Expression Of P-glycoprotein Protein Research Articles

Effects of long-term hepatic ischemia-reperfusion injury on the function of P-glycoprotein in vivo in rats.

Ischemia-reperfusion injury is a common complication in liver surgery with oxidative stress related graft failure as a potential complication. The oxidative stress could affect hepatic drug transporters such as P-glycoprotein, which is crucial in the hepatic clearance of certain immunosuppressant drugs. Thus,, it is important to study its function after ischemia-reperfusion injury in vivo. Rhodamine 123 is a fluorescent substrate of P-glycoprotein and its hepatic disposition can be visualized using multiphoton microscopy in vivo using anaesthetized animals. The aim of this study was to investigate the effect of long-term ischemia-reperfusion injury on P-glycoprotein function in hepatocytes using in vivo multiphoton microscopy. Localized ischemia was induced for 1 hour in rats. The liver was reperfused for 4, 24, 48 hours or 1 week, where-after rhodamine 123 was injected intravenously. Multiphoton microscopy imaged the liver and bile was collected continuously up to 6 hours following drug administration. The liver was harvested for histology and protein expression of P-glycoprotein. Ischemia-reperfusion injury resulted in extensive liver damage, inflammatory cell infiltration and apoptosis in the midzonal and centrilobular regions of the liver acinus. P-glycoprotein protein expression decreased. Cellular concentration of rhodamine 123 increased as visualized by multiphoton microscopy, which was confirmed with decreased excretion of rhodamine 123 in collected bile. This study showed reduced function of P-glycoprotein in ischemia-reperfusion injury as reflected by decreased biliary excretion of Rhodamine 123, as well as reduced protein expression of the transporter. Multiphoton microscopy could be used to visualize and quantitate the intracellular levels of rhodamine 123. These findings stipulate the importance of using multiphoton microscopy to understand transmembrane drug flux and reflect on careful drug dosing after hepatic surgery.

Molecular and cytogenetic changes in multi-drug resistant cancer cells and their influence on new compounds testing

Multi-drug resistance (MDR) is a major obstacle to successful cancer treatment. Therefore, in vitro models are necessary for the investigation of the phenotypic changes provoked by cytotoxic agents and more importantly for preclinical testing of new anticancer drugs. We analyzed chromosomal, numerical, and structural changes after development of MDR, alterations in p53 and PTEN, single nucleotide polymorphisms (SNPs) in the mdr1 gene and corresponding protein expression of P-glycoprotein (P-gp) in three human MDR cancer cell lines: non-small cell lung carcinoma NCI-H460/R, colorectal carcinoma DLD1-TxR, and glioma U87-TxR. In addition, we explored how these molecular and phenotypic alterations influence the anticancer effect of new drugs. Cytogenetic analysis showed polyploidy reduction after development of MDR in U87-TxR. Losses of 6q in all resistant cancer cell lines and inactivation of p53 in U87-TxR and PTEN in DLD1-TxR were also revealed. Overexpression of P-gp was observed in all MDR cancer cell lines. We evaluated the anticancer activities and MDR reversal potential of Akt inhibitor GSK690693, Ras inhibitor Tipifarnib, and two P-gp inhibitors (jatrophane diterpenoids). Their effects vary due to the cell-type differences, existence of MDR phenotype, presence of mdr1 SNP, and tumor suppressors' alterations. Tipifarnib and jatrophane diterpenoids significantly sensitized MDR cancer cells to paclitaxel. In conclusion, investigated MDR cancer cells obtained new molecular and cytogenetic characteristics that may serve as potential clinical prognostic markers. In addition, these MDR cancer cell lines present a valuable model for preclinical evaluation of new anticancer agents.

Podocyte Expression of Membrane Transporters Involved in Puromycin Aminonucleoside-Mediated Injury

Several complex mechanisms contribute to the maintenance of the intricate ramified morphology of glomerular podocytes and to interactions with neighboring cells and the underlying basement membrane. Recently, components of small molecule transporter families have been found in the podocyte membrane, but expression and function of membrane transporters in podocytes is largely unexplored. To investigate this complex field of investigation, we used two molecules which are known substrates of membrane transporters, namely Penicillin G and Puromycin Aminonucleoside (PA).We observed that Penicillin G pre-administration prevented both in vitro and in vivo podocyte damage caused by PA, suggesting the engagement of the same membrane transporters by the two molecules. Indeed, we found that podocytes express a series of transporters which are known to be used by Penicillin G, such as members of the Organic Anion Transporter Polypeptides (OATP/Oatp) family of influx transporters, and P-glycoprotein, a member of the MultiDrug Resistance (MDR) efflux transporter family.Expression of OATP/Oatp transporters was modified by PA treatment. Similarly, in vitro PA treatment increased mRNA and protein expression of P-glycoprotein, as well as its activity, confirming the engagement of the molecule upon PA administration.In summary, we have characterized some of the small molecule transporters present at the podocyte membrane, focusing on those used by PA to enter and exit the cell. Further investigation will be needed to understand precisely the role of these transporter families in maintaining podocyte homeostasis and in the pathogenesis of podocyte injury.

Microglial activation decreases retention of the protease inhibitor saquinavir: implications for HIV treatment

BackgroundActive HIV infection within the central nervous system (CNS) is confined primarily to microglia. The glial cell compartment acts as a viral reservoir behind the blood-brain barrier. It provides an additional roadblock to effective pharmacological treatment via expression of multiple drug efflux transporters, including P-glycoprotein. HIV/AIDS patients frequently suffer bacterial and viral co-infections, leading to deregulation of glial cell function and release of pro-inflammatory mediators including cytokines, chemokines, and nitric oxide.MethodsTo better define the role of inflammation in decreased HIV drug accumulation into CNS targets, accumulation of the antiretroviral saquinavir was examined in purified cultures of rodent microglia exposed to the prototypical inflammatory mediator lipopolysaccharide (LPS).Results[3H]-Saquinavir accumulation by microglia was rapid, and was increased up to two-fold in the presence of the specific P-glycoprotein inhibitor, PSC833. After six or 24 hours of exposure to 10 ng/ml LPS, saquinavir accumulation was decreased by up to 45%. LPS did not directly inhibit saquinavir transport, and did not affect P-glycoprotein protein expression. LPS exposure did not alter RNA and/or protein expression of other transporters including multidrug resistance-associated protein 1 and several solute carrier uptake transporters.ConclusionsThe decrease in saquinavir accumulation in microglia following treatment with LPS is likely multi-factorial, since drug accumulation was attenuated by inhibitors of NF-κβ and the MEK1/2 pathway in the microglia cell line HAPI, and in primary microglia cultures from toll-like receptor 4 deficient mice. These data provide new pharmacological insights into why microglia act as a difficult-to-treat viral sanctuary site.

Upregulation of Drug Transporter Expression by Osteopontin in Prostate Cancer Cells

Multidrug resistance is a major cause of chemotherapy failure. Recent studies indicate that drug resistance can be rapidly induced by some soluble factors, such as cytokines, chemokines, growth factors, and cell adhesion factors in the tumor microenvironment. Osteopontin (OPN), an extracellular matrix protein, has a functional arginine-glycine-aspartic acid (RGD) domain for binding to integrin. Here we found OPN expression to be upregulated by hypoxic condition in PC-3 prostate tumor cells. OPN increased the mRNA and protein expression of p-glycoprotein (P-gp), a subfamily of ATP-binding cassette transporter in a concentration- and time-dependent manner. The increase in P-gp transporter by OPN was mediated by binding to αvβ3 integrin. Daunomycin (DUN), a chemotherapeutic agent with autofluorescence, was used to evaluate the pump activity, and OPN increased the drug pumping-out activity. OPN inhibited DUN-induced cell death, which was antagonized by αvβ3 monoclonal antibody. Long-term treatment with DUN further enhanced the expression of OPN. Knockdown of endogenous OPN potentiated the DUN-induced apoptosis of PC-3 cells. Furthermore, knockdown of OPN enhanced cell death caused by other drugs, including paclitaxel, doxorubicin, actinomycin-D, and rapamycin, which are also P-gp substrates. The animal studies also showed that OPN knockdown enhanced the cytotoxic action of DUN. These results indicate that OPN is a potential therapeutic target for cancer therapy to reduce drug resistance in sensitive tumors.

PXR-mediated P-glycoprotein induction by small molecule tyrosine kinase inhibitors

The rapid development of drug resistance as a result of exposure to small molecule tyrosine kinase inhibitors (TKIs) is an important drawback to the successful use of these agents in the clinic. Although one of the most established mechanisms by which cells acquire drug resistance to anticancer drugs is the up regulation of drug efflux transporters such as P-glycoprotein (PGP), it is currently still unknown whether TKIs have the propensity to induce PGP. The effect of TKIs on the protein expression and activity of PGP was assessed after treatment of LS180 cells with clinically relevant concentrations of the TKIs. In addition, the involvement of the nuclear pregnane X receptor (PXR), a known regulator of PGP expression, was determined. At least five out of the nine tested TKIs (erlotinib, gefitinib, nilotinib, sorafenib, vandetanib) were able to induce the expression of PGP within 48h in LS180 cells. Accordingly, these TKIs were also shown to affect the accumulation of a P-glycoprotein specific probe substrate. Furthermore, we showed that the pregnane X receptor (PXR), which is an important regulator of PGP induction, is involved in the upregulation of PGP protein expression following exposure to these TKIs. Our data show that PXR-mediated upregulation of PGP expression by TKIs might be a possible mechanism underlying acquired drug resistance in cancer cells.

ABC Transporter Function and Regulation at the Blood–Spinal Cord Barrier

We present here an initial characterization of ATP binding cassette (ABC) transporter function and regulation at the blood-spinal cord barrier. We isolated capillaries from rat spinal cords and studied transport function using a confocal microscopy-based assay and protein expression using western blots. These capillaries exhibited transport function and protein expression of P-glycoprotein (Abcb1), multidrug resistance protein 2 (Mrp2, Abcc2), and breast cancer-related protein (Bcrp, Abcg2). Exposing isolated capillaries to dioxin (activates aryl hydrocarbon receptor) increased transport mediated by all three transporters. Brain and spinal cord capillaries from dioxin-dosed rats exhibited increased P-glycoprotein-mediated transport and increased protein expression for all three ABC transporters. These findings indicate similar ABC transporter expression, function, and regulation at the blood-spinal cord and blood-brain barriers.

Reversal of P-glycoprotein-mediated multidrug resistance in human hepatoma cells by hedyotiscone A, a compound isolated from Hedyotis corymbosa

Multidrug resistance is a major problem in hepatocellular carcinoma. Hedyotiscone A, a compound isolated from Chinese herbal medicine Hedyotis corymbosa (HC, family Rubiaceae), was used as the chemical marker to distinguish between HC and an anticancer herb Hedyotis diffusa (HD) in our previous study.The present study aimed to investigate whether HA exhibited antiproliferative activities in multidrug-resistant hepatocellular carcinoma cells R-HepG2 and the parental cells HepG2 using MTT assay and [3H]-thymidine incorporation assay.Our results showed that HA could significantly inhibit cell proliferation in R-HepG2 and HepG2 (IC50 = 43.7 and 56.3 µg/mL, respectively), but not in normal human liver cells WRL-68 (IC50 > 100 µg/mL) cells, suggesting its selective cytotoxic effects. Besides, HA induced apoptosis in R-HepG2 cells, as confirmed by annexin-V & propidium iodide staining, and DNA fragmentation assay. The caspase cascade was activated as shown by a significant increase of cleaved caspases-3, -7 and -9 in HA-treated R-HepG2 cells. The activities and protein expression of P-glycoprotein as well as mRNA expression of MDR1 were also decreased in HA-treated R-HepG2 cells.Our study demonstrated for the first time the antiproliferative activities of hedyotiscone A in multidrug-resistant R-HepG2 cells. The findings revealed the potential of this compound in treating multidrug-resistant tumor.

Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes

Arsenic trioxide (arsenite, As III) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As III on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As III on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As III-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As III were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As III than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As III in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As III-mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As III cytotoxicity between these cells.

Aryl hydrocarbon receptor‐mediated up‐regulation of ATP‐driven xenobiotic efflux transporters at the blood‐brain barrier

Many widespread and persistent organic pollutants, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), activate the aryl hydrocarbon receptor (AhR), causing it to translocate to the cell nucleus, where it transactivates target genes. AhR's ability to target the blood-brain barrier is essentially unexplored. We show here that exposing isolated rat brain capillaries to 0.05-0.5 nM TCDD roughly doubled transport activity and protein expression of P-glycoprotein, an ATP-driven drug efflux pump and a critical determinant of drug entry into the CNS. These effects were abolished by actinomycin D or cycloheximide or by the AhR antagonists resveratrol and α-naphthoflavone. Brain capillaries from TCDD-dosed rats (1-5 μg/kg, i.p.) exhibited increased transport activity and protein expression of 3 xenobiotic efflux pumps, P-glycoprotein, multidrug resistance-associated protein 2, and breast cancer resistance polypeptide, as well as expression of Cyp1a1 and Cyp1b1, both AhR target genes. Consistent with increased P-glycoprotein expression in capillaries from TCDD-dosed rats, in situ brain perfusion indicated significantly reduced brain accumulation of verapamil, a P-glycoprotein substrate. These findings suggest a new paradigm for the field of environmental toxicology: toxicants acting through AhR to target xenobiotic efflux transporters at the blood-brain barrier and thus reduce brain accumulation of CNS-acting therapeutic drugs.

Reversal Effect of A Novel N-sugar Substituted Thalidomide Analogue on Multidrug Resistant Human Nasopharyngeal Carcinoma Cells*

One major problem to successful treatment of cancer is the development of resistance by tumor cells to multiple chemotherapeutic drugs, a phenomenon named multidrug resistance (MDR). Searching for the novel chemotherapeutical agents is one of the important strategies for overcoming MDR. By using a cytotoxicity assay, flow cytometry analysis, Western-blotting and RT-PCR, a drug (Taxol, TAX) resistant human nasopharyngeal carcinoma KB cell line (KB/TAX) was established by addition of the drug to the cell cultures gradually, then a novel N-sugar substituted thalidomide analogue (STA-35) was investigated for its reversal effect on MDR of KB/TAX cells and possible mechanism. The results showed that KB/TAX cells were resistant to several chemotherapeutical agents, and the relative resistance to TAX was 73.1. Compared with parental KB cells, the function and protein expression of P-glycoprotein (P-gp), as well as mdr1 gene in the KB/TAX cells were remarkable reduced. Moreover, both KB and KB/TAX cells were sensitive to STA-35, the relative resistance to TAX on KB/TAX cells was decreased by the addition of STA-35. Furthermore, STA-35 (5 ～20 μmol/L)was capable to reduced the activity of P-gp by increasing the accumulation of rhodamine 123, decreasing P-gp expression in KB/TAX cells in a dose dependent manner , but had no effect on the mdr1 gene expression. These results suggest a potential action of STA-35 as MDR reversing agent, and one of the possible mechanisms could be the suppression of P-gp function and protein expression.

Abcb1a and Abcb1b expression in senescence-accelerated mouse (SAM)

It was recently reported that some strains of senescence-accelerated mouse (SAM) including SAMR1 had a spontaneous retroviral insertional mutation in the ATP-binding cassette, sub-family B, member 1A ( Abcb1a) gene, while other strains including SAMP8 had not. The Abcb1 gene product, P-glycoprotein, is a representative efflux transporter of cerebral vessels. In this study, using brain samples of SAMR1, Abcb1a gene-mutant mice, and of SAMP8 without that mutation, we examined the gene expression of some representative ATP-binding cassettes, such as Abcb1a, Abcb1b, Abcc, and Abcg2, and the protein expression of P-glycoprotein by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical techniques. The gene expression of Abcb1a was decreased in the brain samples of SAMR1 compared with those of SAMP8, while that of Abcb1b was increased in the samples of SAMR1 compared with those of SAMP8. There were no differences in the gene expression of Abcc and Abcg2 between the samples of SAMR1 and SAMP8. The protein expression of P-glycoprotein was decreased in the brain samples of SAMR1 compared with those of SAMP8. Immunosignals of P-glycoprotein were seen in vessels walls, mainly CD34-positive endothelial cells and partially astrocytic cells, in both mice. These findings indicate that SAMR1, Abcb1a-mutant mice, showed decreased expression of Abcb1a gene and P-glycoprotein and increased gene expression of Abcb1b, compared with those of SAMP8 without that mutation, suggesting no clear effect of increased gene expression of Abcb1b on decreased expression of P-glycoprotein. The combination of SAMR1 and SAMP8 may be a good tool to investigate which transporter, Abcb1a or Abcb1b, can be used in drug delivery into the brain.

Correlation between chemosensitivity testing and expression of P-gp,LRP in esophageal carcinoma cell cultured in vitro

Objective To study the correlation between chemotherapy drug sensitivity and the expression of P-glycoprotein protein(P-gp),lung resistant-related protein(LRP)in different esophageal carcinoma patients,which will offer experimental evidence for clinical individualized chemotherapy of esophageal carcinoma patients.Methods Selected fresh esophageal carcinoma's tissue,Common used drugs and chemoscheme for esophageal carcinoma were used for chemosensitivity testing of the primary cultured tumor cells.At the same time,immunohistochemistry method was used to detect the expression of P-gp and LRP of esophageal carcinoma cells.Results The chemotherapy drug sensitivity of single drug was different(P＜0.05).The combination of Tax and DDP had the best effect.DDP and NVB combination had a better effect than DDP and 5-FU combination.The sensitivity of DDP had no significant correlations with expression intensity of P-gp(P＞0.05),and it had significant correlation with expression intensity of LRP(P＜0.05).There were significant correlations between sensitivity of both NVB group and Tax group and expression intensity of P-gp and LRP(P＜0.05).There was no significant correlations between sensitivity of 5-FU and expression intensity of P-gp and LRP(P＞0.05).Conclusion The esophageal carcinoma chemosensitivity in vitro can indicate the sensitive drug of different patients,and guide individualized chemotherapy.The expression of P-gp and LRP has relationship with several chemotherapy drugs,which can provide base for individualized chemotherapy in clinic. Key words: Esophageal neoplasms; Tumor cells,cultured; Antineoplastic agent; P-glyeoprotein; Lung resistant-related protein

Animal Models of Acute Moderate Hypoxia Are Associated with a Down-Regulation of CYP1A1, 1A2, 2B4, 2C5, and 2C16 and Up-Regulation of CYP3A6 and P-glycoprotein in Liver

In humans, indirect evidence suggests that hypoxia reduces the rate of biotransformation of drugs cleared by cytochrome P450 (P450) subfamilies CYP1A, 2B, and 2C. The aim of this study was to assess whether acute moderate hypoxia modulates the expression of CYP2B4, 2C5, and 2C16 in vivo, and to determine whether the changes in hepatic P450 are conveyed by serum mediators. Moreover, because hypoxia increases the expression of P-glycoprotein in vitro, we examined whether in vivo acute moderate hypoxia modulates the expression of several membrane transporters in the liver. Rabbits and rats were exposed to a fractional concentration of oxygen of 8% for 48 h to generate a stable arterial partial pressure of O2 of 34 +/- 1 mm Hg. Compared with rabbits breathing room air, hypoxia in rabbits reduced the amount of CYP1A1, 1A2, 2B4, 2C5, and 2C16 proteins and increased the expression of CYP3A6. Sera of rabbits with hypoxia were fractionated by size exclusion chromatography, the fractions were tested for their ability to modify the expression of P450 isoforms, and serum mediators were identified through neutralization experiments. The serum mediators responsible for the down-regulation of P450 isoforms were interferon-gamma, interleukin-1beta (IL-1beta), and IL-2. In vivo, in rats, hypoxia increased the mRNA and protein expression of P-glycoprotein but did not affect the mRNA of breast cancer resistance protein and organic anion-transporting polypeptide 2. It is concluded that in vivo, hypoxia down-regulates rabbit hepatic CYP1A1, 1A2, 2B4, 2C5, and 2C16 and up-regulates CYP3A6. CYP3A11 and P-glycoprotein were up-regulated in the livers of hypoxic rats.

Gene and Protein Expression of P-Glycoprotein, MRP1, MRP2, and CYP3A4 in the Small and Large Human Intestine

The cytochrome P450 3A4 enzyme and the ABC-transporters may affect the first-pass extraction and bioavailability of drugs and metabolites. Conflicting reports can be found in the literature on the expression levels of efflux transporters in human intestine and how they vary along the intestine. The relative levels of mRNA and protein of CYP3A4 and the ABC tranporters Pgp (ABCB1), MRP1 (ABCC1), and MRP2 (ABCC2) were determined using RT-PCR and Western blot for human intestinal tissues (n = 14) from jejunum, ileum and colon. The expression of mRNA for CYP3A4, Pgp, and MRP2 was highest in jejunum and decreased toward more distal regions, whereas MRP1 was equally distributed in all intestinal regions. For CYP3A4, a more significant correlation could be established between mRNA and protein expression than for the ABC transporters. The samples showed considerable interindividual variability, especially at the protein level. The apically located Pgp and MRP2 showed a similar expression pattern along the human intestine as for CYP3A4. The gene expression of MRP1 exhibited a more uniform distribution.

Expression and functional characteristics of tubular transporters: P-glycoprotein, PEPT1, and PEPT2 in renal mass reduction and diabetes

Renal mass reduction is associated with a compromise in renal excretion, and thus dosages of drugs need to be adjusted to avoid adverse reactions and to ensure their effectiveness. A prototypic example is patients who had undergone transplantation due to a variety of causes, including diabetic nephropathy; the latter appears to be the major cause of renal failure requiring hemodialysis and transplantation. Conceivably, hyperglycemia with reduced renal mass interferes in the delivery of xenobiotics handled by various tubular transporters. In this investigation, effect of renal mass reduction/hyperglycemia on gene and protein expression of P-glycoprotein (Pgp), PEPT1, and PEPT2 was assessed. Also, [H(3)]glycylsarcosine uptake, a prototype of dipeptide, was measured in various groups of rats: sham-operated, uninephrectomized, streptozotocin-induced diabetes, and diabetic + uninephrectomized. An increase in Pgp, PEPT1, and PEPT2 expression was observed in kidneys of uninephrectomy rats, the highest being in the Pgp. Similarly, an increase was observed in diabetic rats who had undergone uninephrectomy, although less than those with nephrectomy alone. No differences were observed between sham-operated and diabetic groups. Increased uptake of [H(3)]glycylsarcosine was also seen in uninephrectomised rats. A modest uptake was observed in diabetic rats who had undergone uninephrectomy. The data suggest that uninephrectomy induces an increase in the expression and activity of transporters localized to renal tubular epithelial brush border. The fact that upregulation and activity of the peptide transporters were less in kidneys of diabetic animals who had undergone uninephrectomy compared with uninephrectomy alone suggests that hyperglycemia interferes in their expression and activity during the compensatory phase.

Relationship between therapeutic efficacy of arterial infusion chemotherapy and expression of P-glycoprotein and p53 protein in advanced hepatocellular carcinoma

To investigate the relationship between the chemotherapeutic drug efficacy and the expression of P-glycoprotein (PGP) and p53 protein in advanced hepatocellular carcinoma (HCC). The study was conducted on 41 patients with advanced HCC who were treated by repeated arterial infusion chemotherapy. Biopsy specimens from the tumor were collected before the start of treatment in all the patients, and the specimens were stored frozen until immunohistochemical staining, which was performed after the start of treatment, to detect PGP and p53 protein expressions. Twenty of the forty-one patients were treated with an anthracycline drug (epirubicin hydrochloride; anthracycline group), and the remaining 21 were treated with a non-anthracycline drug (mitoxantrone hydrochloride in 11 patients and carboplatin in 10 patients; non-anthracycline group). The relationship between the chemotherapeutic efficacy and the results of immunostaining were compared between the two groups. Before the start of the treatment, PGP-positive rate was 90.2% (strongly-positive, 36.6%) and p53 protein-positive rate was 34.1% (strongly-positive, 19.5%). In the anthracycline group, the response rate was 40.0%. The number of patients showing poor response to the treatment was significantly larger in the patients with strongly positive PGP expression (P=0.005), and their prognoses were poor (P=0.001). In the non-anthracycline group, the response rate was 42.9%, and there was no significant relationship between the chemotherapeutic drug efficacy and the PGP or p53 protein expression. When only the data from the 11 patients treated with anthraquinone drug, mitoxantrone, were analyzed, however, the number of patients who showed poor response to treatment was significantly higher among the p53-positive patients (P=0.012), irrespective of the survival outcome. The chemotherapeutic efficacy with an anthracycline drug for advanced HCC can be predicted by immunohistochemical analysis of PGP expression. Similarly, immunostaining to evaluate p53 protein may be useful to predict the response in patients treated with an anthraquinone drug.

IMPACT OF ENDOTOXIN-INDUCED CHANGES IN P-GLYCOPROTEIN EXPRESSION ON DISPOSITION OF DOXORUBICIN IN MICE

P-glycoprotein (PGP) encoded by the Mdr1 gene mediates the excretion of drugs in organs such as the liver and kidney. Inflammation has been shown to suppress the expression and activity of PGP in rodent liver, thus potentially altering the pharmacokinetics of drugs that are substrates of PGP. Here we examined the effect of endotoxin (lipopolysaccharide; LPS)-induced inflammation on the disposition of the PGP substrate doxorubicin (DOX) in the mouse. Male CD-1 mice received 5 mg/kg LPS intraperitoneally. DOX (5 mg/kg) was administered intravenously 24 h after LPS treatment. The time course of DOX levels in plasma, urine, bile, and tissues was analyzed by high performance liquid chromatography. PGP protein and mRNA expression in liver and kidney was measured using Western blots and reverse transcriptase polymerase chain reaction. As compared to controls, LPS-treated mice exhibited a significant decrease (50%) in biliary clearance and 3-fold increased renal clearance of DOX. These changes were associated with strongly reduced PGP protein levels (30% controls, p < 0.05) in the liver and increased PGP levels in the kidney (140% controls, p < 0.05). Hepatic mRNA levels of all Mdr isoforms were reduced in LPS-treated mice, whereas renal Mdr1b levels were increased. In LPS-treated mice, we also measured an increased area under the plasma concentration-time curve and reduced systemic clearance of DOX, as well as a 2- to 5-fold increase in the urinary excretion of the doxorubicin and doxorubicinol aglycones. Our data suggest that endotoxin-induced inflammation in mice causes differential regulation of PGP in liver and kidney, thereby altering the clearance profile of DOX.

Rapid induction of P-glycoprotein expression by high permeability compounds in colonic cells in vitro: a possible source of transporter mediated drug interactions?

P-glycoprotein (PGP) substrates with high membrane permeability, such as propranolol and verapamil, are considered to be essentially “transparent” to PGP since the transporter does not significantly limit their absorption or elimination. However, the question of whether such compounds can modulate PGP expression in epithelial cells following short-term exposure, with potential consequences for drug interactions, has not been addressed. LS180 colonic epithelial cells were exposed to propranolol or verapamil at concentrations (50–300 μM) consistent with those likely to be present in the gut lumen during oral dosing. Both compounds stimulated four to six-fold increases in MDR1 mRNA and PGP protein expression measured by quantitative real-time PCR and immunoblotting, respectively. These changes were accompanied by an induction in transporter activity measured by rhodamine 123 efflux. In contrast, metoprolol, a compound with similar permeability but no affinity for PGP had no effect on PGP expression. The induction of PGP by propranolol and verapamil was rapid with significant increases occurring within 3 h with maximal stimulation after 6 h exposure. Rifampicin, shown to cause clinical drug interactions via a PXR-mediated increase in PGP expression, exhibited a very similar time-course and extent of induction. In conclusion, verapamil and propranolol, whose trans-epithelial permeability are unaffected by PGP, appear to be effective inducers of PGP expression in gut epithelial cells in vitro. While the in vivo significance of these observations is unknown, this questions whether high permeability, “PGP-transparent” compounds, currently favoured in drug selection strategies, should be evaluated in terms of their potential for transporter-mediated drug interactions.

Consequences of bile duct obstruction on intestinal expression and function of multidrug resistance-associated protein 2

Background & aims: Multidrug resistance-associated protein 2 (MRP2), a transporter of organic anions in hepatocytes, renal epithelial cells, and enterocytes, is differentially regulated in liver and kidney during cholestasis, but little is known about its regulation in the intestine. Methods: We investigated duodenal protein expression of MRP2 in male Sprague-Dawley rats with bile duct ligation (BDL) or biliary diversion as well as in 20 cholestatic patients with biliary obstruction. Results: In biliary obstruction, but not biliary depletion, intestinal Mrp2 protein mass was reduced to 9.3% ± 5.5% of controls and mRNA to 40.5% ± 20.8% of controls after 7 days. Binding of RXRα:RARα heterodimers to the Mrp2 promoter element was significantly reduced in BDL rats. Cytokine blockade identified IL-1β as the responsible inducer of Mrp2 down-regulation. In humans with obstructive cholestasis, intestinal MRP2 protein expression was reduced to 27.3% ± 20.3% of control patients; this reduction correlated with the duration of cholestasis and was reversible after reconstitution of bile flow by stenting of the common bile duct. However, no significant differences in MRP2 mRNA levels were detected by RT-PCR in humans. Intestinal protein expression of P-glycoprotein, breast cancer resistance protein (BCRP), and MRP3 was unchanged. In BDL rats, oral bioavailability of the Mrp2 substrate and food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was elevated 2.5 times compared with sham-operated rats. Conclusions: Cholestasis promotes down-regulation of MRP2 expression in the duodenum of rats and humans. Selective down-regulation during cholestasis might be the consequence of species-specific transcriptional and posttranscriptional mechanisms and contributes to higher bioavailability of a food-derived carcinogen.