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In Silico Pharmacology 2021P-glycoprotein (P-gp), which was first identified in cancer cells, is an ATP-dependent efflux transporter that expels a wide variety of cytotoxic compounds out of cells....
P-glycoprotein (P-gp), which was first identified in cancer cells, is an ATP-dependent efflux transporter that expels a wide variety of cytotoxic compounds out of cells. This transporter can decrease the bioavailability of therapeutic drugs by preventing their sufficient intracellular accumulation. Over expression of P-gp in cancer cells lead to multidrug resistance (MDR) phenotype that is one of the main reasons for the failure of chemotherapy. Hence, P-gp inhibition is a favorable method to reverse MDR. In this study, the lignanamides from were docked against P-gp to recognize potential binding affinities of these phytochemicals. Tariquidar and zosuquidar, two well-known P-gp inhibitors, were selected as the control ligands. It was observed that cannabisin M and cannabisin N exhibited higher binding affinities (- 10.2 kcal/mol) to drug-binding pocket of P-gp when compared with tariquidar and zosuquidar that showed binding affinities of - 10.1 and - 9.6 kcal/mol, respectively. Based on these findings, cannabisin M and cannabisin N could be good drug candidates against P-gp.
PubMed: 33442533
DOI: 10.1007/s40203-020-00066-7 -
Journal of Cancer Research and Clinical... Apr 2020Increased ATP-binding-cassette (ABC) transporter activity is a major cause of chemotherapy resistance in cancer. The ABC transporter family member ABCB1 is often...
PURPOSE
Increased ATP-binding-cassette (ABC) transporter activity is a major cause of chemotherapy resistance in cancer. The ABC transporter family member ABCB1 is often overexpressed in colorectal cancer (CRC). Phosphatidylinositol-4,5-bisphosphat (PI(4,5)P)-dependent pathways are involved in the regulation of ABCB1 function. The protein Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) is a pivotal regulator of PI(4,5)P and inactivated in many CRC cancers via genetic deletion or hyperphosphorylation. Therefore, MARCKS may critically impact ABCB1.
METHODS
CRC samples as well as CRC cell lines were tested for a connection between MARCKS and ABCB1 via immunofluorescence and Western-blot analysis. ABCB1 function was studied via calcein influx assay under treatment with known ABCB1 inhibitors (verapamil, tariquidar) as well as the kinase inhibitor bosutinib. ABCB1 internalization and MARCKS translocation was analyzed via confocal microscopy exploiting the endocytosis inhibitors chlorpromazine and dynasore. Abundance of PI(4,5)P was monitored by intramolecular fluorescence resonance energy transfer (FRET). Reproductive cell survival was studied via colorimetric WST-1 and clonogenic assays in combination with exposure to the chemotherapeutics doxorubicin and 5-fuorouracil (5-FU).
RESULTS
We found increased ABCB1 expression in MARCKS negative CRC patient tumor samples and established CRC cell lines. Mechanistically, the reconstitution of MARCKS function via recombinant expression or the pharmacological inhibition of MARCKS phosphorylation led to a substantial decrease in ABCB1 activity. In CRC cells, bosutinib treatment resulted in a MARCKS translocation from the cytosol to the plasma membrane, while simultaneously, ABCB1 was relocated to intracellular compartments. Inhibition of MARCKS phosphorylation via bosutinib rendered cells more sensitive to the chemotherapeutics doxorubicin and 5-FU.
CONCLUSIONS
Cells devoid of MARCKS function showed incomplete ABCB1 internalization, leading to higher ABCB1 activity enhancing chemoresistance. Vice versa our data suggest the prevention of MARCKS inhibition by reversing hyperphosphorylation or genomic restoration after deletion as two promising approaches to overcome tumor cell resistance towards chemotherapeutic ABCB1 substrates.
Topics: ATP Binding Cassette Transporter, Subfamily B; Aniline Compounds; Cell Line, Tumor; Colorectal Neoplasms; Drug Resistance, Neoplasm; Fluoresceins; Fluorescence Resonance Energy Transfer; HT29 Cells; Humans; Microscopy, Confocal; Myristoylated Alanine-Rich C Kinase Substrate; Nitriles; Phosphorylation; Quinolines
PubMed: 32056006
DOI: 10.1007/s00432-020-03149-2 -
Thoracic Cancer Aug 2022Small cell lung cancer (SCLC) is a highly aggressive disease with a poor prognosis. Although most patients initially respond to topoisomerase inhibitors, resistance...
BACKGROUND
Small cell lung cancer (SCLC) is a highly aggressive disease with a poor prognosis. Although most patients initially respond to topoisomerase inhibitors, resistance rapidly emerges. The aim, therefore, is to overcome resistance to topoisomerase I (irinotecan) or II (etoposide) inhibitors in SCLCs.
METHODS
To identify key factors in the chemoresistance of SCLCs, we established four cell lines resistant to etoposide or an active metabolite of irinotecan, SN-38, from SCLC cell lines and evaluated RNA profiles using parental and newly established cell lines.
RESULTS
We found that the drug efflux protein, ATP-binding cassette sub-family B member 1 (ABCB1), was associated with resistance to etoposide, and ATP-binding cassette sub-family G member 2 (ABCG2) was associated with resistance to SN-38 by RNA sequencing. The inhibition of ABCB1 or ABCG2 in each resistant cell line induced synergistic apoptotic activity and promoted drug sensitivity in resistant SCLC cells. The ABC transporter inhibitors, elacridar and tariquidar, restored sensitivity to etoposide or SN-38 in in vitro and in vivo studies, and promoted apoptotic activity and G2-M arrest in resistant SCLC cells.
CONCLUSIONS
ABC transporter inhibitors may be a promising therapeutic strategy for the purpose of overcoming resistance to topoisomerase inhibitors in patients with SCLC.
Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Drug Resistance, Neoplasm; Etoposide; G2 Phase Cell Cycle Checkpoints; Humans; Irinotecan; Lung Neoplasms; Neoplasm Proteins; Small Cell Lung Carcinoma; Topoisomerase Inhibitors
PubMed: 35719112
DOI: 10.1111/1759-7714.14527 -
Journal of Enzyme Inhibition and... Dec 2020Aiming to deepen the structure-activity relationships of the two P-glycoprotein (P-gp) modulators elacridar and tariquidar, a new series of amide and ester derivatives...
Aiming to deepen the structure-activity relationships of the two P-glycoprotein (P-gp) modulators elacridar and tariquidar, a new series of amide and ester derivatives carrying a 6,7-dimethoxy-2-phenethyl-1,2,3,4-tetrahydroisoquinoline scaffold linked to different methoxy-substituted aryl moieties were synthesised. The obtained compounds were evaluated for their P-gp interaction profile and selectivity towards the two other ABC transporters, multidrug-resistance-associated protein-1 and breast cancer resistance protein, showing to be very active and selective versus P-gp. Two amide derivatives, displaying the best P-gp activity, were tested in co-administration with the antineoplastic drug doxorubicin in different cancer cell lines, showing a significant sensitising activity towards doxorubicin. The investigation on the chemical stability of the derivatives towards spontaneous or enzymatic hydrolysis, showed that amides are stable in both models while some ester compounds were hydrolysed in human plasma. This study allowed us to identify two chemosensitizers that behave as non-transported substrates and are characterised by different selectivity profiles.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Amides; Animals; Antineoplastic Agents; Cells, Cultured; Dogs; Dose-Response Relationship, Drug; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Esters; Humans; Molecular Structure; Neoplasms; Structure-Activity Relationship; Tetrahydroisoquinolines
PubMed: 32253945
DOI: 10.1080/14756366.2020.1747449 -
International Journal of Molecular... Nov 2020ABCB1 (P-glycoprotein) and ABCG2 (breast cancer resistance protein) are co-localized at the blood-brain barrier (BBB), where they restrict the brain distribution of many...
BACKGROUND
ABCB1 (P-glycoprotein) and ABCG2 (breast cancer resistance protein) are co-localized at the blood-brain barrier (BBB), where they restrict the brain distribution of many different drugs. Moreover, ABCB1 and possibly ABCG2 play a role in Alzheimer's disease (AD) by mediating the brain clearance of beta-amyloid (Aβ) across the BBB. This study aimed to compare the abundance and activity of ABCG2 in a commonly used β-amyloidosis mouse model (APP/PS1-21) with age-matched wild-type mice.
METHODS
The abundance of ABCG2 was assessed by semi-quantitative immunohistochemical analysis of brain slices of APP/PS1-21 and wild-type mice aged 6 months. Moreover, the brain distribution of two dual ABCB1/ABCG2 substrate radiotracers ([C]tariquidar and [C]erlotinib) was assessed in APP/PS1-21 and wild-type mice with positron emission tomography (PET). [C]Tariquidar PET scans were performed without and with partial inhibition of ABCG2 with Ko143, while [C]erlotinib PET scans were only performed under baseline conditions.
RESULTS
Immunohistochemical analysis revealed a significant reduction (by 29-37%) in the number of ABCG2-stained microvessels in the brains of APP/PS1-21 mice. Partial ABCG2 inhibition significantly increased the brain distribution of [C]tariquidar in APP/PS1-21 and wild-type mice, but the brain distribution of [C]tariquidar did not differ under both conditions between the two mouse strains. Similar results were obtained with [C]erlotinib.
CONCLUSIONS
Despite a reduction in the abundance of cerebral ABCG2 and ABCB1 in APP/PS1-21 mice, the brain distribution of two dual ABCB1/ABCG2 substrates was unaltered. Our results suggest that the brain distribution of clinically used ABCB1/ABCG2 substrate drugs may not differ between AD patients and healthy people.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; Amyloid beta-Peptides; Amyloidosis; Animals; Blood-Brain Barrier; Brain; Disease Models, Animal; Female; Mice; Mice, Inbred C57BL; Mice, Transgenic; Positron-Emission Tomography; Quinolines; Tissue Distribution
PubMed: 33153231
DOI: 10.3390/ijms21218245 -
Biological & Pharmaceutical Bulletin 2022Microfluidic devices are attracting attention for their ability to provide a biomimetic microenvironment wherein cells are arranged in a particular pattern and provided...
Microfluidic devices are attracting attention for their ability to provide a biomimetic microenvironment wherein cells are arranged in a particular pattern and provided fluidic and mechanical forces. In this study, we evaluated drug transport across Caco-2 cell layers in microfluidic devices and investigated the effects of fluid flow on drug transport and metabolism. We designed a microfluidic device that comprises two blocks of polydimethylsiloxane and a sandwiched polyethylene terephthalate membrane with pores 3.0 µm in diameter. When cultured in a dynamic fluid environment, Caco-2 cells were multilayered and developed microvilli on the surface as compared with a static environment. Drugs with higher lipophilicity exhibited higher permeability across the Caco-2 layers, as well as in the conventional method using Transwells, and the fluidic conditions had little effect on permeability. In the Caco-2 cell layers cultured in Transwells and microfluidic devices, the basal-to-apical transport of rhodamine 123, a substrate of P-glycoprotein, was greater than the apical-to-basal transport, and the presence of tariquidar, an inhibitor of P-glycoprotein, completely diminished asymmetric transport. Furthermore, fluidic conditions promoted the metabolism of temocapril by carboxylesterases. On the other hand, we showed that fluidic conditions have little effect on gene expression of several transporters and metabolic enzymes. These results provide useful information regarding the application of microfluidic devices in drug transport and metabolism studies.
Topics: ATP Binding Cassette Transporter, Subfamily B; Caco-2 Cells; Humans; Intestinal Absorption; Intestines; Lab-On-A-Chip Devices; Permeability
PubMed: 36047192
DOI: 10.1248/bpb.b22-00092 -
Proceedings of the National Academy of... Jan 2023ABCG2 is an ATP-binding cassette (ABC) transporter that extrudes a wide range of xenobiotics and drugs from the cell and contributes to multidrug resistance in cancer...
ABCG2 is an ATP-binding cassette (ABC) transporter that extrudes a wide range of xenobiotics and drugs from the cell and contributes to multidrug resistance in cancer cells. Following our recent structural characterization of topotecan-bound ABCG2, here, we present cryo-EM structures of ABCG2 under turnover conditions in complex with a special modulator and slow substrate, tariquidar, in nanodiscs. The structures reveal that similar to topotecan, tariquidar induces two distinct ABCG2 conformations under turnover conditions (turnover-1 and turnover-2). μs-scale molecular dynamics simulations of drug-bound and apo ABCG2 in native-like lipid bilayers, in both topotecan- and tariquidar-bound states, characterize the ligand size as a major determinant of its binding stability. The simulations highlight direct lipid-drug interactions for the smaller topotecan, which exhibits a highly dynamic binding mode. In contrast, the larger tariquidar occupies most of the available volume in the binding pocket, thus leaving little space for lipids to enter the cavity and interact with it. Similarly, when simulating ABCG2 in the apo inward-open state, we also observe spontaneous penetration of phospholipids into the binding cavity. The captured phospholipid diffusion pathway into ABCG2 offers a putative general path to recruit any hydrophobic/amphiphilic substrates directly from the membrane. Our simulations also reveal that ABCG2 rejects cholesterol as a substrate, which is omnipresent in plasma membranes that contain ABCG2. At the same time, cholesterol is found to prohibit the penetration of phospholipids into ABCG2. These molecular findings have direct functional ramifications on ABCG2's function as a transporter.
Topics: Ligands; Topotecan; Drug Resistance, Multiple; ATP-Binding Cassette Transporters; Phospholipids; Cholesterol; Drug Resistance, Neoplasm
PubMed: 36580587
DOI: 10.1073/pnas.2213437120 -
European Journal of Nuclear Medicine... Jul 2020Multidrug resistance (MDR) impedes cancer treatment. Two efflux transporters from the ATP-binding cassette (ABC) family, ABCB1 and ABCG2, may contribute to MDR by...
PURPOSE
Multidrug resistance (MDR) impedes cancer treatment. Two efflux transporters from the ATP-binding cassette (ABC) family, ABCB1 and ABCG2, may contribute to MDR by restricting the entry of therapeutic drugs into tumor cells. Although a higher expression of these transporters has been correlated with an unfavorable response to chemotherapy, transporter expression does not necessarily correlate with function. In this study, we characterized the pharmacological properties of [F]AVT-011, a new PET radiotracer for imaging transporter-mediated MDR in tumors.
METHODS
AVT-011 was radiolabeled with F and evaluated with PET imaging in preclinical models. Transport of [F]AVT-011 by ABCB1 and/or ABCG2 was assessed by measuring its uptake in the brains of wild-type, Abcb1a/b, and Abcg2 mice at baseline and after administration of the ABCB1 inhibitor tariquidar (n = 5/group). Metabolism and biodistribution of [F]AVT-011 were also measured. To measure ABCB1 function in tumors, we performed PET experiments using both [F]AVT-011 and [F]FDG in mice bearing orthotopic breast tumors (n = 7-10/group) expressing clinically relevant levels of ABCB1.
RESULTS
At baseline, brain uptake was highest in Abcb1a/b mice. After tariquidar administration, brain uptake increased 3-fold and 8-fold in wild-type and Abcg2 mice, respectively, but did not increase further in Abcb1a/b mice. At 30 min after injection, the radiotracer was > 90% in its parent form and had highest uptake in organs of the hepatobiliary system. Compared with that in drug-sensitive tumors, uptake of [F]AVT-011 was 32% lower in doxorubicin-resistant tumors with highest ABCB1 expression and increased by 40% with tariquidar administration. Tumor uptake of [F]FDG did not significantly differ among groups.
CONCLUSION
[F]AVT-011 is a dual ABCB1/ABCG2 substrate radiotracer that can quantify transporter function at the blood-brain barrier and in ABCB1-expressing tumors, making it potentially suitable for clinical imaging of ABCB1-mediated MDR in tumors.
Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; Animals; Blood-Brain Barrier; Drug Resistance, Multiple; Mice; Positron-Emission Tomography; Tissue Distribution
PubMed: 31729540
DOI: 10.1007/s00259-019-04589-w -
Drug Resistance Updates : Reviews and... Nov 2023Therapy resistance has long been considered to occur through the selection of pre-existing clones equipped to survive and quickly regrow, or through the acquisition of...
Therapy resistance has long been considered to occur through the selection of pre-existing clones equipped to survive and quickly regrow, or through the acquisition of mutations during chemotherapy. Here we show that following in vitro treatment by chemotherapy, epithelial breast cancer cells adopt a transient drug tolerant phenotype characterized by cell cycle arrest, epithelial-to-mesenchymal transition (EMT) and the reversible upregulation of the multidrug resistance (MDR) efflux transporter P-glycoprotein (P-gp). The drug tolerant persister (DTP) state is reversible, as cells eventually resume proliferation, giving rise to a cell population resembling the initial, drug-naïve cell lines. However, recovery after doxorubicin treatment is almost completely eliminated when DTP cells are cultured in the presence of the P-gp inhibitor Tariquidar. Mechanistically, P-gp contributes to the survival of DTP cells by removing reactive oxygen species-induced lipid peroxidation products resulting from doxorubicin exposure. In vivo, prolonged administration of Tariquidar during doxorubicin treatment holidays resulted in a significant increase of the overall survival of Brca1;p53 mammary tumor bearing mice. These results indicate that prolonged administration of a P-gp inhibitor during drug holidays would likely benefit patients without the risk of aggravated side effects related to the concomitantly administered toxic chemotherapy. Effective targeting of DTPs through the inhibition of P-glycoprotein may result in a paradigm shift, changing the focus from countering drug resistance mechanisms to preventing or delaying therapy resistance.
Topics: Humans; Animals; Mice; Female; ATP Binding Cassette Transporter, Subfamily B, Member 1; Lipid Peroxidation; Pharmaceutical Preparations; Breast Neoplasms; ATP Binding Cassette Transporter, Subfamily B; Doxorubicin
PubMed: 37741091
DOI: 10.1016/j.drup.2023.101007 -
PloS One 2021P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimer's disease (AD). However, previous studies on the...
P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimer's disease (AD). However, previous studies on the ability of P-gp to directly transport the Alzheimer's associated amyloid-β (Aβ) protein have produced contradictory results. Here we use molecular dynamics (MD) simulations, transport substrate accumulation studies in cell culture, and biochemical activity assays to show that P-gp actively transports Aβ. We observed transport of Aβ40 and Aβ42 monomers by P-gp in explicit MD simulations of a putative catalytic cycle. In in vitro assays with P-gp overexpressing cells, we observed enhanced accumulation of fluorescently labeled Aβ42 in the presence of Tariquidar, a potent P-gp inhibitor. We also showed that Aβ42 stimulated the ATP hydrolysis activity of isolated P-gp in nanodiscs. Our findings expand the substrate profile of P-gp, and suggest that P-gp may contribute to the onset and progression of AD.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Adenosine Triphosphate; Alzheimer Disease; Amyloid beta-Peptides; Biocatalysis; Blood-Brain Barrier; Cell Line, Tumor; Disease Progression; Humans; Hydrolysis; Ligands; Molecular Docking Simulation; Molecular Dynamics Simulation; Peptide Fragments; Protein Binding; Protein Conformation, beta-Strand; Protein Domains; Protein Transport; Quinolines; Signal Transduction; Substrate Specificity
PubMed: 33901197
DOI: 10.1371/journal.pone.0250371