P-glycoprotein mRNA Expression Research Articles

Effect of Hydrogel Stiffness on Chemoresistance of Breast Cancer Cells in 3D Culture.

Chemotherapy is one of the most common strategies for cancer treatment, whereas drug resistance reduces the efficiency of chemotherapy and leads to treatment failure. The mechanism of emerging chemoresistance is complex and the effect of extracellular matrix (ECM) surrounding cells may contribute to drug resistance. Although it is well known that ECM plays an important role in orchestrating cell functions, it remains exclusive how ECM stiffness affects drug resistance. In this study, we prepared agarose hydrogels of different stiffnesses to investigate the effect of hydrogel stiffness on the chemoresistance of breast cancer cells to doxorubicin (DOX). Agarose hydrogels with a stiffness range of 1.5 kPa to 112.3 kPa were prepared and used to encapsulate breast cancer cells for a three-dimensional culture with different concentrations of DOX. The viability of the cells cultured in the hydrogels was dependent on both DOX concentration and hydrogel stiffness. Cell viability decreased with DOX concentration when the cells were cultured in the same stiffness hydrogels. When DOX concentration was the same, breast cancer cells showed higher viability in high-stiffness hydrogels than they did in low-stiffness hydrogels. Furthermore, the expression of P-glycoprotein mRNA in high-stiffness hydrogels was higher than that in low-stiffness hydrogels. The results suggested that hydrogel stiffness could affect the resistance of breast cancer cells to DOX by regulating the expression of chemoresistance-related genes.

Non‐nutritive sweeteners competitively inhibit P‐glycoprotein in liver

Non‐nutritive sweeteners (NNS) are popular sugar replacements that provide sweet taste with little to no calories and are used in a wide range of products, including foods, beverages, and medications. Although NNS are recognized as safe for the general population at the Acceptable Daily Intake (ADI) by the Food and Drug Administration, the effects of NNS on specific patient populations and on human development are not completely understood. Previously, our lab has shown that in mice, pups exposed to NNS during early development present alterations in liver detoxification and metabolic enzymes. This led us to question whether NNS may disrupt detoxification in the liver, including transmembrane efflux transporters such as P‐glycoprotein (PGP). PGP plays a major role in the distribution of common xenobiotics and drugs, such as antibiotics, antidepressants, and antivirals, by excreting their conjugated metabolites from liver cells to the general circulation (blood and bile). Mutations in PGP, along with administration of pharmacological inhibitors lead to intracellular accumulation of its substrates and toxicity.We hypothesize that two common NNS, acesulfame potassium (AceK) and sucralose (Sucr), inhibit PGP’s efflux function in liver cells and alter PGP expression, suggesting reduced detoxification capacity in the liver.To investigate the effects of NNS on hepatic PGP, we first exposed the HepG2 liver carcinoma cell line to AceK and Sucr and assessed PGP mRNA expression by qPCR. Cells were treated with varying concentrations of AceK, Sucr, or combined NNS (AceK+Sucr) that spanned levels below and above the ADI for each sweetener for either 24h acute exposure or 72h chronic exposure. In parallel, PGP protein levels were assessed by Western blot. 24h combined exposure of AceK and Sucr, at concentrations above the ADI, increased PGP transcript expression, but had no effect at concentrations below or equal to the ADI. Only levels above the ADI of AceK and Sucr treatment at 72h led to increased PGP mRNA levels. Combined NNS treatment for 72h, however, increased PGP transcript levels at lower concentrations than acute exposure. In western blot analysis of protein expression, concentrations of combined NNS below the ADI increased PGP levels, in contrast to the qPCR data.As PGP inhibitor drugs have been shown to similarly affect its expression, we then wished to understand whether NNS impact PGP’s efflux action. Therefore, a cell‐free assay measuring PGP’s ATPase activity was used. Complementarily, in vitro assays of substrate retention and substrate efflux in HepG2 were performed to measure cellular efflux. We found that physiologically relevant NNS concentrations led to significant stimulation of ATPase activity, similar to potent PGP competitive inhibitors such as Verapamil. Similarly, low concentrations of AceK, Sucr, or combined NNS, comparable to 1/8th to 1 times the ADI for each, significantly retain PGP substrate Calcein‐AM intracellularly, or reduce efflux of PGP substrate Rhodamine 123, confirming PGP inhibition by NNS.Our results provide evidence that two common NNS, AceK and Sucr, are both substrates and inhibitors of PGP, and that both acute and chronic exposure to those NNS impacts PGP expression. Most importantly, altered PGP efflux function was observed after exposure of hepatic cells to sub‐ADI concentrations of NNS within expected exposure to common foods and beverages, suggesting risks for NNS consumers when taking medications that utilize the PGP as a main detoxification transporter.

Decreased sensitivity of several anticancer drugs in TMEPAI knockout triple-negative breast cancer cells

BACKGROUND Transmembrane prostate androgen-induced protein (TMEPAI) was reported to be highly amplified in the majority of patients with triple-negative breast cancer (TNBC). TMEPAI is related to poorer prognosis, limited treatment options, and prone to drug resistance compared with other proteins. One of the established markers to determine cancer resistance to drugs is the increased expression levels of drug efflux transporters. However, the role of TMEPAI in cancer resistance to drugs has not been elucidated. This study was aimed to investigate whether TMEPAI participates in cancer resistance to drugs by regulating drug efflux transporters.
 METHODS TMEPAI knockout (KO) cells were previously developed from a TNBC cell line, Hs578T (wild-type/WT), using a CRISPR-Cas9 system. The expression levels of drug efflux transporters were determined in Hs578T-KO and Hs578-WT by quantitative reverse transcriptase polymerase chain reaction. Cytotoxic concentration 50% (CC50) of several anticancer drugs (doxorubicin, cisplatin, and paclitaxel) were determined in the two cell lines via 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay.
 RESULTS The results showed that the mRNA expression of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) was significantly increased in Hs578T-KO compared with that in Hs578T-WT cells. CC50 of several anticancer drugs investigated (doxorubicin, paclitaxel, and cisplatin) in Hs578T-KO cells was higher than that in Hs678-WT.
 CONCLUSIONS TMEPAI participated in the regulation of mRNA expression levels in drug efflux transporters (P-gp, BCRP, and multidrug resistance-associated protein 1). Further studies are necessary to confirm whether this finding might be dependent on the development of cancer cell sensitivity to anticancer agents.

Effects of a common pharmaceutical, atorvastatin, on energy metabolism and detoxification mechanisms of a marine bivalve Mytilus edulis

Biologically active compounds from pharmaceuticals cause concern due to their common occurrence in water and sediments of urbanized coasts and potential threat to marine organisms. Atorvastatin (ATO), a globally prescribed drug, is environmentally stable and bioavailable to marine organisms; however, the physiological and toxic effects of this drug on ecologically important coastal species are yet to be elucidated. We studied the effect of ATO (˜1.2 μg L−1) on bioenergetics (including whole-organism and mitochondrial respiration, as well as tissue energy reserves and mRNA expression of genes involved in mitochondrial biogenesis and fatty acid metabolism in the gills and the digestive gland) of a keystone bivalve Mytulis edulis (the blue mussel) from the Baltic Sea. Xenobiotic detoxification systems including activity and mRNA expression of P-glycoprotein, and Phase I and II biotransformation enzymes (cytochrome P450 monooxygenase CYP1A and glutathione transferase, GST) were also assessed in the gill and digestive gland of the mussels. Exposure to ATO caused rapid uptake and biotransformation of the drug by the mussels. Standard metabolic rate of ATO-exposed mussels increased by 56% indicating higher maintenance costs, yet no changes were detected in the respiratory capacity of isolated mitochondria. ATO exposure led to ˜60% decrease in the lysosomal membrane stability of hemocytes and ˜3-fold decrease in the whole-organism P-glycoprotein-driven and diffusional efflux of xenobiotics indicating altered membrane properties. The digestive gland was a major target of ATO toxicity in the mussels. Exposure of mussels to ATO led to depletion of lipid, carbohydrate and protein pools, and suppressed transcription of key enzymes involved in mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator 1-alpha PGC-1α) and fatty acid metabolism (acetyl-CoA carboxylase and CYP4Y1) in the digestive gland. No bioenergetic disturbances were observed in the gills of ATO-exposed mussels, and elevated GST activity indicated enhanced ATO detoxification in this tissue. These data demonstrate that ATO can act as a metabolic disruptor and chemosensitizer in keystone marine bivalves and warrant further investigations of statins as emerging pollutants of concern in coastal marine ecosystems.

THE MODULATION OF DRUG EFFLUX TRANSPORTER BY CURCUMIN IN MCF7 BREAST CANCER CELLS AFTER REPEATED EXPOSURE OF ENDOXIFEN AND ESTRADIOL

Objective: The aim of the present study was to determine whether curcumin (CM) can prevent drug sensitivity of breast cancer (BC) cells when E andβ-E2 are administered together and whether the underlying mechanism involves modulation of drug efflux transporters.Methods: MCF7 BC cells were treated with the vehicle only, E+β-E2, or E+β-E2+CM repeatedly for 8 weeks. Afterward, the cells were harvested,counted, and isolated for total RNA extraction. Total RNA was then processed into cDNA and further processed for the determination of mRNAexpression patterns of drug efflux transporters (P-glycoprotein, BCRP, and MRP1).Results: Decreased sensitivity of BC cells was shown by the increased cell viability of MCF7 cells after 8 weeks. This condition was accompanied withincreased mRNA expression of P-glycoprotein, BCRP, and MRP1 in cells treated with E+β-E2, as compared with the vehicle only. CM, administered incombination with E+β-E2, resulted in decreased cell viability versus E and β-E2 and also decreased in mRNA expression of P-glycoprotein, BCRP, andMRP1.Conclusion: CM partially reversed the sensitivity loss of BC cells to E in the presence of β-E2 by modulating drug efflux transporters.

PIPERINE IN THE PREVENTION OF THE DECREASED TAMOXIFEN SENSITIVITY IN MCF-7 BREAST CANCER CELL LINE

Objective: This study was designed to analyze the role of piperine in modulating P-glycoprotein mRNA expression when added in combination withtamoxifen to breast cancer cells in culture.Methods: MCF-7 breast cancer cells were treated with 1 μM tamoxifen with or without piperine (12.5, 25, or 50 μM) or verapamil 50 μM (P-glycoproteininhibitor positive control) for up to 12 days. We assessed the cell viability and isolated total RNA from them. We quantified P-glycoprotein expressionsusing quantitative reverse transcription polymerase chain reaction.Results: Administration of various doses of piperine decreased MCF-7 breast cancer cell viability. Piperine, when given in combination with tamoxifen,decreased the expression of P-glycoprotein mRNA in cells compared with the expression in cells treated with tamoxifen only. The effects were shownto be dose dependent.Conclusion: Piperine prevents the development of breast cancer cell tamoxifen resistance, probably through its inhibition of P-glycoprotein expression.

Apiaceous vegetable intake modulates expression of DNA damage response genes and microRNA in the rat colon

We examined chemo-preventive mechanisms of apiaceous vegetable intake against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Rats fed either a purified diet, or purified diet supplemented with 21% apiaceous vegetables (API), were injected with PhIP after 6 d of feeding and colons harvested 24 h later. There was a trend for increased colonic mRNA expression of P-glycoprotein, a PhIP efflux transporter. No DNA damage response genes were up-regulated by API but 13 of these genes were down-regulated, compared to the positive control group, which may have been due to formation of fewer PhIP-DNA adducts. In colonic miRNA profiling, 11 out of 421 miRNAs were modulated in the API group, 3 of which target 19 mRNAs involved in cancer signaling pathways. API increased miR-19a, which targets Ccnd1, a potential oncogene responsible for G1-S cell cycle progression. In conclusion, apiaceous vegetable intake may be chemopreventive through modification of miRNA expression as well as P-glycoprotein expression in colon.

Multivalent methionine-functionalized biocompatible block copolymers for targeted small interfering RNA delivery and subsequent reversal effect on adriamycin resistance in human breast cancer cell line MCF-7/ADR.

Cationic polymers are outstanding representatives of the most efficient small interfering RNA (siRNA) vectors. Low cytotoxicity and siRNA protecting effect can be obtained with these cationic polymers via a variety of structural modifications. Nevertheless, the gap between their efficiency and the requirement for therapeutic processes is still noticeable. A cationic polymer vector was synthesized via the copolymerization of N-(1,3-dihydroxy propan-2-yl)methacrylamide (DHPMA) and N-(3-aminopropyl)methacrylamide (APMA). APMA provides amine functionality that allows the conjugation of guanidine and methionine groups. Attributed to the hydroxy groups of DHPMA, the synthesized guanidine and methionine grafted DHPMA-b-APMA block copolymer (mDG) is water soluble and has good biocompatibility. The obtained mDG has high zeta potential, narrow molecular weight distribution, better membrane-penetrating ability, high transfection efficiency, tumor cell targeting ability and high stability. The synthesized polymer vector can deliver siRNA molecules into tumor cells and then reverse drug resistance by down regulation of P-glycoprotein mRNA expression.

Targeting P-glycoprotein function, p53 and energy metabolism: Combination of metformin and 2-deoxyglucose reverses the multidrug resistance of MCF-7/Dox cells to doxorubicin.

Multidrug resistance(MDR) is a major obstacle to efficiency of breast cancer chemotherapy. We investigated whether combination of metformin and 2-deoxyglucose reverses MDR of MCF-7/Dox cells and tried to elucidate the possible mechanisms. The combination of metformin and 2-deoxyglucose selectively enhanced cytotoxicity of doxorubicin against MCF-7/Dox cells. Combination of the two drugs resumed p53 function via inhibiting overexpression of murine doubleminute 2(MDM2) and murine doubleminute 4(MDM4) leading to G2/M arrest and apoptosis in MCF-7/Dox cells. Combination of the two drugs had no effect on P-glycoprotein mRNA expression and P-glycoprotein ATPase activity but increased doxorubicin accumulation in MCF-7/Dox cells. The increased doxorubicin accumulation maybe associate with metabolic stress. Combination of metformin and 2-deoxyglucose initiated a strong metabolic stress in MCF-7/Dox cells via inhibiting glucose uptake, lactate, fatty acid, ATP production and protein kinase B(AKT)/ mammalian target of rapamycin(mTOR) pathway. Taken together, combination of metformin and 2-deoxyglucose reverses MDR of MCF-7/Dox cells by recovering p53 function and increasing doxorubicin accumulation. Furthermore, doxorubicin selectively increases MCF-7/Dox apoptosis via aggravating metabolic stress induced by metformin plus 2-deoxyglucose. The mutually reinforcing effect made the combination of metformin and 2DG had a better effect on reversing MDR.

Co-treatment by docetaxel and vinblastine breaks down P-glycoprotein mediated chemo-resistance.

Chemoresistance remains the main causes of treatment failure and mortality in cancer patients. There is an urgent need to investigate novel approaches to improve current therapeutic modalities and increase cancer patients' survival. Induction of drug efflux due to overexpression of P-glycoproteins is considered as an important leading cause of multidrug resistance. In this study, we investigated the role of combination treatments of docetaxel and vinblastine in overcoming P-glycoprotein mediated inhibition of apoptosis and induction of cell proliferation in human non-small cell lung carcinoma cells. Cell proliferation and apoptosis were assessed using MTT assay and DAPI staining, respectively. P-glycoprotein expression was evaluated in gene and protein levels by Real-time RT-PCR and Western blot analysis, respectively. Combination treatment of the cells with docetaxel and vinblastine decreased the IC50 values for docetaxel from (30±3.1) to (15±2.6) nM and for vinblastine from (30±5.9) to (5±5.6) nM (P≤0.05). P-glycoprotein mRNA expression level showed a significant up-regulation in the cells incubated with each drug alone (P≤0.001). Incubation of the cells with combined concentrations of both agents neutralized P-glycoprotein overexpression (P≤0.05). Adding verapamil, a P-glycoprotein inhibitor caused a further increase in the percentage of apoptotic cells when the cells were treated with both agents. Our results suggest that combination therapy along with P-glycoprotein inhibition can be considered as a novel approach to improve the efficacy of chemotherapeutics in cancer patients with high P-glycoprotein expression.

The effect of pomelo mix ethyl acetate extract on CYP3A6 and P-glycoprotein gene transcripts in rabbits.

Pomelo fruit juice and pomelo ethylacetate extract have been shown to increase the bioavailability of some CYP3A substrates. The purpose of this study was to investigate if this effect might be contributed to by changes in CYP3A and p-glycoprotein mRNAs levels in the liver and proximal small intestine. The ethyl acetate extract of pomelo mix was administered for 7 days to 10 rabbits. Nine rabbits were administered tap water for 7 days. The administration was through oral intubation to the stomach. On the 8(th) day, the rabbits were sacrificed, and the liver and the proximal 15 cm of the small intestine were dissected. Total RNA was extracted from the specimens and cDNA was prepared by quantitative real-time-polymerase chain reaction (RT-PCR) using specific primers. The ethyl acetate extract of pomelo mix reduced the mRNA expression of CYP3A6 almost 5-folds in the intestine and 2-folds in the liver. In contrast, a 1-fold increase to the p-glycoprotein mRNA expression was observed under the same experimental conditions. In conclusion, the ethyl acetate extract of pomelo mix reduced the mRNA expression of CYP3A6 in both intestine and liver but to different degrees, while the p-glycoprotein mRNA expression was not reduced.

Ginsenoside Metabolites Inhibit P-Glycoprotein In Vitro and In Situ Using Three Absorption Models

P-glycoprotein, an ATP-dependent transporter expressed in the gastrointestinal tract and tumor cells, mediates the efflux transport of multiple drugs. Inhibition or induction of P-glycoprotein by herbal ingredients can lead to herb-drug interactions and thus influence the activities of P-glycoprotein substrate drugs. The present study aimed to explore the effect of nine naturally occurring ginsenosides and their intestinal bacterial metabolites on P-glycoprotein-mediated transport. The results showed that three ginsenoside metabolites (CK, Ppd, and Ppt) formed by intestinal bacteria significantly enhanced rhodamine 123 retention in Caco-2 cells, increased the absorptive permeability of rhodamine 123, and decreased the efflux ratio of digoxin in two absorption models, which were comparable to the effects of the known P-glycoprotein inhibitor verapamil. However, the prototype ginsenosides such as Rb1, Rb2, and Re showed no inhibitory effect on P-glycoprotein activity. In situ intestinal perfusion experiments also showed that CK, Ppd, and Ppt increased the absorption rate constant and permeability coefficient of rhodamine 123. Long-term treatment with CK, Ppd, and Ppt had no effect on P-glycoprotein mRNA expression in Caco-2 cells. In conclusion, CK, Ppd, and Ppt are potent P-glycoprotein inhibitors, indicating an unpredictable herb-drug interaction when ginsenosides are coadministered orally with P-glycoprotein substrate drugs.

Tissue accumulation and toxicity of isothiazolinone in Ctenopharyngodon idellus (grass carp): Association with P-glycoprotein expression and location within tissues

Isothiazolinone is widely used as a broad-spectrum fungicide in various industries, such as oil, paper, pesticide, dyes, tanning and cosmetics. There is an increasing concern over protection of the aquatic environment due to its large-scale use. The acute toxicity (LC50) of isothiazolinone in Ctenopharyngodon idellus was investigated. The residual time and accumulation in tissues, P-glycoprotein mRNA level and localization of P-glycoprotein in the liver and kidney were also analyzed. The LC50 (48h) values of isothiazolinone to C. idellus were 0.53±0.17mg/L and 0.41±0.08mg/L at 15°C and 25°C, respectively. The LC50 values decreased as the temperature increased. The accumulation of isothiazolinone in livers and kidneys in the high temperature group (25°C) was significantly greater than that of the low temperature group (15°C). Prolonged tissue residual time of isothiazolinone was seen in all the groups. There were significant differences in P-glycoprotein mRNA expression between isothiazolinone-treated groups and control samples (P<0.05–0.01). Temperature affected accumulation and toxicity of isothiazolinone.

Intercellular Transfer of P-Glycoprotein from the Drug Resistant Human Bladder Cancer Cell Line BIU-87 Does Not Require Cell-to-Cell Contact

The efflux activity of transmembrane P-glycoprotein prevents various therapeutic drugs from reaching lethal concentrations in cancer cells, resulting in multidrug resistance. We investigated whether drug resistant bladder cancer cells could transfer functional P-glycoprotein to sensitive parental cells. Drug sensitive BIU-87 bladder cancer cells were co-cultured for 48 hours with BIU-87/ADM, a doxorubicin resistant derivative of the same cell line, in a Transwell® system that prevented cell-to-cell contact. The presence of P-glycoprotein in recipient cell membranes was established using fluorescein isothiocyanate, laser scanning confocal microscopy and Western blot. P-glycoprotein mRNA levels were compared between cell types. Rhodamine 123 efflux assay was done to confirm that P-glycoprotein was biologically active. The amount of P-glycoprotein protein in BIU-87 cells co-cultured with BIU-87/ADM was significantly higher than in BIU-87 cells (0.44 vs 0.25) and BIU-87/H33342 cells (0.44 vs 0.26, each p <0.001), indicating P-glycoprotein transfer. P-glycoprotein mRNA expression was significantly higher in BIU-87/ADM cells than in co-cultured BIU-87 cells (1.28 vs 0.30), BIU-87/H33342 (0.28) and BIU-87 cells (0.25, each p <0.001), ruling out a genetic mechanism. After 30 minutes of efflux, rhodamine 123 fluorescence intensity was significantly lower in BIU-87/ADM cells (5.55 vs 51.45, p = 0.004) and co-cultured BIU-87 cells than in BIU-87 cells (14.22 vs 51.45, p <0.001), indicating that P-glycoprotein was functional. Bladder cancer cells can acquire functional P-glycoprotein through a nongenetic mechanism that does not require direct cell contact. This mechanism is consistent with a microparticle mediated process.

Impact of drug transporters on cellular resistance towards saquinavir and darunavir

Highly active antiretroviral therapy is complicated by drug-drug interactions and the development of viral resistance. Drug interactions involve transporters that may critically affect the pharmacokinetics of many antiretroviral drugs and contribute to the formation of functional sanctuary sites. We therefore investigated the effect of saquinavir and darunavir on drug transporter expression and functional consequences for cellular resistance towards these compounds. Induction of transporters was investigated in LS180 cells over a period of 4 weeks by means of RT-PCR, and for some transporters also at the protein and functional levels. Cellular resistance was measured by growth inhibition assays. Incubation with 10 µM darunavir for 1 week significantly increased mRNA expression of P-glycoprotein (P-gp/MDR1/ABCB1) 3.8-fold and of organic anion-transporting polypeptide 2B1 (SLCO2B1) 1.9-fold. In contrast, 10 µM saquinavir significantly increased mRNA expression of P-gp 5.7-fold, multidrug resistance-associated protein 1 (MRP1/ABCC1) 2.3-fold, MRP2/ABCC2 4.5-fold, MRP3/ABCC3 2.0-fold, MRP4/ABCC4 1.8-fold, MRP5/ABCC5 3.8-fold, breast cancer resistance protein (BCRP/ABCG2) 4.1-fold, SLCO1B1 4.6-fold, SLCO2B1 1.8-fold and SLCO3A1 1.8-fold. P-gp induction was also confirmed at the protein and functional levels. Induction by darunavir caused an increase in cellular resistance towards this compound, as measured in growth inhibition assays; however, saquinavir treatment did not cause reduced sensitivity of cells, indicating unchanged intracellular concentration. Hence, induction by darunavir increased drug efflux and might therefore lead to a suboptimal intracellular concentration of darunavir. The study revealed substantial induction of several drug transporters by saquinavir and darunavir, possibly leading to decreased efficacy of antiretrovirals and drugs used to treat co-morbidity.

Paclitaxel-resistance Conferred by Altered Expression of Efflux and Influx Transporters for Paclitaxel in the Human Hepatoma Cell Line, HepG2

Paclitaxel-resistant HepG2 (PR-HepG2) cells were established by long-term exposure of HepG2 cells to paclitaxel and expression and function of efflux (P-glycoprotein, MRP2) and influx (OATP1B3) transporters for paclitaxel were examined to understand the mechanisms underlying the resistance. mRNA expression of P-glycoprotein (P-gp) increased in PR-HepG2 more than in HepG2 cells, while that of MRP2 did not change. Interestingly, mRNA expression of OATP1B3 drastically decreased in PR-HepG2 cells. [(3)H]Paclitaxel uptake was less in PR-HepG2 than in HepG2 cells and the uptake in both cells increased by metabolic inhibition. The uptake of [(3)H]paclitaxel and rhodamine 123 increased by verapamil, a P-gp inhibitor. Probenecid, an MRP inhibitor, did not affect [(3)H]paclitaxel uptake in both cells. Sulfobromophthalein, an OATP1B3 inhibitor, inhibited [(3)H]paclitaxel uptake in HepG2 but not in PR-HepG2 cells. Cytotoxicity studies showed that the resistance of PR-HepG2 cells to paclitaxel was reversed by verapamil. PR-HepG2 cells showed cross-resistance to doxorubicin, a P-gp substrate, but not to cisplatin. These results indicate that enhanced expression and function of P-gp may be a predominant mechanism of paclitaxel resistance in PR-HepG2 cells and the reduced influx via OATP1B3 may also serve to lower intracellular paclitaxel concentration in cooperation with P-gp-mediated efflux.

Effect of endotoxin on P-glycoprotein-mediated biliary and renal excretion of rhodamine-123 in rats.

The effects of Klebsiella pneumoniae endotoxin on the biliary excretion and renal handling of rhodamine-123 were investigated in rats at different times after intraperitoneal injection (1 mg/kg of body weight). The typical substrates for P glycoprotein, i.e., cyclosporine, colchicine, and erythromycin, inhibited the biliary clearance of rhodamine-123, whereas a substrate for organic cation transporter, cimetidine, did not inhibit clearance, suggesting that rhodamine-123 is transported mainly by P glycoprotein. The biliary, renal, and tubular secretory clearances of rhodamine-123 and the glomerular filtration rate significantly decreased 6 h after injection of endotoxin but returned to control levels by 24 h. These results suggest that endotoxin-induced decreases in P-glycoprotein-mediated biliary excretion and renal handling of rhodamine-123 were probably due to impairment of P-glycoprotein-mediated transport ability. Pretreatment with pentoxifylline (50 mg/kg) significantly inhibited endotoxin-induced increases in tumor necrosis factor alpha (TNF-alpha) levels in plasma, which ameliorated the endotoxin-induced reduction of the biliary excretion of rhodamine-123. It is likely that endotoxin-induced impairment of the transport of rhodamine-123 is caused, in part, by overproduction of TNF-alpha. The effect of endotoxin on the expression of P-glycoprotein mRNA in liver and kidneys of rats was investigated by using a reverse transcriptase PCR. The expression of Mdr1a mRNA in both liver and kidney decreased 6 h after endotoxin injection and returned to control levels after 24 h, whereas the expression of Mdr1b mRNA in liver increased at both times and that in kidney decreased at 24 h. These findings suggest that K. pneumoniae endotoxin dramatically decreases P-glycoprotein-mediated biliary and renal excretion of rhodamine-123 probably by decreasing the expression of Mdr1a, which is likely due to increased plasma TNF-alpha levels.

Effect of chronic renal failure on the expression and function of rat intestinal P-glycoprotein in drug excretion.

In chronic renal failure, the renal excretion of certain drugs is dramatically reduced. To determine whether other routes of drug elimination, such as secretion through the intestinal barrier by intestinal P-glycoprotein can be altered, we compared P-glycoprotein activity, P-glycoprotein protein content, and P-glycoprotein mRNA levels in intestine of control and chronic renal failure rats. Chronic renal failure was surgically induced in rats by partial (7/8) nephrectomy. After 5 weeks, intestinal transport of rhodamine 123, a P-glycoprotein substrate, was carried out using an in vitro model of everted gut sacs. P-glycoprotein protein content was quantified by enzyme-linked immunosorbent assay and P-glycoprotein mRNA expression was evaluated by semi-quantitative reverse transcriptase polymerase chain reaction. A decrease of intestinal rhodamine 123 transport was observed in chronic renal failure rats, pointing to an inhibition of P-glycoprotein activity. Transport was inhibited in both sham-operated rats and rats with chronic renal failure by verapamil and cyclosporin A, but relative inhibition vs baseline was less marked in chronic renal failure than in sham-operated rats. In contrast, no significant differences in levels of P-glycoprotein protein or mRNA were observed between the two groups. Intestinal secretion of rhodamine 123 is mainly mediated by P-glycoprotein. It was reduced in rats with chronic renal failure, reflecting reduced intestinal drug elimination via a decrease in P-glycoprotein transport activity rather than via protein underexpression.

Prognostic value of P-glycoprotein expression in bone and soft-tissue sarcoma

Background. The purpose of this study was to investigate the correlation between P-glycoprotein status and outcome in adult patients with high-grade osteosarcomas and soft-tissue sarcomas. Methods. P-glycoprotein status was determined im-munohistochemically in specimens from 28 patients with osteosarcoma and 34 patients with soft-tissue sarcoma. The polyclonal antibody mdr(Ab-1) was used for either decalcified or undecalcified tissue samples which were formalin-fixed and paraffin-embedded. The expression of P-glycoprotein mRNA was also determined by the polymerase chain reaction in 23 fresh sarcoma specimens. P-glycoprotein status was analyzed in relation to the duration of event-free survival. Results. Positivity for P-glycoprotein was found in 29% of the osteosarcomas and 34% of the soft-tissue sarcomas. Consistent results were obtained at both the immunohistochemical and reverse transcriptase-polymerase chain reaction (RT-PCR) levels in 19 of 23 sarcomas (83%). In patients with osteosarcoma, the presence of increased levels of P-glycoprotein was significantly associated with a decreased probability of event-free survival after diagnosis (P = 0.022). In contrast, in patients with soft-tissue sarcoma there was no correlation between the level of P-glycoprotein and prognosis. Conclusions. In patients with high-grade osteosarcomas, the presence of increased levels of P-glycoprotein detected by polyclonal antibody mdr(Ab-1) was associated with a significantly increased risk of adverse events. This association was not found in patients with soft-tissue sarcomas.

Sigma receptors: recent advances and new clinical potentials

Several recent advances are leading to a better understanding of sigma receptors. Here we focus on our recent findings regarding cellular functions of sigma-2 receptors and discuss their possible clinical implications. Agonists at sigma-2 receptors induced changes in cell morphology and apoptosis in various cell types. Sigma-2 receptor activation produced both transient and sustained increases in [Ca++]i, derived from different intracellular stores. These changes in [Ca++]i and cytotoxic effects are mediated by intracellular sigma-2 receptors. Sigma-2 agonists induced apoptosis in drug-resistant cancer cells, enhanced the potency of DNA damaging agents, and down-regulated expression of p-glycoprotein mRNA. Thus, sigma-2 receptor agonists may be useful in treatment of drug-resistant cancers. Sigma radioligands have been used in tumor imaging. We also discuss how sigma-2 antagonists might prevent the irreversible motor side effects of typical neuroleptics. Sigma-2 receptors may subserve a novel signalling pathway to apoptosis, involved in regulation of cell proliferation and/or viability.