Induction of P-glycoprotein expression and activity by prolactin in female rat liver.

AIM: P-glycoprotein (P-gp) plays a critical role in the excretion of xenobiotics into bile. Previous studies have demonstrated that prolactin (PRL) regulates biotransformation and bile salt transport. Here we investigate whether the capability of the liver to transport xenobiotics into bile is altered in hyperprolactinemic states studying the modulation of hepatic P-gp by PRL. METHODS: We used lactating post-partum rats (PP), as a model of physiological hyperprolactinemia (15 and 21 days after delivery: PP15 and PP21, respectively), and ovariectomized rats treated with PRL (300 µg/day, 7 days, via osmotic minipumps, OVX + PRL). Hepatic P-gp expression and activity were evaluated by western blotting and using rhodamine 123 as substrate in vivo, respectively. Since P-gp is encoded by Mdr1a and Mdr1b in rodents, we quantified their expression by qPCR in primary hepatocyte cultures exposed to 0.1 µg/ml of PRL after 12 h. To further study the mechanism of hepatic P-gp modulation by PRL, hepatocytes were pretreated with actinomycin D and then exposed to PRL (0.1 µg/ml) for 12 h. KEY FINDINGS: We found increased hepatic P-gp protein expression and activity in PP15 and OVX + PRL. Also, a significant increase in Mdr1a and Mdr1b mRNA levels was observed in primary hepatocyte cultures exposed to PRL, pointing out the hormone direct action. Actinomycin D prevented these increases, confirming a transcriptional up-regulation of P-gp by PRL. SIGNIFICANCE: These findings suggest the possibility of an increased biliary excretion of xenobiotics substrates of P-gp, including therapeutic agents, affecting their pharmaco/toxicokinetics in hyperprolactinemic situations.

Downregulated expression of intestinal P-glycoprotein in rats with cisplatin-induced acute kidney injury causes amplification of its transport capacity to maintain "gatekeeper" function.

The expression of transporters on the apical and basal membranes of renal proximal tubular cells are down- or upregulated to various extents under cisplatin (CDDP)-induced acute kidney injury (AKI). However, little is known about the changes in transporters in tissues other than the kidney under CDDP-induced AKI. This study aimed to investigate the modulation of the expression/function of intestinal efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), in CDDP-induced AKI rats. On day 3 after the intraperitoneal administration of CDDP (5 mg/kg) to rats, the expression levels of P-gp and Bcrp were compared with those of normal rats. Further, the absorption of three P-gp substrates (6α-methylprednisolone, rhodamine 123, and gatifloxacin) was evaluated in both groups using conventional loop techniques. In the CDDP-induced AKI rats, P-gp expression in the ileum was markedly decreased to approximately 38% of that in the normal rats. However, no significant changes in Bcrp expression were observed in the AKI rats. In contrast with the reduction in P-gp expression in the AKI rats, the absorption of the three P-gp substrates remained almost the same or decreased in the AKI group. The addition of verapamil (a potent P-gp inhibitor) increased the absorption of the three P-gp substrates to the values obtained from the normal rats. In conclusion, our results suggested that P-gp expression is downregulated in rats with CDDP-induced AKI but that P-gp maintains its potency as a "gatekeeper" against the absorption of xenobiotics by amplifying its individual transport capacity under these conditions.

CAR directs T cell adaptation to bile acids in the small intestine.

Bile acids are lipid-emulsifying metabolites synthesized in hepatocytes and maintained in vivo through enterohepatic circulation between the liver and small intestine1. As detergents, bile acids can cause toxicity and inflammation in enterohepatic tissues2. Nuclear receptors maintain bile acid homeostasis in hepatocytes and enterocytes3, but it is unclear how mucosal immune cells tolerate high concentrations of bile acids in the small intestine lamina propria (siLP). CD4+ T effector (Teff) cells upregulate expression of the xenobiotic transporter MDR1 (encoded by Abcb1a) in the siLP to prevent bile acid toxicity and suppress Crohn's disease-like small bowel inflammation4. Here we identify the nuclear xenobiotic receptor CAR (encoded by Nr1i3) as a regulator of MDR1 expression in T cells that can safeguard against bile acid toxicity and inflammation in the mouse small intestine. Activation of CAR induced large-scale transcriptional reprogramming in Teff cells that infiltrated the siLP, but not the colon. CAR induced the expression of not only detoxifying enzymes and transporters in siLP Teff cells, as in hepatocytes, but also the key anti-inflammatory cytokine IL-10. Accordingly, CAR deficiency in T cells exacerbated bile acid-driven ileitis in T cell-reconstituted Rag1-/- or Rag2-/- mice, whereas pharmacological activation of CAR suppressed it. These data suggest that CAR acts locally in T cells that infiltrate the small intestine to detoxify bile acids and resolve inflammation. Activation of this program offers an unexpected strategy to treat small bowel Crohn's disease and defines lymphocyte sub-specialization in the small intestine.

Bile duct ligation causes opposite impacts on the expression and function of BCRP and P-gp in rat brain partly via affecting membrane expression of ezrin/radixin/moesin proteins.

Breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) are co-located at blood-brain barrier (BBB) cells, preventing their substrates from entering brain. Accumulating evidence demonstrates that liver failure impairs P-gp and BCRP expression and function in the brain. In the current study, we investigated how liver failure influenced the expression and function of brain BCRP and P-gp in rats subjected to bile duct ligation (BDL). The function of BCRP, P-gp and BBB integrity was assessed using distribution of prazosin, rhodamine 123 and fluorescein, respectively. We showed that BDL significantly decreased BCRP function, but increased P-gp function without affecting BBB integrity. Furthermore, we found that BDL significantly downregulated the expression of membrane BCRP and upregulated the expression of membrane P-gp protein in the cortex and hippocampus. In human cerebral microvascular endothelial cells, NH4Cl plus unconjugated bilirubin significantly decreased BCRP function and expression of membrane BCRP protein, but upregulated P-gp function and expression of membrane P-gp protein. The decreased expression of membrane BCRP protein was linked to the decreased expression of membrane radixin protein, while the increased expression of membrane P-gp protein was related to the increased location of membrane ezrin protein. Silencing ezrin impaired membrane location of P-gp, whereas silencing radixin impaired membrane location of BCRP protein. BDL rats showed the increased expression of membrane ezrin protein and decreased expression of membrane radixin protein in the brain. We conclude that BDL causes opposite effects on the expression and function of brain BCRP and P-gp, attributing to the altered expression of membrane radixin and ezrin protein, respectively, due to hyperbilirubinemia and hyperammonemia.

Type 2 diabetes mellitus decreases systemic exposure of clopidogrel active metabolite through upregulation of P-glycoprotein in rats.

Patients with diabetic mellitus tend to have a poor response to clopidogrel (Clop) due to reduced generation of active metabolite (Clop-AM). However, the underlying mechanism is not elucidated. A type 2 diabetic mellitus (T2DM) rat model was established by combining high-fat diet feeding and low-dose streptozotocin (STZ) injection. The reduced Clop-AM exposure was observed in T2DM rats after oral administration of Clop. However, in vitro liver microsomes incubated with Clop exhibited increased Clop-AM levels in T2DM rats due to a significant decrease in carboxylesterase (CES)1 expression and activity and a significant increase in the expression or activity of CYP1A2 and CYP3A. Interestingly, different from oral administration, the significantly increased Cmax of Clop-AM was observed in T2DM rats after intravenous injection, with no difference in AUC0-t and t1/2 values between the two strains. Meanwhile, in situ single -pass intestinal perfusion study showed lower absorption rate constant (Ka) and effective apparent permeability values (Peff) of Clop in T2DM rats than in control rats. It is explained by the increased expression or function of P-glycoprotein (P-gp) and pregnane X receptor (PXR) in duodenum and jejunum of T2DM rats. Moreover, the decreased Clop-AM level in T2DM rats was eliminated by the pretreatment of cyclosporin A, a P-gp inhibitor. It suggests that intestinal absorption, not hepatic metabolism is responsible for the reduced Clop-AM exposure in T2DM rats. P-gp might be the key factor causing the reduction of Clop absorption, consequently making less Clop available for Clop-AM formation.

Suppression of P-glycoprotein by cigarette smoke extract in human lung-derived A549/P-gp cells.

Effect of long-term treatment with cigarette smoke extract (CSE) on the function and expression of P-glycoprotein (P-gp) in lung alveolar epithelial cells was examined using A549/P-gp cell line expressing P-gp. CSE treatment suppressed P-gp activity in a concentration- and treatment time-dependent manner. The suppression of P-gp activity by CSE was irreversible for at least 96 h after removal of CSE. In addition, CSE treatment suppressed the expression of P-gp mRNA and protein. In order to understand the mechanisms underlying P-gp suppression by CSE, the role of reactive oxygen species (ROS) was examined. CSE treatment increased intracellular ROS level, and suppressed catalase activity. α-Tocopherol suppressed ROS production by CSE, and ameliorated the suppression of P-gp activity by CSE, suggesting that ROS is involved in CSE-induced suppression of P-gp. The role of intracellular signaling pathways such as the nuclear factor κB and mitogen-activated protein kinase pathways was also examined. Among these pathways, the involvement of extracellular signal-regulated kinase (ERK) pathway was suggested. Taken together, long-term CSE treatment may suppress P-gp via modulation of ROS level and ERK pathway in alveolar epithelial cells.

Irinotecan-induced gastrointestinal damage impairs the absorption of dabigatran etexilate.

Irinotecan causes serious gastrointestinal damage. Dabigatran etexilate (DABE), an oral anticoagulant and substrate of P-glycoprotein (P-gp), is poorly absorbed and exhibits low bioavailability in humans. The aim of this study was to evaluate the effects of irinotecan-induced gastrointestinal damage on the pharmacokinetics/pharmacodynamics (PK/PD) of DABE. Irinotecan was administered intravenously to rats for 4 days to induce gastrointestinal damage. To investigate the PK profile of dabigatran (DAB), an active moiety of DABE, DABE was administered orally on day 5, and then DAB was administered intravenously on day 6. To evaluate the PD profile of DAB, the activated partial thromboplastin time (APTT) was measured. The protein expression level of intestinal P-gp was evaluated. In the irinotecan-treated rats, the area under the concentration-time curve of DAB after the oral administration of DABE and the bioavailability of DABE were decreased significantly. The APTT ratio also decreased, suggesting that the impaired efficacy of DABE was attributable to a reduction in its bioavailability. The expression of intestinal P-gp was higher in the irinotecan-treated rats. Taking into consideration the histological damage caused to the intestinal epithelium, both the increased P-gp expression and the reduced passive diffusion were considered to be responsible for the reduction in the bioavailability of DABE.

MicroRNA-302c enhances the chemosensitivity of human glioma cells to temozolomide by suppressing P-gp expression.

Increasing evidence indicates that microRNAs (miRNAs) participate in the regulation of chemoresistance in a variety of cancers including glioma. However, the molecular mechanism underlying the development of chemoresistance in glioma is not well understood. The aim of the present study was to explore the role of miRNAs in the chemosensitivity of glioma cells and the underlying mechanism. By microarray and qRT-PCR, we observed significant down-regulation of microRNA-302c (miR-302c) in the temozolomide (TMZ)-resistant human glioma tissues/cells. The low expression of miR-302c was closely associated with poor prognosis and chemotherapy resistant in patients. miR-302c up-regulation re-sensitized U251MG-TMZ cells and LN229-TMZ cells to TMZ treatment, as evidenced by inhibition of the cell viability, cell migration, and invasion capacity, and promotion of the apoptosis after TMZ treatment. Furthermore, P-glycoprotein (P-gp) was identified as a functional target of miR-302c and this was validated using a luciferase reporter assay. In addition, P-gp was found to be highly expressed in U251MG-TMZ cells and there was an inverse correlation between P-gp and miR-302c expression levels in clinical glioma specimens. Most importantly, we further confirmed that overexpression of P-gp reversed the enhanced TMZ-sensitivity induced by miR-302c overexpression in U251MG-TMZ and LN229-TMZ cells. Our finding showed that up-regulation of miR-302c enhanced TMZ-sensitivity by targeting P-gp in TMZ-resistant human glioma cells, which suggests that miR-302c would be potential therapeutic targets for chemotherapy-resistant glioma patients.

Expression of multidrug transporter P-glycoprotein in Pichia pastoris affects the host's methanol metabolism.

Pichia pastoris KM71H (MutS ) is an efficient producer of hard-to-express proteins such as the membrane protein P-glycoprotein (Pgp), an ATP-powered efflux pump which is expressed properly, but at very low concentration, using the conventional induction strategy. Evaluation of different induction strategies indicated that it was possible to increase Pgp expression by inducing the culture with 20% media containing 2.5% methanol. By quantifying methanol, formaldehyde, hydrogen peroxide and formate, and by measuring alcohol oxidase, catalase, formaldehyde dehydrogenase, formate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenases, it was possible to correlate Pgp expression to the induction strategy. Inducing the culture by adding methanol with fresh media was associated with decreases in formaldehyde and hydrogen peroxide, and increases in formaldehyde dehydrogenase, formate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenases. At these conditions, Pgp expression was 1400-fold higher, an indication that Pgp expression is affected by increases in formaldehyde and hydrogen peroxide. It is possible that Pgp is responsible for this behaviour, since the increased metabolite concentrations and decreased enzymatic activities were not observed when parental Pichia was subjected to the same growth conditions. This report adds information on methanol metabolism during expression of Pgp from P. pastoris MutS strain and suggests an expression procedure for hard-to-express proteins from P. pastoris.

Brain region-specific regulation of histone acetylation and efflux transporters in mice.

Multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) protect the brain by restricting the passage of chemicals across the blood-brain barrier. Prior studies have demonstrated the epigenetic regulation of MDR1 and BCRP in cancer cells treated with histone deacetylase (HDAC) inhibitors that enhance histone acetylation and gene transcription. In the present study, we tested the in vivo effects of two HDAC inhibitors, valproic acid (VPA; 400 mg/kg) and apicidin (5 mg/kg), on Mdr1 and Bcrp transporter expression in brain regions of adult male mice injected intraperitoneally daily for 7 days. VPA increased Mdr1 protein expression in the striatum (70%) and Bcrp protein in the midbrain (30%). Apicidin enhanced striatal Mdr1 protein (30%) and hippocampal Bcrp protein (20%). Transporter induction correlated with increased histone H3 acetylation in discrete brain regions. In conclusion, HDAC inhibitors upregulate transporter proteins in vivo, which may be important in regulating regional xenobiotic disposition within the brain.

Enhanced expression of ten-eleven translocation 1 reverses gemcitabine resistance in cholangiocarcinoma accompanied by a reduction in P-glycoprotein expression.

Increasing evidence revealed that ten-eleven translocation 1 (TET1) plays an important role in tumorigenesis and chemoresistance, but its functions in gemcitabine resistance in cholangiocarcinoma (CCA) remain unknown. This study aims to investigate the effect of TET1 on gemcitabine resistance in CCA and the possible effect on P-glycoprotein (P-gp) expression encoded by multidrug resistance (MDR) genes. We established two kinds of gemcitabine-resistant CCA cell lines and confirmed its specific features. The expression of TET1 and P-gp was evaluated in gemcitabine-resistant CCA cells and their parental cells at mRNA and protein level by quantitative RT-PCR and western blot analysis. After transfecting the gemcitabine-resistant CCA cell lines with TET1 gene or siRNA, the cell viability test was obtained to verify the effect of TET1 on the sensitivity of CCA cells to gemcitabine. And then, the possible effect of TET1 on the expression of P-gp was examined by western blot analysis. Xenograft tumor experiment was conducted to confirm the association between TET1 and P-gp expression under gemcitabine chemoresistance. The associations between clinical outcomes of CCA patients with chemotherapy and TET1 expression were analyzed in 82 patients. The results showed that TET1 expression was significantly decreased, and P-gp expression was increased in gemcitabine-resistant CCA cells. Additionally, overexpression of TET1 augmented the sensitivity of CCA cells to gemcitabine and induced the decreased expression of P-gp in gemcitabine-resistant CCA cells. Furthermore, multivariate Cox regression analysis indicated that TET1 expression and TNM stage were independent risk factors (P < 0.001) for the clinical outcomes of CCA patients with chemotherapy. Additionally, Kaplan-Meier survival and the log-rank test showed that decreased expression of TET1 was associated with poorer prognosis of CCA patients with chemotherapy. These findings suggest that TET1 expression reverses gemcitabine resistance in CCA accompanied by a reduction in P-gp expression. Thus, TET1 may be a promising target to overcome chemoresistance in CCA.

The effect of endurance training and testosterone supplementation on the expression of blood spinal cord barrier proteins in rats.

The present study aimed to estimate the effect of endurance training, two doses of testosterone, and the combination of these stimuli on the level of the endothelial proteins claudin, occludin, JAM-1, VE-cadherin, ZO-1, ZO-2, and P-glycoprotein in rat spinal cords. Adult male Wistar rats were trained using a motor-driven treadmill for 6 weeks (40-60 min, 5 times per week) and/or were treated for 6 weeks with two doses of testosterone (i.m.; 8 mg/kg or 80 mg/kg body weight). Spinal cords were collected 48 hours after the last training cycle and stored at -80°C. The levels of selected proteins in whole tissue lysates of the spinal cord were measured by western blot. Testosterone-treated trained rats had significantly lower claudin levels than vehicle-treated trained rats. High doses of testosterone resulted in a significant decrease in claudin-5 in untrained rats compared to the control group. Both doses of testosterone significantly reduced occludin levels compared to those in vehicle-treated untrained rats. The JAM-1 level in the spinal cords of both trained and untrained animals receiving testosterone was decreased in a dose-dependent manner. The JAM-1 level in the trained group treated with high doses of testosterone was significantly higher than that in the untrained rats treated with 80 mg/kg of testosterone. VE-cadherin levels were decreased in all groups receiving testosterone regardless of endurance training and were also diminished in the vehicle-treated group compared to the control group. Testosterone treatment did not exert a significant effect on ZO-1 protein levels. Testosterone and/or training had no significant effects on ZO-2 protein levels in the rat spinal cords. Endurance training increased P-glycoprotein levels in the rat spinal cords. The results suggest that an excessive supply of testosterone may adversely impact the expression of endothelial proteins in the central nervous system, which, in turn, may affect the blood-brain barrier function.

Baicalin reduces ciclosporin bioavailability by inducing intestinal p-glycoprotein in rats.

OBJECTIVES: To investigate the effects of multiple doses of baicalin (BG) on the pharmacokinetics of ciclosporin (CsA) in rats and the potential mechanisms. METHODS: Pharmacokinetic parameters of CsA were determined in male rats after administration of CsA (3 mg/kg, i.g. or i.v.) to rats in the presence and absence of BG (80 mg/kg, i.g. or i.v.) for 7 days. The livers and intestines of rats were isolated and the CYP3A and p-glycoprotein (P-gp) expression were analysed. The effect of BG on the intestinal absorptive behaviour of CsA was also investigated using in-vitro everted rat gut sac model. KEY FINDINGS: Baicalin (80 mg/kg, i.v., 7 days) had no effect on the intravenously administered CsA. However, BG (80 mg/kg, i.g., 7 days) significantly decreased the Cmax , AUC0-t and AUC0-∞ of orally administered CsA by 38, 26 and 25%, respectively (P < 0.01 or P < 0.05). Further study revealed that the expression of P-gp in intestine increased in oral multiple doses of BG-treated rats. The in-vitro everted rat gut sac model demonstrated BG (10 µm) significantly decreased the absorption of CsA (10 µm) in intestine (P < 0.05). CONCLUSIONS: Multiple doses of BG decreased the oral bioavailability of CsA in rats significantly, which may be mainly attributable to inhibition of absorption of CsA in intestine and induction of P-gp. The interaction between BG and CsA may occur when BG and CsA were co-administered for long-term use. The dosage adjustment and blood concentration monitoring of CsA may be required in clinic.

The effect of micro-sized titanium dioxide on WM-266-4 metastatic melanoma cell line.

Titanium dioxide (TiO2) is widely used as an inorganic UV-filter in cosmetic products; however, it has been classified as possibly carcinogenic to humans. While numerous studies demonstrated cytotoxic and genotoxic effects of nano-sized TiO2 in different cell lines, including human skin cells, studies investigating the effects of micro-TiO2 on human keratinocytes and melanocytes, in healthy and cancer cells, are scarce. Adenosine triphosphate (ATP) binding cassette subfamily B member 5 (ABCB5) is a plasma membrane protein known for its role in the tumorigenicity, progression, and recurrence of melanoma. Here, we investigated the effect of micro-TiO2 (average particle size ≤5 µm) on the metabolic activity, cytotoxicity and ABCB5 mRNA expression in metastatic melanoma cells. Metastatic melanoma cell line WM-266-4 was treated with different concentrations of micro-TiO2 for different incubation times to obtain dose- and time-dependent responses. Untreated WM-266-4 cells, cultured under the same conditions, were used as control. The cell metabolic activity was determined by MTT assay. Cytotoxicity of micro-TiO2 was analyzed by lactate dehydrogenase (LDH) cytotoxicity assay. The ABCB5 mRNA expression in melanoma cells was analyzed using quantitative reverse transcription polymerase chain reaction (RT-qPCR). After 120 hours of exposure to micro-TiO2 the metabolic activity of melanoma cells decreased, especially at the two highest micro-TiO2 concentrations. Comparably, the cytotoxicity of micro-TiO2 on melanoma cells increased after 48 and 120 hours of exposure, in a time-dependent manner. The ABCB5 mRNA expression in micro-TiO2-treated melanoma cells also decreased significantly after 24 and 48 hours, in a time-dependent manner. Overall, our results suggest inhibitory effects of micro-TiO2 on the metabolic activity and ABCB5 mRNA expression in metastatic melanoma cells, indicating its potential use as an anticancer agent.

Bisphenol A (BPA) and bisphenol S (BPS) alter the promoter activity of the ABCB1 gene encoding P-glycoprotein in the human placenta in a haplotype-dependent manner.

Exposure to bisphenols (BPA and BPS) during pregnancy can significantly affect fetal development and increase risk of adverse health consequences, however the underlying mechanisms are not fully elucidated. In human placenta, the efflux transporter P-glycoprotein (P-gp), encoded by the ABCB1 gene, extrudes its substrates from the trophoblasts back into the maternal circulation. Alterations in levels of placental P-gp could therefore significantly affect fetal exposure to xenobiotics that are P-gp substrates. The ABCB1 promoter contains many single nucleotide polymorphisms (SNPs). In the genome, SNPs are not arrayed as independent variants but as combinations forming defined haplotypes. Recently, we determined the haplotype sequences encompassing the ABCB1 promoter SNPs and found that promoter haplotypes differentially affect ABCB1 promoter activity. Here we investigate the effect of BPA and BPS on ABCB1 promoter activity by testing the hypothesis that BPA and BPS exposure affect ABCB1 promoter activity in a haplotype-dependent manner. Our data indicate that acute exposure to 50 nM BPA induced a significant haplotype-dependent increase in ABCB1 promoter activity (P < .05). However, acute exposure to 0.5 nM BPS induced a significant decrease (P < .05) in promoter activity that was haplotype-dependent. Chronic exposure to BPA and BPS individually (5 nM and 0.3 nM, respectively) or as a mixture (5 nM BPA:1.5 nM BPS) induced significant haplotype-dependent increases (P < .01) in ABCB1 promoter activity. Our data indicate that BPA and BPS significantly alter ABCB1 promoter activity in a haplotype- and exposure type- dependent manners. Such alteration could significantly impact placental P-gp levels and alter fetal exposure to many therapeutic and environmental xenobiotics.

A new approach for Alzheimer's disease treatment through P-gp regulation via ibuprofen.

This study was aimed to investigate whether ibuprofen could alter the P-glycoprotein expression and function under Alzheimer's Disease condition and whether this alteration was induced by the inhibition of inflammatory reaction. APP/PS1 mice were used as AD model mice and ibuprofen-treated AD mice were given ibuprofen for 5 months. Then, Abcb1a/1b mRNA levels and P-gp expression were evaluated by qRT-PCR and western blot. Abcb1 mRNA levels were significantly reduced in AD mice compared to control mice, and it could be restored by ibuprofen treatment. Meanwhile, P-gp expression result showed a similar trend. Aß plaques in cerebral cortices and hippocampus were investigated via immunohistochemical, and the results revealed that Aß plaques were reduced in ibuprofen-treated AD mice compared with the AD mice, indicated that P-gp function may be recovered by ibuprofen treatment. qRT-PCR and ELISA were used to determined TNF-α, IL-1ß, IL-6 and NF-κB levels. The results demonstrated that TNF-α, IL-1ß mRNA levels and NF-κB expression were all significantly upregulated in AD mice in comparison with the control mice, and ibuprofen treatment could suppress the increase of inflammatory factors. In conclusion, the P-gp expression and function were suppressed in AD condition by activating inflammatory reaction, and then causing the Aß efflux decreased. However, upregulating P-gp could increase the Aß efflux in further to treat AD via inhibiting the inflammatory factors expression.

The effects of DMARDs on the expression and function of P-gp, MRPs, BCRP in the treatment of autoimmune diseases.

The ATP-binding cassette (ABC) transporter family is a large class of ATP energy-dependent transmembrane proteins, and its primary function is to use the energy produced by ATP hydrolysis to transfer the substrate bound to the plasma membrane. This family is also closely related to multidrug resistance (MDR) in various diseases. Among the ABC transporter proteins, P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and breast cancer resistance protein (BCRP) are the main members associated with MDR. At present, the roles of these transporters in therapeutic failures have been extensively studied and reviewed in cancer; however, they have rarely been described in autoimmune diseases (AIDs). AID is a group of chronic inflammatory diseases of unknown aetiology. AID's basic feature is the production of a large number of autoantibodies, which leads to extensive damage to multiple systems and multiple organs. Disease-modifying anti-rheumatic drugs (DMARDs) are commonly used in the treatment of AID, but a considerable number of patients have no response or develop resistance to these drugs over time. This phenomenon may be related to the abnormal expression of the ABC transporter, which leads to a decrease in the amount of drug entering cells that produce MDR. This article reviews the effects of DMARDs on the expression and function of P-gp, MRPs, and BCRP and the related molecular mechanism in the treatment of AID.

Differential expression of ABCB5 in BRAF inhibitor-resistant melanoma cell lines.

BACKGROUND: More than 50% of metastatic melanoma patients have a specific mutation in the serine/threonine kinase BRAF. This results in constitutive activation of the RAS-RAF-MEK-ERK-MAP kinase pathway, which causes uncontrolled cell growth. Vemurafenib (PLX4032) is an oral chemotherapeutic agent that targets the specific mutation V600E in the BRAF protein. Initial response rates are high in patients with BRAF mutant melanoma treated with a BRAF inhibitor such as vemurafenib, but resistance nearly always develops and disease progression ensues. There are several different mechanisms by which melanoma develops BRAF inhibitor resistance. One potential component of resistance is increased drug efflux. Overexpressed ABCB5 (ATP-binding cassette transporter, subfamily B, member 5) has been shown to efflux anti-cancer drugs from cancer cells. The purpose of this study is to determine whether ABCB5 is highly expressed in BRAF inhibitor-resistant melanoma cells and to evaluate whether ABCB5 is involved in the development of resistance to BRAF inhibitors in cutaneous melanoma. METHODS: We established three BRAF inhibitor-resistant melanoma cell lines with BRAF mutation. The expression level of ABCB5 in PLX-resistant cell lines was checked by real-time PCR and Western blot analysis. SK-MEL-2 melanoma cells with wild-type BRAF were used for comparison. The association of different levels of ABCB5 with the changes of ERK, p-ERK, Akt and p-Akt was also assessed by Western blotting. Re-sensitization of melanoma cells to PLX was tested by p-ERK inhibitor PD58059 and ABCB5 knockdown by ABCB5 siRNA, respectively. RESULTS: We showed that ABCB5 was overexpressed in SK-MEL-28PLXr and A2058PLXr cells but not in A375PLXr cells. ABCB5 overexpression is associated with activation of p-ERK status but not Akt. Inhibition of p-ERK re-sensitized SK-MEL-28PLXr and A2058PLXr cells to PLX treatment, but knockdown of ABCB5 did not re-sensitize A2058 PLXr and SK-MEL-28 PLXr cells to PLX treatment. CONCLUSION: These results confirm that, even though ABCB5 was overexpressed in SK-MEL-28 and A2058 melanoma cells that develop resistance to BRAF inhibitors, ABCB5 may not be a major targetable contributor to BRAF resistance. p-ERK inhibition may play important roles in BRAF resistance in these two melanoma cell lines.

Characterization of carfilzomib-resistant non-small cell lung cancer cell lines.

PURPOSE: We previously showed that carfilzomib (CFZ) has potent anti-proliferative and cytotoxic activity in a broad range of lung cancer cell lines. Here we investigate possible mechanisms of CFZ acquired resistance in lung cancer cell lines. METHODS: CFZ-resistant non-small cell lung cancer (NSCLC) cell lines were developed by exposing A549 and H520 cells to stepwise increasing concentrations of CFZ. Resistance to CFZ and cross-resistance to bortezomib and other chemotherapy drugs was measured using the MTT assay. Cytotoxicity to CFZ was determined using a CytoTox assay. Western blot was used to measure apoptosis, autophagy, and drug efflux transporter-related proteins. Quantitative targeted whole transcriptome sequencing and quantitative RT-PCR was used to measure gene expression. Flow cytometry was used to analyze intracellular accumulation of doxorubicin. RESULTS: The CFZ IC50 value of the resistant cells increased versus parental lines (2.5-fold for A549, 122-fold for H520). Resistant lines showed reduced expression of apoptosis and autophagy markers and reduced death versus parental lines following CFZ treatment. Both resistant lines exhibited higher P-glycoprotein (Pgp) gene (TempO-Seq® analysis, increased 1.2-fold in A549, > 9000-fold in H520) and protein expression levels versus parental lines. TempO-Seq® analysis indicated other drug resistance pathways were upregulated. The resistant cell lines demonstrated less accumulation of intracellular doxorubicin, and were cross-resistant to other Pgp client drugs: bortezomib, doxorubicin, and paclitaxel, but not cisplatin. CONCLUSIONS: Upregulation of Pgp appears to be an important, but not the only, mechanism of CFZ resistance in NSCLC cell lines.

Expression of MATE1, P-gp, OCTN1 and OCTN2, in epithelial and immune cells in the lung of COPD and healthy individuals.

BACKGROUND: Several inhaled drugs are dependent on organic cation transporters to cross cell membranes. To further evaluate their potential to impact on inhaled drug disposition, the localization of MATE1, P-gp, OCTN1 and OCTN2 were investigated in human lung. METHODS: Transporter proteins were analysed by immunohistochemistry in lung tissue from healthy subjects and COPD patients. Transporter mRNA was analysed by qPCR in lung tissue and in bronchoalveolar lavage (BAL) cells from smokers and non-smokers. RESULTS: We demonstrate for the first time MATE1 protein expression in the lung with localization to the apical side of bronchial and bronchiolar epithelial cells. Interestingly, MATE1 was strongly expressed in alveolar macrophages as demonstrated both in lung tissue and in BAL cells, and in inflammatory cells including CD3 positive T cells. P-gp, OCTN1 and OCTN2 were also expressed in the alveolar epithelial cells and in inflammatory cells including alveolar macrophages. In BAL cells from smokers, MATE1 and P-gp mRNA expression was significantly lower compared to cells from non-smokers whereas no difference was observed between COPD patients and healthy subjects. THP-1 cells were evaluated as a model for alveolar macrophages but did not reflect the transporter expression observed in BAL cells. CONCLUSIONS: We conclude that MATE1, P-gp, OCTN1 and OCTN2 are expressed in pulmonary lung epithelium, in alveolar macrophages and in other inflammatory cells. This is important to consider in the development of drugs treating pulmonary disease as the transporters may impact drug disposition in the lung and consequently affect pharmacological efficacy and toxicity.