ABSTRACT
Elesclomol (ES), a copper-binding ionophore, forms an ES-Cu complex with copper ions (Cu(II)). ES-Cu has been proven to induce mitochondrial oxidative stress and copper-dependent cell death (cuprotosis). However, ES-Cu is poorly water-soluble, and its delivery to various cancer cells is a challenge. Herein, we designed a d-α-tocopherol polyethylene glycol 1000 succinate/chondroitin sulfate-cholic acid (TPGS/CS-CA)-based micellar nanoparticle for delivering the ES-Cu complex to various cancer cell lines to demonstrate its efficacy as an anticancer agent. The ES-Cu nanoparticles exerted high encapsulation efficiency and excellent serum stability. The anticancer efficacy of ES-Cu nanoparticles was evaluated in various drug-sensitive cell lines (DU145, PC3, and A549) and drug-resistant cell lines (DU145TXR, PC3TXR, and A549TXR). The results showed that ES-Cu nanoparticles exerted potent anticancer activities in both drug-sensitive and drug-resistant cell lines. The Western blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), and molecular docking results suggested that ES-Cu is not a substrate for P glycoprotein (P-gp), which is an efflux transporter potentially causing multidrug resistance (MDR) in cancer cells. ES-Cu nanoparticles could bypass P-gp without compromising their activity, indicating that they may overcome MDR in cancer cells and provide a novel therapeutic strategy. Additionally, the extracellular matrix of ES-Cu nanoparticles-pretreated drug-resistant cells could polarize Raw 264.7 macrophages into the M1 phenotype. Therefore, our TPGS/CS-CA-based ES-Cu nanoparticles provide an effective method of delivering the ES-Cu complex, a promising strategy to overcome MDR in cancer therapy with potential immune response stimulation.
Subject(s)
Antineoplastic Agents , Hydrazines , Nanoparticles , Neoplasms , Copper/chemistry , Molecular Docking Simulation , Antineoplastic Agents/chemistry , Nanoparticles/chemistry , Drug Resistance, Multiple , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , Drug Resistance, Neoplasm , Cell Line, TumorABSTRACT
The chemical transformation of lathyrane nucleus through reduction and oxidation reactions using Euphorbia Factor L1 (EFL1) and Euphorbia Factor L1 (EFL3) as examples were investigated, along with a co-modification strategy of lathyrane nucleus and its side ester chain. A total of 38 lathyrane derivatives (5-42) including 34 new compounds were obtained, which greatly enriched the structural diversity of the lathyrane-type diterpenoids. Cytotoxicity against drug-sensitive and drug (adriamycin, ADM) resistant MCF-7 cells showed that 23 out of 38 transformed derivatives possessed obvious cytotoxic activity with IC50 values ranging from 7.0 to 41.1 µM and 3.2 to 45.5 µM, respectively, against both cells, compared to the noncytotoxic EFL1 and EFL3. The multidrug resistance (MDR) reversing activities of these lathyrane derivatives were further evaluated in MCF-7/ADM. Three transformed compounds (reversal fold, RF = 151.33, 62.94 and 47.3 for 27, 37 and 42) showed markedly higher activity than EFL1 (RF = 32.92) and EFL3 (RF = 39.68). Structure-activity relationship study revealed an essential role of C-6/17 and C-12/13 double bonds on lathyrane nucleus for exerting MDR reversal activity. Western blotting analysis showed that 42 could reduce the expression level of P-glycoprotein (P-gp) in MCF-7/ADM cells; however, the most active compound 27 with an unnatural 5/7/7/4 fused-ring diterpenoid skeleton, had no inhibitory effect on P-gp expression.
Subject(s)
Diterpenes , Euphorbia , Phenylpropionates , Molecular Structure , Euphorbia/chemistry , Drug Resistance, Multiple , Diterpenes/pharmacology , Diterpenes/chemistry , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , ATP Binding Cassette Transporter, Subfamily BABSTRACT
PURPOSE: PET imaging using [11C]metoclopramide revealed the importance of P-glycoprotein (P-gp, ABCB1) in mediating the brain-to-blood efflux of substrates across the blood-brain barrier (BBB). In this work, the elimination rate constant from the brain (kE,brain), calculated from dynamic PET images without the need for arterial blood sampling, was evaluated as an outcome parameter for the interpretation of [11C]metoclopramide PET data. PROCEDURES: kE,brain parameter was obtained by linear regression of log-transformed brain time-activity curves (TACs). kE,brain values (h-1) obtained under baseline conditions were compared with values obtained after complete P-gp inhibition using tariquidar in rats (n = 4) and baboons (n = 4) or after partial inhibition using cyclosporine A in humans (n = 10). In baboons, the sensitivity of kE,brain to measure complete P-gp inhibition was compared with outcome parameters derived from kinetic modeling using a 1-tissue compartment model (1-TCM). Finally, kE,brain-maps were generated in each species using PMOD software. RESULTS: The linear part of the log-transformed brain TACs occurred from 10 to 30 min after radiotracer injection in rats, from 15 to 60 min in baboons, and from 20 to 60 min in humans. P-gp inhibition significantly decreased kE,brain values by 39 ± 12% in rats (p < 0.01), by 32 ± 6% in baboons (p < 0.001), and by 37 ± 22% in humans (p < 0.001). In baboons, P-gp inhibition consistently decreased the brain-to-plasma efflux rate constant k2 (36 ± 9%, p < 0.01) leading to an increase in the total brain volume of distribution (VT, 101 ± 12%, p < 0.001). In all studied species, brain kE,brain-maps displayed decreased P-gp-mediated efflux across the BBB. CONCLUSIONS: kE,brain of [11C]metoclopramide provides a simple outcome parameter to describe P-gp function in the living brain when arterial input function data are unavailable, although less sensitive than VT. kE,brain-maps represent easy to compute parametric images reflecting the effect of P-gp on [11C]metoclopramide elimination from the brain.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1 , Blood-Brain Barrier , Humans , Rats , Animals , Blood-Brain Barrier/diagnostic imaging , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , Metoclopramide , Brain/diagnostic imaging , Brain/metabolism , ATP Binding Cassette Transporter, Subfamily B/metabolism , Positron-Emission Tomography/methods , Papio/metabolismABSTRACT
Context: Treatment failure due to multidrug resistance (MDR) is a crucial hurdle during chemotherapy. MDR is generally correlated with an upregulation of adenosine triphosphate (ATP)-binding cassette (ABC) transport proteins. Also, aberrant activation of the phosphoinositide 3-kinase (PI3K)/ protein kinase B (Akt) pathway can counteract chemotherapeutic induction. Identification of safe and functioning MDR-reversing compounds is necessary in gastric-cancer therapy. Objective: The study intended to examine the role of Quercetin (Qur) in the mediation of osmotic glycoprotein (P-gp) expression and activity as an ABC transporter in the PI3K/Akt/ P-gp cascade in the oxaliplatin (OxR)-resistant, gastric-cancer cell line KATOIII/OxR. Design: The research team performed a laboratory study. Setting: The study took place at Nantong Haimen People's Hospital. Outcome Measures: The research team: (1) determined the impact of OxR on cell viability after treatment with Qur using trypan blue and "3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide" (MTT) assays; (2) employed a rhodamine 123 (Rh123) assay to detect the activity of P-gp; (3) used quantitative reverse transcription polymerase chain reaction (RT-qPCR) to measure mRNA expression of P-gp; and (4) detected apoptosis using an enzyme-linked immunoassay (ELISA) cell-death assay. Results: Qur: (1) increased the cytotoxicity of OxR; (2) downregulated the expression level and activity of P-gp and reversed MDR through the enhancement of the cytotoxicity of intracellular OxR in KATOIII/OxR cells; and (3) enhanced the apoptosis rate in KATOIII/OxR cells. Conclusions: Qur induced a dramatic reduction in the survival rate of KATOIII/OxR cells and may reverse OxR resistance through a decrease in P-gp expression and activity. These data imply that exposure of KATOIII/OxR cells in the dose-dependent manner to Qur can circumvent MDR by improving the intracellular accumulation of OxR. Qur might provide a new treatment strategy and improve patients' survival after chemotherapy for gastric cancer.
Subject(s)
Antineoplastic Agents , Stomach Neoplasms , Humans , Oxaliplatin/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Quercetin/pharmacology , Quercetin/therapeutic use , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol 3-Kinases/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/pharmacology , Stomach Neoplasms/drug therapy , Drug Resistance, Neoplasm , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , Cell Line, Tumor , DoxorubicinABSTRACT
BACKGROUND: Based on the effect of breviscapine (BRE) on reversing drug resistance of human breast cancer cell line MCF-7/doxorubicin (Dox), the mechanism was preliminarily explored. METHODS: The methyl thiazolyl tetrazolium (MTT) method was introduced to detect inhibitory effect of Dox alone or in combination with BRE on MCF-7 (M) and MCF-7/Dox (MD) cells, and the inhibitory concentration (IC50 ) was obtained. Cell apoptosis and Dox concentration was assessed by flow cytometry. The drug resistance multiple and reversal fold were calculated. Western blot was performed to evaluate the expression of Bcl-2, Bax, EGFR, p-EGFR, P-gp, caspase-3, and cleaved-caspase-3 protein in cells. The efflux of Rho-123 was measured by flow cytometry and fluorescence microscopy. RESULTS: The IC50 of Dox on MD and M cells was 16.67 and 0.71 µg/mL, respectively, with a drug resistance ratio of 23.48 times. The IC50 of Dox combined with BRE on MD cells was 5.62 µg/mL, with a reversal ratio of 2.97 times. BRE greatly enhanced Dox-induced apoptosis of MD cells. Bax and cleaved-caspase-3 (proapoptotic protein) expression were obviously increased, while Bcl-2 (antiapoptotic protein) expression was significantly decreased after BRE treatment. BRE inhibited EGFR activation and P-gp expression. BRE increased the intracellular accumulation of Dox in MD cells by P-gp. CONCLUSION: BRE could increase the MD sensitivity to Dox via increasing Bax and cleaved-caspase-3 expression and inhibiting Bcl-2 expression, thereby promoting cell apoptosis. BRE reversed Dox resistance of MD cells by increasing Dox intracellular accumulation through inhibiting P-gp expression via EGFR.
Subject(s)
Breast Neoplasms , Humans , Female , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Caspase 3/metabolism , Antibiotics, Antineoplastic/pharmacology , Antibiotics, Antineoplastic/therapeutic use , Drug Resistance, Multiple , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , bcl-2-Associated X Protein/metabolism , bcl-2-Associated X Protein/pharmacology , bcl-2-Associated X Protein/therapeutic use , Drug Resistance, Neoplasm , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , ErbB Receptors/metabolism , Apoptosis , Cell Line, TumorABSTRACT
The emergence of multidrug resistance (MDR) in malignant tumors is one of the leading threats encountered currently in many chemotherapeutic agents. The overexpression of the ATP-binding cassette (ABC) transporters is involved in MDR. P-glycoprotein (P-gp)/ABCB1 is a member of the ABC transporter family that significantly increases the efflux of various anticancer drugs from tumor cells. Therefore, targeting P-gp with small molecule inhibitors is an effective therapeutic strategy to overcome MDR. Over the past four decades, diverse compounds with P-gp inhibitory activity have been identified to sensitize drug-resistant cells, but none of them has been proven clinically useful to date. Research efforts continue to discover an effective approach for circumventing MDR. This review has provided an overview of the most recent advances (last three years) in various strategies for circumventing MDR mediated by P-gp. It may be helpful for the scientists working in the field of drug discovery to further synthesize and discover new chemical entities/therapeutic modalities with less toxicity and more efficacies to overcome MDR in cancer chemotherapy.
Subject(s)
Antineoplastic Agents , Neoplasms , Humans , Drug Resistance, Neoplasm , Drug Resistance, Multiple , ATP Binding Cassette Transporter, Subfamily B , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Neoplasms/drug therapy , ATP-Binding Cassette Transporters , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/therapeutic useABSTRACT
Multidrug resistance (MDR) caused by ATP-Binding Cassette Subfamily B Member 1 (ABCB1, P-glycoprotein, P-gp) is a major barrier for the success of chemotherapy in clinics. In this study, we designed and synthesized a total of 19 Lissodendrins B analogues and tested their ABCB1-mediated MDR reversal activity in doxorubicin (DOX)-resistant K562/ADR and MCF-7/ADR cells. Among all derivatives, compounds D1, D2, and D4 with a dimethoxy-substituted tetrahydroisoquinoline fragment possessed potent synergistic effects with DOX and reversed ABCB1-mediated drug resistance. Notably, the most potent compound D1 merits multiple activities, including low cytotoxicity, the strongest synergistic effect, and effectively reversing ABCB1-mediated drug resistance of K562/ADR (RF = 1845.76) and MCF-7/ADR cells (RF = 207.86) to DOX. As a reference substance, compound D1 allows for additional mechanistic studies on ABCB1 inhibition. The synergistic mechanisms were mainly related to the increased intracellular accumulation of DOX via inhibiting the efflux function of ABCB1 rather than from affecting the expression level of ABCB1. These studies suggest that compound D1 and its derivatives might be potential MDR reversal agents acting as ABCB1 inhibitors in clinical therapeutics and provide insight into a design strategy for the development of ABCB1 inhibitors.
Subject(s)
Antineoplastic Agents , Drug Resistance, Neoplasm , Humans , Drug Resistance, Multiple , ATP Binding Cassette Transporter, Subfamily B/metabolism , Doxorubicin/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Cell Line, TumorABSTRACT
Chemotherapy resistance in tumours is due to complex processes and is responsible for about half of all cancer deaths. In my thesis, I have investigated multiple different resistance mechanisms, most in depth the effect of multidrug resistance (MDR) caused by expression and function of P-glycoprotein (Pgp), and the MDR-selective compounds (such as NSC297366) effectively targeting it. The mechanism was investigated using cell models with different Pgp expression. Seeking the mechanism of action of the MDR-selective NSC297366, we showed that the intracellular iron-binding chelator molecule is able to reduce the amount of free iron available within the cell. Furthermore, by active efflux through Pgp in MDR cells, the compounds can lead to intracellular iron deficiency, upregulation of iron-demanding processes such as cell cycle and apoptosis, and selective death of MDR cancer cells. Our results raise the possibility of targeted killing of MDR phenotypic cancer cells resistant to other therapies, which in combination with conventional chemotherapeutic approaches may form the basis of a strategy of long-term control of the disease.
Subject(s)
Drug Resistance, Neoplasm , Neoplasms , Humans , Neoplasms/drug therapy , Drug Resistance, Multiple , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , Iron/metabolism , Iron/pharmacology , Iron/therapeutic useABSTRACT
BACKGROUND: Methotrexate (MTX) is a commonly used chemotherapeutic agent; however, its clinical use is challenged by various types of injuries, including hepatotoxic side effects. Therefore, finding new protective drugs against MTX-induced toxicities is a critical need. Moreover, the different mechanisms mediating such effects are still not clear. The current study aimed to evaluate the possible ameliorative action of nicorandil (NIC) in MTX-induced hepatotoxicity and examine the roles of the ATP-sensitive potassium channel (KATP), endothelial nitric oxide synthase (eNOS), and P-glycoprotein (P-gp). MATERIALS AND METHODS: Thirty-six male Wistar albino rats were used. NIC (3 mg/kg/day) was given orally for 2 weeks, and hepatotoxicity was induced by a single intraperitoneal injection of MTX (20 mg/kg) on the 11th day of the experiment. We confirmed the role of KATP by co-administering glimepiride (GP) (10 mg/kg/day) 30 min before NIC. The measured serum biomarkers were [alanine transaminase (ALT) and aspartate transaminase (AST)], total antioxidant capacity (TAC), malondialdehyde (MDA), nitric oxide (NOx), tumor necrosis factor-alpha (TNFα), superoxide dismutase (SOD), and P-gp. Histopathology, eNOS, and caspase-3 immunoexpression were evaluated. RESULTS: The MTX group displayed hepatotoxicity in the form of elevations of ALT, AST, MDA, NOx, and caspase-3 immunoexpression. Furthermore, the histopathological examination showed marked liver injury. TAC, SOD, P-gp, and eNOS immunoexpression showed significant inhibition. In the protective group, all parameters improved (P value < 0.05). CONCLUSION: NIC has an ameliorative action against MTX-induced hepatotoxicity, most probably via its antioxidant, anti-inflammatory, and anti-apoptotic functions together with the modulation of the KATP channel, eNOS, and P-glycoprotein.
Subject(s)
Chemical and Drug Induced Liver Injury, Chronic , Chemical and Drug Induced Liver Injury , Rats , Male , Animals , Antioxidants/pharmacology , Methotrexate/toxicity , Rats, Wistar , Nicorandil/pharmacology , Caspase 3/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , Oxidative Stress , Nitric Oxide Synthase Type III/metabolism , KATP Channels/metabolism , KATP Channels/pharmacology , Chemical and Drug Induced Liver Injury, Chronic/metabolism , Chemical and Drug Induced Liver Injury, Chronic/pathology , Liver , ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP Binding Cassette Transporter, Subfamily B/pharmacology , Superoxide Dismutase/metabolism , Adenosine Triphosphate , Chemical and Drug Induced Liver Injury/drug therapy , Chemical and Drug Induced Liver Injury/prevention & control , Chemical and Drug Induced Liver Injury/metabolismABSTRACT
Cryptosporidium parvum is a globally distributed zoonotic protozoan parasite that causes moderate to severe, sometime deadly, watery diarrhea in humans and animals, for which fully effective treatments are yet unavailable. In studying the mechanism of action of drugs against intracellular pathogens, it is important to validate whether the observed anti-infective activity is attributed to the drug action on the pathogen or host target. For the epicellular parasite Cryptosporidium, we have previously developed a concept that the host cells with significantly increased drug tolerance by transient overexpression of the multidrug resistance protein-1 (MDR1) could be utilized to evaluate whether and how much the observed anti-cryptosporidial activity of an inhibitor was attributed to the inhibitor's action on the parasite target. However, the transient transfection model was only applicable to evaluating native MDR1 substrates. Here we report an advanced model using stable MDR1-transgenic HCT-8 cells that allows rapid development of novel resistance to non-MDR1 substrates by multiple rounds of drug selection. Using the new model, we successfully validated that nitazoxanide, a non-MDR1 substrate and the only FDA-approved drug to treat human cryptosporidiosis, killed C. parvum by fully (100%) acting on the parasite target. We also confirmed that paclitaxel acted fully on the parasite target, while several other inhibitors including mitoxantrone, doxorubicin, vincristine and ivermectin acted partially on the parasite targets. Additionally, we developed mathematical models to quantify the proportional contribution of the on-parasite-target effect to the observed anti-cryptosporidial activity and to evaluate the relationships between several in vitro parameters, including antiparasitic efficacy (ECi), cytotoxicity (TCi), selectivity index (SI) and Hill slope (h). Owning to the promiscuity of the MDR1 efflux pump, the MDR1-transgenic host cell model could be applied to assess the on-parasite-target effects of newly identified hits/leads, either substrates or non-substrates of MDR1, against Cryptosporidium or other epicellular pathogens.
Subject(s)
Cryptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Parasites , Animals , Humans , Cryptosporidium parvum/genetics , Cryptosporidiosis/parasitology , Paclitaxel/pharmacology , Paclitaxel/therapeutic use , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/therapeutic use , AlgorithmsABSTRACT
Multidrug resistance (MDR) hinders treatment efficacy in cancer therapy. One typical mechanism contributing to MDR is the overexpression of permeability-glycoprotein (P-gp) encoded by ATP-binding cassette subfamily B member 1 (ABCB1). Basic helix-loop-helix family member e40 (BHLHE40) is a well-known transcription factor that has pleiotropic effects including the regulation of cancer-related processes. However, whether BHLHE40 regulates MDR is still unknown. Chromatin immunoprecipitation-seq study revealed BHLHE40 occupancy in the promoter of ABCB1 gene. Adriamycin (ADM)-resistant human chronic myeloid leukemia cells (K562/A) and human breast cancer cells (MCF-7/A) were established. BHLHE40 expression was downregulated in the ADM-resistant cell lines. Overexpression of BHLHE40 resensitized resistant cells to ADM, promoted cell apoptosis in vitro and suppressed tumor growth in vivo, whereas BHLHE40 knockdown induced resistance to ADM in parental cells. Moreover, we found that BHLHE40 regulated drug resistance by directly binding to the ABCB1 promoter (-1605 to -1597) and inactivating its transcription. In consistence, the expression of BHLHE40 was negatively correlated with ABCB1 in various cancer cells, while positively with cancer cell chemosensitivity and better prognosis of patients with breast cancer. The study reveals the role of BHLHE40 as a transcriptional suppressor on the expression of ABCB1, major ABC transporter in chemoresistance. The findings extend the function of BHLHE40 in tumor progression and provides a novel mechanism for the reversal of multidrug resistance.
Subject(s)
Breast Neoplasms , Transcription Factors , Humans , Female , Transcription Factors/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , Drug Resistance, Neoplasm/genetics , Drug Resistance, Multiple/genetics , Doxorubicin/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Cell Line, Tumor , Homeodomain Proteins/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , ATP Binding Cassette Transporter, Subfamily B/geneticsABSTRACT
BACKGROUND: . Transplant recipients may develop rejection despite having adequate tacrolimus whole blood predose concentrations (C 0 ). The intra-immune cellular concentration is potentially a better target than C 0 . However, little is known regarding intracellular tacrolimus concentration in T-lymphocytes and monocytes. We investigated the tacrolimus concentrations in both cell types and their relation with the expression and activity of FK-binding protein (FKBP)-12 and P-glycoprotein (P-gp). METHODS: . T-lymphocytes and monocytes were isolated from kidney transplant recipients followed by intracellular tacrolimus concentration measurement. FKBP-12 and P-gp were quantified with Western blot, flow cytometry, and the Rhodamine-123 assay. Interleukin-2 and interferon-γ in T-lymphocytes were measured to quantify the effect of tacrolimus. RESULTS: . Tacrolimus concentration in T-lymphocytes was lower than in monocytes (15.3 [8.5-33.4] versus 131.0 [73.5-225.1] pg/million cells; P < 0.001). The activity of P-gp (measured by Rhodamine-123 assay) was higher in T-lymphocytes than in monocytes. Flow cytometry demonstrated a higher expression of P-gp (normalized mean fluorescence intensity 1.5 [1.2-1.7] versus 1.2 [1.1-1.4]; P = 0.012) and a lower expression of FKBP-12 (normalized mean fluorescence intensity 1.3 [1.2-1.7] versus 1.5 [1.4-2.0]; P = 0.011) in T-lymphocytes than monocytes. Western blot confirmed these observations. The addition of verapamil, a P-gp inhibitor, resulted in a 2-fold higher intra-T-cell tacrolimus concentration. This was accompanied by a significantly fewer cytokine-producing cells. CONCLUSIONS: . T-lymphocytes have a higher activity of P-gp and lower concentration of the FKBP-12 compared with monocytes. This explains the relatively lower tacrolimus concentration in T-lymphocytes. The addition of verapamil prevents loss of intracellular tacrolimus during the cell isolation process and is required to ensure adequate intracellular concentration measurement.
Subject(s)
Kidney Transplantation , Tacrolimus , Humans , Tacrolimus/pharmacology , Immunosuppressive Agents/pharmacology , T-Lymphocytes/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , Kidney Transplantation/adverse effects , Kidney Transplantation/methods , Monocytes/metabolism , Carrier Proteins/metabolism , Carrier Proteins/pharmacology , Transplant Recipients , Tacrolimus Binding Protein 1A/metabolism , Tacrolimus Binding Protein 1A/pharmacology , ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP Binding Cassette Transporter, Subfamily B/pharmacology , Verapamil/pharmacology , Rhodamines/metabolism , Rhodamines/pharmacologyABSTRACT
N-methylpretrichodermamide B (NB) is a biologically active epidithiodiketopiperazine isolated from several strains of the algae-derived fungus Penicillium sp. Recently, we reported the first data on its activity in human cancer cells lines in vitro. Here, we investigated the activity, selectivity, and mechanism of action of NB in human prostate cancer cell lines, including drug-resistant subtypes. NB did not reveal cross-resistance to docetaxel in the PC3-DR cell line model and was highly active in hormone-independent 22Rv1 cells. NB-induced cell death was stipulated by externalization of phosphatidylserine and activation of caspase-3. Moreover, inhibition of caspase activity by z-VAD(OMe)-fmk did not affect NB cytotoxicity, suggesting a caspase-independent cell death induced by NB. The compound has a moderate p-glycoprotein (p-gp) substrate-like affinity and can simultaneously inhibit p-gp at nanomolar concentrations. Therefore, NB resensitized p-gp-overexpressing PC3-DR cells to docetaxel. A kinome profiling of the NB-treated cells revealed, among other things, an induction of mitogen-activated protein kinases JNK1/2 and p38. Further functional analysis confirmed an activation of both kinases and indicated a prosurvival role of this biological event in the cellular response to the treatment. Overall, NB holds promising anticancer potential and further structure-activity relationship studies and structural optimization are needed in order to improve its biological properties.
Subject(s)
Antineoplastic Agents , Prostatic Neoplasms , Humans , Male , Antineoplastic Agents/pharmacology , Apoptosis , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , Caspase 3/metabolism , Cell Line, Tumor , Docetaxel/pharmacology , Drug Resistance, Neoplasm , Hormones/pharmacology , Phosphatidylserines/pharmacology , Prostatic Neoplasms/drug therapyABSTRACT
Pesticides remain one of the most effective ways of controlling agricultural and public health insects, but much is still unknown regarding how these compounds reach their targets. Specifically, the role of ABC transporters in pesticide absorption and excretion is poorly understood, especially compared to the detailed knowledge about mammalian systems. Here, we present a comprehensive characterization of pesticide transporters in the model insect Drosophila melanogaster. An RNAi screen was performed, which knocked down individual ABCs in specific epithelial tissues and examined the subsequent changes in sensitivity to the pesticides spinosad and fipronil. This implicated a novel ABC drug transporter, CG4562, in spinosad transport, but also highlighted the P-glycoprotein orthologue Mdr65 as the most impactful ABC in terms of chemoprotection. Further characterization of the P-glycoprotein family was performed via transgenic overexpression and immunolocalization, finding that Mdr49 and Mdr50 play enigmatic roles in pesticide toxicology perhaps determined by their different subcellular localizations within the midgut. Lastly, transgenic Drosophila lines expressing P-glycoprotein from the major malaria vector Anopheles gambiae were used to establish a system for in vivo characterization of this transporter in non-model insects. This study provides the basis for establishing Drosophila as a model for toxicology research on drug transporters.
Subject(s)
Anopheles , Insecticides , Malaria , Pesticides , ATP Binding Cassette Transporter, Subfamily B/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , ATP-Binding Cassette Transporters , Animals , Animals, Genetically Modified , Drosophila melanogaster , Insecticide Resistance/genetics , Insecticides/toxicity , Mammals , Mosquito Vectors , Pesticides/toxicityABSTRACT
A novel folic acid mediated chitosan oligosaccharide-grafted disulfide-containing polyethylenimine copolymer-based silica nanohybrids were fabricated for co-delivering paclitaxel and P-shRNA. These nanoparticles could efficiently protect P-shRNA against degradation, and exhibited well redox-responsive P-shRNA release and pH-responsive drug release behaviors. Folic acid as the targeting head, could improve cellular uptake of nanoparticles by multidrug-resistant breast cancer cells. Moreover, these nanoparticles showed excellent delivery P-shRNA into cells and displayed high gene silencing efficiency at the targeted mRNAs to downregulate the expression of P-gp which induced up to 63% decrease. Finally, nanoparticles could completely reverse the resistance of breast cancer cells to paclitaxel and the resistance reversion index was 50.59. These results suggested that our nanoparticles could efficiently co-deliver paclitaxel and P-shRNA into cancer cells to exert its synergistic antitumor effect, and opened up a new avenue for overcoming multidrug resistance.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Drug Carriers/chemistry , Drug Resistance, Multiple/drug effects , Drug Resistance, Neoplasm/drug effects , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , Chitosan/analogs & derivatives , Chitosan/chemistry , Drug Liberation , Folic Acid/chemistry , Humans , Hydrogen-Ion Concentration , MCF-7 Cells , Nanoparticles/chemistry , Oxidation-Reduction , Paclitaxel/pharmacology , Polyethyleneimine/analogs & derivatives , Polyethyleneimine/chemistry , RNA, Small Interfering/pharmacologyABSTRACT
Multidrug resistance (MDR) remains one of the major impediments for efficacious cancer chemotherapy. Increased efflux of multiple chemotherapeutic drugs by transmembrane ATP-binding cassette (ABC) transporter superfamily is considered one of the primary causes for cancer MDR, in which the role of P-glycoprotein (P-gp/ABCB1) has been most well-established. The clinical co-administration of P-gp drug efflux inhibitors, in combination with anticancer drugs which are P-gp transport substrates, was considered to be a treatment modality to surmount MDR in anticancer therapy by blocking P-gp-mediated multidrug efflux. Extensive attempts have been carried out to screen for sets of nontoxic, selective, and efficacious P-gp efflux inhibitors. In this review, we highlight the recent achievements in drug design, characterization, structure-activity relationship (SAR) studies, and mechanisms of action of the newly synthetic, potent small molecules P-gp inhibitors in the past 5 years. The development of P-gp inhibitors will increase our knowledge of the mechanisms and functions of P-gp-mediated drug efflux which will benefit drug discovery and clinical cancer therapeutics where P-gp transporter overexpression has been implicated in MDR.
Subject(s)
Antineoplastic Agents , Neoplasms , ATP Binding Cassette Transporter, Subfamily B/pharmacology , ATP Binding Cassette Transporter, Subfamily B/therapeutic use , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/therapeutic use , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Humans , Neoplasms/drug therapyABSTRACT
The emergence of drug resistance is a major concern for combating against Cutaneous Leishmaniasis, a neglected tropical disease affecting 98 countries including India. Miltefosine is the only oral drug available for the disease and Miltefosine transporter proteins play a pivotal role in the emergence of drug-resistant Leishmania major. The cause of resistance is less accumulation of drug inside the parasite either by less uptake of the drug due to a decrease in the activity of P4ATPase-CDC50 complex or by increased efflux of the drug by P-glycoprotein (P-gp, an ABC transporter). In this paper, we are trying to allosterically modulate the behavior of resistant parasite (L. major) towards its sensitivity for the existing drug (Miltefosine, a phosphatidylcholine analog). We have used computational approaches to deal with the conservedness of the proteins and apparently its three-dimensional structure prediction through ab initio modeling. Long scale membrane-embedded molecular dynamics simulations were carried out to study the structural interaction and stability. Parasite-specific motifs of these proteins were identified based on the machine learning technique, against which a peptide library was designed. The protein-peptide docking shows good binding energy of peptides Pg5F, Pg8F and PC2 with specific binding to the motifs. These peptides were tested both in vitro and in vivo, where Pg5F in combination with PC2 showed 50-60% inhibition in resistant L. major's promastigote and amastigote forms and 80-90% decrease in parasite load in mice. We posit a model system wherein the data provide sufficient impetus for being novel therapeutics in order to counteract the drug resistance phenotype in Leishmania parasites.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , Allosteric Regulation/drug effects , Leishmania major/metabolism , Phosphorylcholine/analogs & derivatives , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/toxicity , ATP-Binding Cassette Transporters/metabolism , ATP-Binding Cassette Transporters/pharmacology , ATP-Binding Cassette Transporters/toxicity , Animals , Antiprotozoal Agents/metabolism , Antiprotozoal Agents/pharmacology , Cell Line , Computational Biology/methods , Drug Resistance/drug effects , Leishmania major/drug effects , Leishmaniasis, Cutaneous/drug therapy , Machine Learning , Mice , Molecular Docking Simulation/methods , Molecular Dynamics Simulation , Peptide Library , Peptides/chemical synthesis , Phosphorylcholine/metabolism , Phosphorylcholine/pharmacologyABSTRACT
Multidrug resistance is the major problem in cancer treatment nowadays. Compounds from plants are the new targets to solve this problem. Quercetin (QCT), quercetrin (QTR), and rutin (RUT) are potential anticancer flavonoids but their poor water solubility leads to less efficacy. In this study, the polymeric micelles of benzoylated methoxy-poly (ethylene glycol)-b-oligo(ε-caprolactone) or mPEG-b-OCL-Bz loading with the flavonoids were prepared to solve these problems. The flavonoid-loaded micelles showed an average size of 13-20 nm and maximum loading capacity of 35% (w/w). The release of QCT (21%, 3 h) was lower than that of QTR (51%, 3 h) and RUT (58%, 3 h). QCT (free and micelle forms) exhibited significantly higher cytotoxicity against P-glycoprotein-overexpressing leukemia (K562/ADR) cells than QTR and RUT (p < 0.05). The results demonstrated that QCT-loaded micelles effectively reversed cytotoxicity of both doxorubicin (multidrug resistant reversing (δ) values up to 0.71) and daunorubicin (δ values up to 0.74) on K562/ADR cells. It was found that QCT-loaded micelles as well as empty polymeric micelles inhibited P-gp efflux of tetrahydropyranyl Adriamycin. Besides, mitochondrial membrane potential was decreased by QCT (in its free form and micellar formation). Our results suggested that the combination effects of polymeric micelles (inhibiting P-gp efflux) and QCT (interfering mitochondrial membrane potential) might be critical factors contributing to the reversing multidrug resistance of K562/ADR cells by QCT-loaded micelles. We concluded that QCT-loaded mPEG-b-OCL-Bz micelles are the attractive systems for overcoming multidrug-resistant cancer cells.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/chemical synthesis , Antineoplastic Agents/chemical synthesis , Flavonoids/chemical synthesis , Micelles , Polyethylene Glycols/chemical synthesis , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/physiology , Dose-Response Relationship, Drug , Flavonoids/pharmacology , Humans , K562 Cells , Polyethylene Glycols/pharmacology , Polymers/chemical synthesis , Polymers/pharmacology , SolubilityABSTRACT
The enhanced endo-lysosomal sequestration still remains a big challenge in overcoming multidrug resistance (MDR). Herein, a dual-responsive polyplex with effective endo-lysosomal escape based on methoxypoly(ethylene glycol)-polylactide-polyhistidine-ss-oligoethylenimine (mPEG- b-PLA-PHis-ssOEI) was developed for codelivering MDR1 siRNA and doxorubicin (DOX). The polyplex showed good encapsulation of DOX and siRNA as well as triggered payload release in response to pH/redox stimuli because of the PHis protonation and the disulfide bond cleavage. The polyplex at an N/P ratio of 7 demonstrated a much higher payload delivery efficiency, MDR1 gene silence efficiency, cytotoxicity against MCF-7/ADR cell, and stronger MCF-7/ADR tumor growth inhibition than the polyplexes at higher N/P ratios. This was attributed to the stronger electrostatic attraction between siRNA and OEIs at higher N/P ratios that suppressed the release of MDR1 siRNA and OEIs, which played a dominant role in overcoming payload endo-lysosomal sequestration by the OEI-induced membrane permeabilization effect. Consequently, the polyplex with effective endo-lysosomal escape provides a rational approach for codelivery of siRNAs and chemotherapy agents for MDR reversal.