Changes in P-glycoprotein activity are mediated by the growth of a tumour cell line as multicellular spheroids

Background  

Expression of P-glycoprotein (P-gp), the multidrug resistance (MDR) 1 gene product, can lead to multidrug resistance in tumours. However, the physiological role of P-gp in tumours growing as multicellular spheroids is not well understood. Recent evidence suggests that P-gp activity may be modulated by cellular components such as membrane proteins, membrane-anchoring proteins or membrane-lipid composition. Since, multicellular spheroids studies have evidenced alterations in numerous cellular components, including those related to the plasma membrane function, result plausible that some of these changes might modulate P-gp function and be responsible for the acquisition of multicellular drug resistance. In the present study, we asked if a human lung cancer cell line (INER-51) grown as multicellular spheroids can modify the P-gp activity to decrease the levels of doxorubicin (DXR) retained and increase their drug resistance.     

Effects of Sertraline and Fluoxetine on P-Glycoprotein at Barrier Sites: In Vivo and In Vitro Approaches

Background and Purpose

Retention of substances from systemic circulation in the brain and testes are limited due to high levels of P-glycoprotein (P-gp) in the luminal membranes of brain and testes capillary endothelial cells. From a clinical perspective, P-gp rapidly extrudes lipophilic therapeutic agents, which then fail to reach efficacious levels. Recent studies have demonstrated that acute administration of selective serotonin reuptake inhibitors (SSRI) can affect P-gp function, in vitro and in vivo. However, little is known concerning the time-course of these effects or the effects of different SSRI in vivo.

Experimental Approach

The P-gp substrate, tritiated digoxin ([³H] digoxin), was co-administered with fluoxetine or sertraline to determine if either compound increased drug accumulation within the brains and testes of mice due to inhibition of P-gp activity. We undertook parallel studies in endothelial cells derived from brain microvessels to determine the dose-response and time-course of effects.

Key Results

In vitro, sertraline resulted in rapid and potent inhibition of P-gp function in brain endothelial cells, as determined by cellular calcein accumulation. In vivo, a biphasic effect was demonstrated. Brain accumulation of [³H] digoxin was increased 5 minutes after treatment with sertraline, but by 60 minutes after sertraline treatment, brain accumulation of digoxin was reduced compared to control. By 240 minutes after sertraline treatment brain digoxin accumulation was elevated compared to control. A similar pattern of results was obtained in the testes. There was no significant effect of fluoxetine on P-gp function, in vitro or in vivo.

Conclusions and Implications

Acute sertraline administration can modulate P-gp activity in the blood-brain barrier and blood-testes barrier. This clearly has implications for the ability of therapeutic agents that are P-gp substrates, to enter the brain when co-administered with SSRI.     

Nitric oxide reverses drug resistance by inhibiting ATPase activity of p-glycoprotein in human multi-drug resistant cancer cells

Background

Development of resistance to chemotherapy drugs is a significant problem in treating human malignancies in the clinic. Overexpression of drug efflux proteins, including P-170 glycoprotein (P-gp), an ATP-dependent efflux protein, is one of the main mechanisms responsible for multi-drug resistance (MDR). Because our previous studies have shown that nitric oxide (^˙NO) or its related species inhibit the ATPase activities of topoisomerase II, we hypothesized that ^˙NO should also inhibit the ATPase activity of P-gp and increase drug accumulation in MDR cells, causing a reversal of drug resistance.

Evaluation of the activity of CYP2C19 in Gujrati and Marwadi subjects living in Mumbai (Bombay)

Background  

Inherited differences in the metabolism and disposition of drugs, and genetic polymorphisms in the targets of drug therapy (e.g., receptors), can greatly influence efficacy and toxicity of medications. Marked interethnic differences in CYP2C19 (a member of the cytochrome P-450 enzyme superfamily catalyzing phase I drug metabolism) which affects the metabolism of a number of clinically important drugs have been documented. The present study evaluated the activity of CYP2C19 in normal, healthy Gujrati and Marwadi subjects by phenotyping (a western Indian population).     

Regulation of Biotransformation Systems and ABC Transporters by Benznidazole in HepG2 Cells: Involvement of Pregnane X-Receptor

Background

Benznidazole (BZL) is the only antichagasic drug available in most endemic countries. Its effect on the expression and activity of drug-metabolizing and transporter proteins has not been studied yet.

Methodology/Principal Findings

Expression and activity of P-glycoprotein (P-gp), Multidrug resistance-associated protein 2 (MRP2), Cytochrome P450 3A4 (CYP3A4), and Glutathione S-transferase (GST) were evaluated in HepG2 cells after treatment with BZL. Expression was estimated by immunoblotting and real time PCR. P-gp and MRP2 activities were estimated using model substrates rhodamine 123 and dinitrophenyl-S-glutathione (DNP-SG), respectively. CYP3A4 and GST activities were evaluated through their abilities to convert proluciferin into luciferin and 1-chloro-2,4-dinitrobenzene into DNP-SG, respectively. BZL (200 µM) increased the expression (protein and mRNA) of P-gp, MRP2, CYP3A4, and GSTπ class. A concomitant enhancement of activity was observed for all these proteins, except for CYP3A4, which exhibited a decreased activity. To elucidate if pregnane X receptor (PXR) mediates BZL response, its expression was knocked down with a specific siRNA. In this condition, the effect of BZL on P-gp, MRP2, CYP3A4, and GSTπ protein up-regulation was completely abolished. Consistent with this, BZL was able to activate PXR, as detected by reporter gene assay. Additional studies, using transporter inhibitors and P-gp-knock down cells, demonstrated that P-gp is involved in BZL extrusion. Pre-treatment of HepG2 cells with BZL increased its own efflux, as a consequence of P-gp up-regulation.

Conclusions/Significance

Modifications in the activity of biotransformation and transport systems by BZL may alter the pharmacokinetics and efficiency of drugs that are substrates of these systems, including BZL itself.     

Consequences of cell-to-cell P-glycoprotein transfer on acquired multidrug resistance in breast cancer: a cell population dynamics model

Background  

Cancer is a proliferation disease affecting a genetically unstable cell population, in which molecular alterations can be somatically inherited by genetic, epigenetic or extragenetic transmission processes, leading to a cooperation of neoplastic cells within tumoural tissue. The efflux protein P-glycoprotein (P-gp) is overexpressed in many cancer cells and has known capacity to confer multidrug resistance to cytotoxic therapies. Recently, cell-to-cell P-gp transfers have been shown. Herein, we combine experimental evidence and a mathematical model to examine the consequences of an intercellular P-gp trafficking in the extragenetic transfer of multidrug resistance from resistant to sensitive cell subpopulations.     

The contribution of peroxynitrite generation in HIV replication in human primary macrophages

Background

The discovery of diketoacid-containing derivatives as inhibitors of HIV-1 Integrase (IN) (IN inhibitors, IINs) has played a major role in validating this enzyme as an important target for antiretroviral therapy. Since the in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether the IINs are recognized by the multidrug transporter MDR1-P-glycoprotein (P-gp) thereby reducing their intracellular accumulation. To address the effect of IINs on drug transport, nine quinolonyl diketo acid (DKA) derivatives active on the HIV-1 IN strand transfer (ST) step and with EC50 ranging from 1.83 to >50 μm in cell-based assays were tested for their in vitro interaction with P-gp in the CEM-MDR cell system. IINs were investigated for the inhibition and induction of the P-gp function and expression as well as for multidrug resistance (MDR) reversing ability.

Results

The HIV-1 IINs act as genuine P-gp substrates by inhibiting doxorubicin efflux and inducing P-gp functional conformation changes as evaluated by the modulation of UIC2 mAb epitope. Further, IINs chemosensitize MDR cells to vinblastine and induce P-gp expression in drug sensitive revertants of CEM-MDR cells.

Conclusion

To our knowledge, this is the first demonstration that HIV-1 IINs are P-gp substrates. This biological property may influence the absorption, distribution and elimination of these novels anti HIV-1 compounds.     

Astroglial expression of the P-glycoprotein is controlled by intracellular CNTF

Background  

The P-glycoprotein (P-gp), an ATP binding cassette transmembrane transporter, is expressed by astrocytes in the adult brain, and is positively modulated during astrogliosis. In a search for factors involved in this modulation, P-gp overexpression was studied in long-term in vitro astroglial cultures.     

Inhibition of P-Glycoprotein by HIV Protease Inhibitors Increases Intracellular Accumulation of Berberine in Murine and Human Macrophages

Background

HIV protease inhibitor (PI)-induced inflammatory response in macrophages is a major risk factor for cardiovascular diseases. We have previously reported that berberine (BBR), a traditional herbal medicine, prevents HIV PI-induced inflammatory response through inhibiting endoplasmic reticulum (ER) stress in macrophages. We also found that HIV PIs significantly increased the intracellular concentrations of BBR in macrophages. However, the underlying mechanisms of HIV PI-induced BBR accumulation are unknown. This study examined the role of P-glycoprotein (P-gp) in HIV PI-mediated accumulation of BBR in macrophages.

Methodology and Principal Findings

Cultured mouse RAW264.7 macrophages, human THP-1-derived macrophages, Wild type MDCK (MDCK/WT) and human P-gp transfected (MDCK/P-gp) cells were used in this study. The intracellular concentration of BBR was determined by HPLC. The activity of P-gp was assessed by measuring digoxin and rhodamine 123 (Rh123) efflux. The interaction between P-gp and BBR or HIV PIs was predicated by Glide docking using Schrodinger program. The results indicate that P-gp contributed to the efflux of BBR in macrophages. HIV PIs significantly increased BBR concentrations in macrophages; however, BBR did not alter cellular HIV PI concentrations. Although HIV PIs did not affect P-gp expression, P-gp transport activities were significantly inhibited in HIV PI-treated macrophages. Furthermore, the molecular docking study suggests that both HIV PIs and BBR fit the binding pocket of P-gp, and HIV PIs may compete with BBR to bind P-gp.

Conclusion and Significance

HIV PIs increase the concentration of BBR by modulating the transport activity of P-gp in macrophages. Understanding the cellular mechanisms of potential drug-drug interactions is critical prior to applying successful combinational therapy in the clinic.     

<Emphasis Type="Italic">In Silico</Emphasis> Quantitative Structure-Activity Relationship Studies on P-gp Modulators of Tetrahydroisoquinoline-Ethyl-Phenylamine Series

Background  

Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. The drug efflux by a transport protein is the main reason for MDR. In humans, MDR mainly occurs when the ATP-binding cassette (ABC) family of proteins is overexpressed simultaneously. P-glycoprotein (P-gp) is most commonly associated with human MDR; it utilizes energy from adenosine triphosphate (ATP) to transport a number of substrates out of cells against concentration gradients. By the active transport of substrates against concentration gradients, intracellular concentrations of substrates are decreased. This leads to the cause of failure in cancer chemotherapy.     

Penetration of verapamil across blood brain barrier following cerebral ischemia depending on both paracellular pathway and P-glycoprotein transportation

Background

Blood brain barrier (BBB) dysfunction is a common facet of cerebral ischemia, and the alteration of drug transporter, P-glycoprotein (P-gp), has been documented.

Aims

This study explores influence of damaged BBB and elevated P-gp on cerebral verapamil penetration after ischemia both in vivo and in vitro.

Methods

Middle cerebral artery occlusion (MCAO) induced ischemia/reperfusion (I/R) of rats, and Na₂S₂O₄ induced hypoxia/reoxygenation (H/R) damage of rat brain mirovessel endothelial cells (RBMECs) respectively, served as BBB breakdown model in vivo and in vitro. Evans-Blue (EB) extravagation and ¹²⁵I-albumin were used to quantify BBB dysfunction; UPLC–MS/MS analytical method was performed to determine accurately the concentration of verapamil in brain tissue and cell. Flow cytometry, immunohistochemistry and western blotting were applied to evaluate transport function and protein expression of P-gp.

Results

Overexpressed ICAM-1 and MMP-9 mediated BBB dysfunction after ischemia, which induced EB leakage and ¹²⁵I-albumin uptake increase. Enhanced accumulation of verapamil in brain tissue, but intracellular concentration reduced evidently after H/R injury. Transcellular transportation of verapamil elevated when P-gp function or expression was inhibited after H/R injury.

Conclusion

These data indicated that BBB penetration of verapamil under ischemia condition was not only depending on BBB breakdown, but also regulated by P-gp.     

Synthesis,biological evaluation and 3D-QSAR studies of new chalcone derivatives as inhibitors of human P-glycoprotein

P-glycoprotein (P-gp) is an ATP-dependent multidrug resistance efflux transporter that plays an important role in anticancer drug resistance and in pharmacokinetics of medicines. Despite a large number of structurally and functionally diverse compounds, also flavonoids and chalcones have been reported as inhibitors of P-gp. The latter share some similarity with the well studied class of propafenones, but do not contain a basic nitrogen atom. Furthermore, due to their rigidity, they are suitable candidates for 3D-QSAR studies. In this study, a set of 22 new chalcone derivatives were synthesized and evaluated in a daunomycin efflux inhibition assay using the CCRF.CEM.VCR1000 cell line. The compound 10 showed the highest activity (IC₅₀ = 42 nM), which is one order of magnitude higher than the activity for an equilipohillic propafenone analogue. 2D- and 3D-QSAR studies indicate the importance of H-bond acceptors, methoxy groups, hydrophobic groups as well as the number of rotatable bonds as pharmacophoric features influencing P-gp inhibitory activity.     

Prediction of promiscuous p-glycoprotein inhibition using a novel machine learning scheme

Background

P-glycoprotein (P-gp) is an ATP-dependent membrane transporter that plays a pivotal role in eliminating xenobiotics by active extrusion of xenobiotics from the cell. Multidrug resistance (MDR) is highly associated with the over-expression of P-gp by cells, resulting in increased efflux of chemotherapeutical agents and reduction of intracellular drug accumulation. It is of clinical importance to develop a P-gp inhibition predictive model in the process of drug discovery and development.

Methodology/Principal Findings

An in silico model was derived to predict the inhibition of P-gp using the newly invented pharmacophore ensemble/support vector machine (PhE/SVM) scheme based on the data compiled from the literature. The predictions by the PhE/SVM model were found to be in good agreement with the observed values for those structurally diverse molecules in the training set (n = 31, r ² = 0.89, q ² = 0.86, RMSE = 0.40, s = 0.28), the test set (n = 88, r ² = 0.87, RMSE = 0.39, s = 0.25) and the outlier set (n = 11, r ² = 0.96, RMSE = 0.10, s = 0.05). The generated PhE/SVM model also showed high accuracy when subjected to those validation criteria generally adopted to gauge the predictivity of a theoretical model.

Conclusions/Significance

This accurate, fast and robust PhE/SVM model that can take into account the promiscuous nature of P-gp can be applied to predict the P-gp inhibition of structurally diverse compounds that otherwise cannot be done by any other methods in a high-throughput fashion to facilitate drug discovery and development by designing drug candidates with better metabolism profile.     

Curcumin induces apoptosis of multidrug-resistant human leukemia HL60 cells by complex pathways leading to ceramide accumulation

Most anti-cancer agents induce apoptosis, however, a development of multidrug resistance in cancer cells and defects in apoptosis contribute often to treatment failure. Here, the mechanism of curcumin-induced apoptosis was investigated in human leukemia HL60 cells and their HL60/VCR multidrug-resistant counterparts. In both cell lines curcumin induced a bi-phasic ceramide generation with a slow phase until 6 h followed by a more rapid one. The level of the ceramide accumulation correlated inversely with the cell viability. We found that the ceramide elevation resulted from multifarious changes of the activity of sphingolipid-modifying enzymes. In both cell lines curcumin induced relatively fast activation of neutral sphingomyelinase (nSMase), which peaked at 3 h, and was followed by inhibition of sphingomyelin synthase activity. In addition, in HL60/VCR cells the glucosylceramide synthase activity was diminished by curcumin. This process was probably due to curcumin-induced down-regulation of P-gp drug transporter, since cyclosporine A, a P-gp blocker, also inhibited the glucosylceramide synthase activity. Inhibition of nSMase activity with GW4869 or silencing of SMPD3 gene encoding nSMase2 reversed the curcumin-induced inhibition of sphingomyelin synthase without affecting the glucosylceramide synthase activity. The early ceramide generation by nSMase was indispensable for the later lipid accumulation, modulation of Bax, Bcl-2 and caspase 3 levels, and for reduction of cell viability in curcumin-treated cells, as all these events were inhibited by GW4869 or nSMase2 depletion. These data indicate that the early ceramide generation by nSMase2 induced by curcumin intensifies the later ceramide accumulation via inhibition of sphingomyelin synthase, and controls pro-apoptotic signaling.     

Naphthalenyl derivatives for hitting P-gp/MRP1/BCRP transporters

Substituted naphthalenyl derivatives bearing oxazole, or thiazole or furyl heteronuclei have been carried out as bioisosters of aryl-oxazoles and -thiazoles derivatives previously reported in order to investigate the role of the hindrance on the activity towards P-gp/BCRP/and MRP1 transporters. In addition, the role of naphthalenyl group to modulate P-gp intrinsic activity of these compounds was ascertained.The results demonstrated that all naphthalenyl derivatives displayed comparable P-gp activity with respect to lead compounds previously characterized in our SAR studies but were less active towards BCRP and MRP1 pumps. In terms of intrinsic activity, the replacement of aryl with naphthalenyl moiety led to P-gp inhibitors, unambiguous or ambiguous substrates on the base of the heteronucleus and the substituent on the naphthalenyl fragment. Indeed, oxazole derivatives were: inhibitors (R = H, F, OH), unambiguous substrates (R = OCH₃), or ambiguous substrate (R = Br); thiazole derivatives were: unambiguous substrates (R = OCH₃, Br), or ambiguous substrates (R = H, F). Finally furyl derivatives were ambiguous substrates.     

Nucleotide binding domain 1 pharmacophore modeling for visualization and analysis of P-glycoprotein–flavonoid molecular interactions

Background

P-glycoprotein (P-gp) is a 170-kDa membrane protein. It provides a barrier function and help to excrete toxins from the body as a transporter. Some bioflavonoids have been shown to block P-gp activity.

Objective

To evaluate the important amino acid residues within nucleotide binding domain 1 (NBD1) of P-gp that play a key role in molecular interactions with flavonoids using structure-based pharmacophore model.

Methods

In the molecular docking with NBD1 models, a putative binding site of flavonoids was proposed and compared with the site for ATP. The binding modes for ligands were achieved using LigandScout to generate the P-gp–flavonoid pharmacophore models.

Results

The binding pocket for flavonoids was investigated and found these inhibitors compete with the ATP for binding site in NBD1 including the NBD1 amino acid residues identified by the in silico techniques to be involved in the hydrogen bonding and van der Waals (hydrophobic) interactions with flavonoids.

Conclusion

These flavonoids occupy with the same binding site of ATP in NBD1 proffering that they may act as an ATP competitive inhibitor.

     

Monkey hybrid stem cells develop cellular features of Huntington's disease

Background  

Pluripotent stem cells that are capable of differentiating into different cell types and develop robust hallmark cellular features are useful tools for clarifying the impact of developmental events on neurodegenerative diseases such as Huntington's disease. Additionally, a Huntington's cell model that develops robust pathological features of Huntington's disease would be valuable for drug discovery research.     

Autophagy regulated by lncRNA HOTAIR contributes to the cisplatin-induced resistance in endometrial cancer cells

Objectives

To identify whether lncRNAs (long non-coding RNA) participate in the regulation of cisplatin-resistant induced autophagy in endometrial cancer cells.

Results

Autophagy activity was significantly boosted in cisplatin-resistant Ishikawa cells, a human endometrial cancer cell line, compared with that in parental Ishikawa cells. After analyzing the overall long noncoding RNA (lncRNA) profiling, a meaningful lncRNA, HOTAIR, was identified. It was down-regulated simultaneously in cisplatin-resistant Ishikawa cells and parental Ishikawa cells treated with cisplatin. RNA interference of HOTAIR reduced the proliferation of cisplatin-resistant Ishikawa cells and enhanced the autophagy activity of cisplatin-resistant Ishikawa cells with or without cisplatin treatment, in addition, beclin-1, multidrug resistance (MDR), and P-glycoprotein (P-gp) were mediated by lncRNA HOTAIR.

Conclusions

It is clear that lncRNAs, specifically HOTAIR, can regulate the cisplatin-resistance ability of human endometrial cancer cells through the regulation of autophagy by influencing Beclin-1, MDR, and P-gp expression.

     

Infliximab therapy increases body fat mass in early rheumatoid arthritis independently of changes in disease activity and levels of leptin and adiponectin: a randomised study over 21 months

Introduction  

Rheumatoid arthritis (RA) is associated with changes in body composition and bone mineral density (BMD). The purpose of the present study was to evaluate whether anti-TNF treatment in early RA has an impact on body composition and BMD besides that which could be achieved by intensive disease-modifying anti-rheumatic drug (DMARD) combination therapy.