Modulation Of P-glycoprotein Activity Research Articles

Mechanisms involved in the cytotoxic action of Brazilian propolis and caffeic acid against HEp-2 cells and modulation of P-glycoprotein activity

The effects of propolis and phenolic compounds (caffeic acid - Caf; dihydrocinnamic acid - Cin; p-coumaric acid - Cou) in the same quantity found in our propolis sample were investigated on human laryngeal epidermoid carcinoma (HEp-2) cells. Cell viability, apoptosis/necrosis and cell cycle arrest, P53 and CASPASE-3 gene expression, generation of reactive oxygen species (ROS) and the ability of propolis to induce doxorubicin (DOX) efflux using a P-glycoprotein (P-gp) inhibitor (verapamil) were assayed. Propolis exerted a cytotoxic effect on HEp-2 cells, whereas isolated compounds had no effect on cell viability. Higher concentrations were tested and Caf induced late apoptosis or necrosis in HEp-2 cells, while propolis induced apoptosis, both probably due to ROS generation. P53 expression was downregulated by propolis but not by Caf. CASPASE-3 expression was correlated with induction of both early and late apoptosis, with both propolis and Caf alone upregulating its expression. Propolis induced cell cycle arrest at G2/M phase and Caf at S phase. Propolis but not Caf may act as a P-gp inhibitor by modulating P-gp activity and inhibiting DOX efflux. Propolis exerted cytotoxic effects on HEp-2 cells, and the mechanisms are discussed, showing its potential as an antitumour drug.

Modulation of P-glycoprotein activity by novel synthetic curcumin derivatives in sensitive and multidrug-resistant T-cell acute lymphoblastic leukemia cell lines

BackgroundMultidrug resistance (MDR) and drug transporter P-glycoprotein (P-gp) represent major obstacles in cancer chemotherapy. We investigated 19 synthetic curcumin derivatives in drug-sensitive acute lymphoblastic CCRF–CEM leukemia cells and their multidrug-resistant P-gp-overexpressing subline, CEM/ADR5000. Material and methodsCytotoxicity was tested by resazurin assays. Doxorubicin uptake was assessed by flow cytometry. Binding modes of compounds to P-gp were analyzed by molecular docking. Chemical features responsible for bioactivity were studied by quantitative structure activity relationship (QSAR) analyses. A 7-descriptor QSAR model was correlated with doxorubicin uptake values, IC50 values and binding energies. ResultsThe compounds displayed IC50 values between 0.7±0.03 and 20.2±0.25μM. CEM/ADR5000 cells exhibited cross-resistance to 10 compounds, collateral sensitivity to three compounds and regular sensitivity to the remaining six curcumins. Molecular docking studies at the intra-channel transmembrane domain of human P-gp resulted in lowest binding energies ranging from −9.00±0.10 to −6.20±0.02kcal/mol and pKi values from 0.24±0.04 to 29.17±0.88μM. At the ATP-binding site of P-gp, lowest binding energies ranged from −9.78±0.17 to −6.79±0.01kcal/mol and pKi values from 0.07±0.02 to 0.03±0.03μM. CEM/ADR5000 cells accumulated approximately 4-fold less doxorubicin than CCRF–CEM cells. The control P-gp inhibitor, verapamil, partially increased doxorubicin uptake in CEM/ADR5000 cells. Six curcumins increased doxorubicin uptake in resistant cells or even exceeded uptake levels compared to sensitive one. QSAR yielded good activity prediction (R=0.797 and R=0.794 for training and test sets). ConclusionSelected derivatives may serve to guide future design of novel P-gp inhibitors and collateral sensitive drugs to combat MDR.

Modulation of P-Glycoprotein activity: Insights from docking studies

Modulation of P-Glycoprotein activity: Insights from docking studies

Modulation of P-glycoprotein activity by the substituted quinoxalinone compound QA3 in adriamycin-resistant K562/A02 cells

QA3 is a derivative of the substituted 1,3-dimethyl-1H-quinoxalin-2-ones, which are compounds that may selectively antagonize P-glycoprotein (P-gp) in multidrug resistance (MDR) cancer cells. Our previous work identified QA3 as a candidate compound for reversing MDR in cancer cells. In the present study, we found that QA3 significantly decreases the intracellular level of ATP, stimulates ATPase activity in membrane microsomes and decreases protein kinase C (PKC) activity. These results indicated that QA3 inhibits P-gp activity by blocking ATP hydrolysis and ATP regeneration. Furthermore, QA3 triggered and increased adriamycin-induced K562/A02 cell apoptosis as evidenced by Annexin V-FITC plus PI staining. Western blot analysis showed that the levels of cleaved caspase-9 and cleaved caspase-3 proteins increased, and similarly, the levels of procaspase-9 and procaspase-3 decreased after QA3 treatment. Consequently, poly ADP-ribose polymerase (PARP) activity increased as evidenced by the presence of the PARP cleavage product in K562/A02 cells. QA3 also enhanced the potency of adriamycin against K562/A02 cells as demonstrated by increased apoptosis and activation of caspase-9,-3 and PARP. These data support the observation that P-gp activity is inhibited after QA3 treatment. Moreover, these results indicate that QA3 is a novel MDR reversal agent with potent inhibitory action against P-gp MDR cancer cells.

Transport of HIV-protease inhibitors across 1 alpha,25di-hydroxy vitamin D3-treated Calu-3 cell monolayers: modulation of P-glycoprotein activity.

The presence of P-glycoprotein (P-gp) within the lipid bi-layers of the absorptive cells greatly influences drug entry into the HIV-infected sanctuary sites. The objective of this study was to acces the potential role of pulmonary cells expressing high levels of P-gp in the efflux of potent anti-HIV drugs such as protease inhibitors. Human airway epithelium-derived Calu-3 cells grown in the presence of 0.025 mM 1alpha,25di-hydroxy Vitamin D3 (di-OH vit D3) were used as a model to evaluate the effects of p-glycoprotein efflux of HIV protease inhibitors. Cells used as controls were not treated with di-OH vit D3. The anti-HIV agents 3H Ritonavir and Saquinavir (50 microM) were used as model compounds for influx and efflux studies Di-OH vit D3 treatment enhanced the differentiation c Calu-3 cells indicated by more cilia and mucus secretion. It also caused elevated P-gp expression as demonstrated by Western Bla analysis and enhanced basal to apical transport of cyclosporine as compared with untreated cells. The amount of Saquinavir transported, after 3 h, across untreated Calu-3 cells (A-B) was 3-fold higher (1.62 microg; Papp = 2A (+/- 0.79) x 10(-6) cm/s) than di-OH vi D3-treated cells (0.57 microg with the Papp = 5.02 (+/- 0.62) x 10(-7) cm/s) Similar transport profiles were obtained for 3H ritonavir and a significant increase (p < 0.05) in the A-B transport (2.5-fold) of 3H ritonavir was observed when the cell monolayers were preincubated with testosterone prior to transport studies. However, transport of AZT remained unaltered in di-OH vit D3 treated monolayers. Modulation of P-gp activity may be necessary to increase the therapeutic efficacy of protease inhibitors against HIV-1 reservoirs across alveolar lining cells and fluids.

Modulation of P-glycoprotein activity in Calu-3 cells using steroids and β-ligands

The purpose of this work was to investigate if P-glycoprotein (Pgp) efflux pump activity could be inhibited in the sub-bronchial epithelial cell line, Calu-3, by glucocorticosteroids and β-ligands. The Pgp modulation efficiency of each compound was determined by its ability to increase the accumulation of the Pgp substrate rhodamine 123 (Rh123) accumulation in these cells. Pgp inhibition was observed at ≥100 μM steroids and β-ligand. The modulation effectiveness of the β-ligands increased with increasing hydrophobicity (log P octanol/aqueous) whereas an obvious correlation was not obtained with the complete set of steroids tested. Steroidal Pgp substrates did not affect Rh123 accumulation (e.g. aldosterone, dexamethasone, 11β, 17α, 21-OH progesterone). In contrast, two hydrophobic non-Pgp steroidal substrates (testosterone and progesterone) displayed different effects on Rh123 accumulation, with progesterone being the more potent modulator. The most hydrophobic β-ligand, propranolol, a known Pgp substrate, gave the largest increase in Rh123 accumulation in this therapeutic class. The β-ligand modulation efficiency could also be correlated to Pgp structural recognition elements such as hydrogen bonding potential, the presence of a basic nitrogen and planar aromatic ring. No effect on Rh123 accumulation was observed with the formulation additives tested (ethanol, glycerol and palmitoyl carnitine) at concentrations previously reported to be non-toxic to Calu-3 cells.

Role of formulation vehicles in taxane pharmacology.

The non-ionic surfactants Cremophor EL (CrEL) and Tween 80, both used as formulation vehicles of many (anticancer) agents including paclitaxel and docetaxel, are not physiological inert compounds. We describe their biological properties, especially the toxic side effects, and their pharmacological properties, such as modulation of P-glycoprotein activity. In detail, we discuss their influence on the disposition of the solubilized drugs, with focus on CrEL and paclitaxel, and of concomitantly administered drugs. The ability of the surfactants to form micelles in aqueous solution as well as biological fluids (e.g. plasma) appears to be of great importance with respect to the pharmacokinetic behavior of the formulated drugs. Due to drug entrapment in the micelles, plasma concentrations and clearance of free drug change significant leading to alteration in pharmacodynamic characteristics. We conclude with some perspectives related to further investigation and development of alternative methods of administration.

Modulation of P-glycoprotein activity by glial factors and retinoic acid in an immortalized rat brain microvessel endothelial cell line

P-glycoprotein (P-gp), a product of the multidrug-resistant (mdr) genes, is expressed in the endothelial cells of the blood–brain barrier (BBB). Effects of glial factors and retinoic acid (RA) on P-gp activity and level were investigated in the immortalized rat brain endothelial cell line RBE4, which expressed immunodetectable P-gp associated with a decrease in accumulation of the P-gp substrates, vinblastine and colchicine. When RBE4 cells were cultured either in the presence of C6-conditioned medium or on C6- or astrocyte-extracellular matrix, intracellular vinblastine and colchicine concentrations were decreased. When the cells were treated with RA, increases in P-gp activities were correlated with increases in P-gp levels. Effects of simultaneous treatments with glial factors and RA were studied in RBE4 cells cultured on astrocyte-extracellular matrix and were shown to be additive on P-gp activity and level. RBE4 cells may serve as a useful in vitro model for basic research on P-gp regulation at the level of the BBB.

Modulation of P-glycoprotein activity by estramustine is limited by binding to plasma proteins.

Estramustine previously has been shown to interact with P-glycoprotein and to restore intracellular accumulation of vinblastine and paclitaxel in cells overexpressing this drug transporter. However, the ability of estramustine to potentiate the cytotoxicities of several drugs was less than that expected. To resolve this apparent discordance, the authors examined the effects of serum on the actions of estramustine. The cytotoxicities of anticancer drugs with or without estramustine or verapamil toward MCF-7 breast carcinoma cells and a P-glycoprotein-overexpressing subline MCF-7/ADR were determined using the sulforhodamine-binding assay. The extent of intracellular accumulation of [3H]vinblastine and [3H]paclitaxel was determined for each using standard methods, and the binding of radiolabeled drugs to plasma proteins was characterized by equilibrium dialysis. Without serum, the sensitivities of MCF-7/ADR cells to several P-glycoprotein-transported drugs were increased by estramustine and verapamil. Conversely, when the cells were treated with a 10% serum, the cytotoxicities of these drugs were increased by verapamil, but not by estramustine. Without serum, intracellular accumulation of [3H]vinblastine and [3H]paclitaxel by MCF-7/ADR cells was increased markedly by verapamil and estramustine; however, serum suppressed the effects of estramustine much more strongly than those of verapamil. Equilibrium dialysis experiments demonstrated that [3H]estramustine binds to plasma proteins, predominantly albumin, whereas [3H]paclitaxel binds to albumin and alpha 1-acid-glycoprotein, and [3H]vinblastine binds predominantly to alpha 1-acid-glycoprotein. Although estramustine can bind to P-glycoprotein, its effectiveness as a reversing agent in vivo likely is limited by binding to plasma proteins.