflux transporters may be a useful strategy for increasing the intracellular concentration and thereby enhancing the clinical efficacy of lopinavir.

Use of genetically modified bakers yeast in high sensitivity screening of antifungal and multidrug resistance modulatory activities in extracts of plants used in traditional medicine

The majority of human fungal infections are caused by two yeast species Candida albicans and Candida glabrata, with the prevalence rate of about 70% and 15% respectively. Although less frequent, Candida glabrata infections are difficult to treat and are associated with high mortality rates due to innate increased tolerance towards azole antifungal drugs. Azoles constitute the most important of the limited number of antifungals classes suitable for treatment of systemic infections. The incidence of Candida albicans infections resistant to azoles treatment also increases. The underlying mechanism frequently found in azole resistant strains is the overproduction of efflux transporters Cdr1p and Cdr2p from the ATP binding cassette superfamily that actively extrude azoles out of cells. In face of the raising resistance there is an urgent need for the identification of novel antifungal compounds not affected by efflux transporters as well as inhibitors of these proteins able to sensitize resistant strains towards currently available antifungals. Bakers yeast Saccharomyces cerevisiae due to its well characterized network of genes involved in multidrug resistance, close similarity to pathogenic yeast and ease of genetic manipulation is well suited for antifungal screening purposes. In this study we have screened a panel of 63 crude extracts of plants used in traditional medicine, mainly from South and Southeast African regions, for their ability to inhibit growth of strains showing different levels of expression of endogenous multidrug efflux transporters. We have also used a strain heterologously overproducing Cdr1p to screen for modulators of its efflux activity. Extracts were also assayed against Candida species. Our results indicate the advantage of genetic modulation of activity of drug efflux transporters in the process of antifungal activity screening in natural products.

Severe frequent ventricular ectopy after exercise as a predictor of death in patients with heart failure

Drug efflux transporters can influence the absorption, tissue distribution and elimination of many therapeutic agents. Modulation of drug efflux transporter activity is being explored as a means for improving the pharmacokinetic and pharmacodynamic properties of various drugs. In this regard, several polymer formulations have been shown to inhibit drug efflux transporters such as P-glycoprotein (P-gp). The current review will focus on Pluronic® block copolymers in particular, the mechanisms involved in the effects of Pluronic® on drug efflux transporters, and the optimal polymer compositions required for inhibition of drug efflux transporters. Special emphasis will be placed on the potential applications of Pluronic® in enhancing the blood–brain barrier (BBB) penetration of drugs.

Effect of pluronic P85 on ATPase activity of drug efflux transporters

Effect of pluronic P85 on ATPase activity of drug efflux transporters

Optimal structure requirements for pluronic block copolymers in modifying P-glycoprotein drug efflux transporter activity in bovine brain microvessel endothelial cells.

Pluronic block copolymer P85 was shown to inhibit the P-glycoprotein (Pgp) drug efflux system and to increase the permeability of a broad spectrum of drugs in the blood-brain barrier (BBB). However, there is an entire series of Pluronics varying in lengths of propylene oxide and ethylene oxide and overall lipophilicity. This study identifies those structural characteristics of Pluronics required for maximal impact on drug efflux transporter activity in bovine brain microvessel endothelial cells (BBMECs). Using a wide range of block copolymers, differing in hydrophilic-lipophilic balance (HLB), this study shows that lipophilic Pluronics with intermediate length of propylene oxide block (from 30 to 60 units) and HLB <20 are the most effective at inhibiting Pgp efflux in BBMECs. The methods used included 1) cellular accumulation studies with the Pgp substrate rhodamine 123 in BBMECs to assess Pgp activity; 2) luciferin/luciferase ATP assay to evaluate changes in cellular ATP; 3) 1,6-diphenyl-1,3,5-hexatriene membrane microviscosity studies to determine alterations in membrane fluidity; and 4) Pgp ATPase assays using human Pgp-expressing membranes. Pluronics with intermediate lipophilic properties showed the strongest fluidization effect on the cell membranes along with the most efficient reduction of intracellular ATP synthesis in BBMEC monolayers. The relationship between the structure of Pluronic block copolymers and their biological response-modifying effects in BBMECs are useful for determining formulations with maximal efficacy for increasing BBB permeability.

In vitro and in vivo models for assessing drug efflux transporter activity

Determining the activity of drug efflux transport proteins has important implications in the identification of substrates and/or inhibitors of the various transport systems, as well as mechanistic determination of localization, and functional role of the transporters in absorption, distribution and elimination of compounds in the body. This review examines both in vitro and in vivo approaches used to determine drug efflux transporter activity, their applications, and advantages and potential limitations.

Pluronic ® block copolymers as modulators of drug efflux transporter activity in the blood–brain barrier

Drug efflux transporters can influence the absorption, tissue distribution and elimination of many therapeutic agents. Modulation of drug efflux transporter activity is being explored as a means for improving the pharmacokinetic and pharmacodynamic properties of various drugs. In this regard, several polymer formulations have been shown to inhibit drug efflux transporters such as P-glycoprotein (P-gp). The current review will focus on Pluronic ® block copolymers in particular, the mechanisms involved in the effects of Pluronic ® on drug efflux transporters, and the optimal polymer compositions required for inhibition of drug efflux transporters. Special emphasis will be placed on the potential applications of Pluronic ® in enhancing the blood–brain barrier (BBB) penetration of drugs.

Relationship between cytotoxic drug response patterns and activity of drug efflux transporters mediating multidrug resistance

Drug activity patterns in 10 human tumor cell lines representing defined mechanisms of drug resistance, including cell lines with high expression of P-glycoprotein and multidrug resistance associated protein (MRP), have previously been used for prediction of mechanism of drug action. In the present study, this cell line panel was analyzed for cellular accumulation of the fluorescent probe calcein/AM [4′5′-bis( N, N-bis (carboxymethyl) aminomethyl) fluorescein acetoxymethyl ester] and compared with drug response patterns of 20 standard chemotherapeutic drugs. According to degree of correlation with the ability to exclude calcein/AM, topoisomerase II inhibitors and tubulin actives were at the top of the list although the correlations were of lower magnitude than those obtained from the drug response patterns of mechanistically similar drugs. There was a significant relationship between the rank-order of drugs based on correlation with calcein/AM accumulation and Pgp/MRP mediated drug resistance suggesting that compounds being substrates for these pumps were identified. In simulated drug response profiles, the impact of Pgp and MRP expressing cell lines on the mechanistic prediction was found to be marginal. The results indicate that the differential molecular function/expression in the cell line panel may identify drugs interacting with specific biochemical pathways. Furthermore, the presence of cell lines overexpressing drug efflux mechanisms in the panel do not significantly influence the mechanistic predictions.