Pgp mRNA Research Articles

Effect of air pre-exposure on Tetrabromobisphenol A resistance in the clam Ruditapes philippinarum

The present study was conducted to evaluate how air pre-exposure influences the responses in gills and digestive gland of Ruditapes philippinarum on subsequent Tetrabromobisphenol A (TBBPA) exposure. Firstly, clams were maintained in seawater or pre-exposed to air for 24 h, and then exposed to 0 or 100 μg/L TBBPA. Clam tissues were sampled after 1 day (T1) and 7 days (T7) of exposure to TBBPA. The results showed that in comparison with TBBPA exposure alone, air pre-exposure following TBBPA exposure reduced TBBPA accumulation in both tissues, up-regulated mRNA levels of CAT, Hsp70 and pi-GST in gills, and induced GST activity and P-glycoprotein (Pgp) mRNA level in digestive gland of clams at T1, whereas increased lipid peroxidation at T7. Overall, the findings indicate that 24-h exposure to air can activate a priming mechanism withstanding the subsequent TBBPA exposure, which is transient and may change after prolonged subsequent exposure.

Citrobacter rodentium infection inhibits colonic P-glycoprotein expression

P-glycoprotein (Pgp/MDR1) serves as a biological barrier that protects intestinal epithelial cells (IECs) by transporting out xenobiotics and bacterial toxins. Decreased Pgp function and expression has been seen in mouse models of inflammatory colitis and also in patients with IBD. Pgp knockout mice spontaneously develop severe colitis, which is also seen in human patients with ulcerative colitis. However, whether Pgp is also altered in infectious colitis is not known. Citrobacter rodentium (CR), a murine pathogen has been shown to cause colonic hyperplasia and colitis in mice by attaching to IECs. The current study investigated the direct effects of Citrobacter rodentium infection on intestinal Pgp expression in mice. Mice were challenged with a single dose of C. rodentium (1 × 109 CFU) by oral gavage for 9 days and Pgp expression in the ileum and colon was measured by real time qRT-PCR and immunofluorescence studies. Our results showed that C. rodentium infection significantly decreased Pgp mRNA and protein expression in the colon, although no significant change was observed in the ileum of mice. These findings suggest that inhibition of the efflux protein, Pgp by C. rodentium can cause perturbations in the intestinal epithelial integrity, which could further lead to the pathogenesis of intestinal inflammation as observed in infectious colitis.

Dual-functional Brij-S20-modified nanocrystal formulation enhances the intestinal transport and oral bioavailability of berberine.

IntroductionBerberine (BBR) is a plant-derived benzylisoquinoline alkaloid and has been demonstrated to be a potential treatment for various chronic diseases. The poor water solubility and P-glycoprotein (Pgp)-mediated drug efflux are the main challenges for its further application in a clinical setting.Materials and methodsIn this study, a Brij-S20 (BS20)-modified nanocrystal formulation (BBR-BS20-NCs) has been developed and investigated with the purpose of improving the intestinal absorption of BBR. The physicochemical properties of the developed BBR-BS20-NCs were characterized and the enhancement of the BBR-BS20-NCs on BBR absorption were investigated both in vitro and in vivo.ResultsThe results indicated that BS20 could significantly enhance the intracellular uptake of BBR in MDCK-MDR1 cells via a short-term and reversible modulation on the Pgp function, accompanied by a marked increase in Pgp mRNA expression but without significant influence on the Pgp protein expression. Moreover, the morphology of the prepared BBR-BS20-NCs was observed to be prism-like, with a smooth surface and an average diameter of 148.0 ± 3.2 nm. Compared to raw BBR and physical mixture, BBR-BS20-NCs facilitated the dissolution rate and extent of release of BBR in aqueous solution, and further increased the absorption of BBR in MDCK-MDR1 monolayer by overcoming the Pgp-mediated secretory transport (Papp[BL-AP] values of 2.85 ± 0.04 × 10−6 cm/s, 2.21 ± 0.14 × 10−6 cm/s, and 2.00 ± 0.07 × 10−6 cm/s for pure BBR, physical mixture, and BBR-BS20-NCs, respectively). Significant improvements in the maximum concentration observed (Cmax) and area under drug concentration-time curve (AUC0–t) of BBR-BS20-NCs were obtained in pharmacokinetic studies compared to pure BBR, and the relative bioavailability of BBR-BS20-NCs to pure BBR was 404.1%.ConclusionThe developed BBR-BS20-NCs combine the advantages of nanocrystal formulation and functional excipient. The novel pharmaceutical design provides a new strategy to improve the oral bioavailability of those drugs with both poor water solubility and Pgp-mediated efflux.

Oxidative Stress Promotes Doxorubicin-Induced Pgp and BCRP Expression in Colon Cancer Cells Under Hypoxic Conditions.

P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) are ATP binding cassette (ABC) transporters that are overexpressed in different drug-resistant cancer cell lines. In this study, we investigated whether doxorubicin promotes Pgp and/or BCRP expression to induce drug resistance in colon cancer cells under hypoxic conditions. We analyzed HIF-1α activity via ELISA, Pgp, and BCRP expression by qRT-PCR and the relationship between doxorubicin uptake and ABC transporter expression via confocal microscopy in HT-29WT and HT-29 doxorubicin-resistant colon cancer cells (HT-29DxR). These cells were treated with doxorubicin and/or CoCl2 (chemical hypoxia), and reactive oxygen species inductors. We found that the combination of chemically induced hypoxia and doxorubicin promoted Pgp mRNA expression within 24 h in HT-29WT and HT-29DxR cells. Both doxorubicin and CoCl2 alone or in combination induced Pgp and BCRP expression, as demonstrated via confocal microscopy in each of the above two cell lines. Thus, we surmised that Pgp and BCRP expression may result from synergistic effects exerted by the combination of doxorubicin-induced ROS production and HIF-1α activity under hypoxic conditions. However, HIF-1α activity disruption via the administration of E3330, an APE-1 inhibitor, downregulated Pgp expression and increased doxorubicin delivery to HT-29 cells, where it served as a substrate for Pgp, indicating the existence of an indirect relationship between Pgp expression and doxorubicin accumulation. Thus, we concluded that Pgp and BCRP expression can be regulated via cross-talk between doxorubicin and hypoxia, promoting drug resistance in HT-29 WT, and HT-29DxR cells and that this process may be ROS dependent. J. Cell. Biochem. 118: 1868-1878, 2017. © 2017 Wiley Periodicals, Inc.

Regulation of pregnane-X-receptor, CYP3A and P-glycoprotein genes in the PCB-resistant killifish (Fundulus heteroclitus) population from New Bedford Harbor

Killifish survive and reproduce in the New Bedford Harbor (NBH) in Massachusetts (MA), USA, a site severely contaminated with polychlorinated biphenyls (PCBs) for decades. Levels of 22 different PCB congeners were analyzed in liver from killifish collected in 2008. Concentrations of dioxin-like PCBs in liver of NBH killifish were ∼400 times higher, and the levels of non-dioxin-like PCBs ∼3000 times higher than in killifish from a reference site, Scorton Creek (SC), MA. The NBH killifish are known to be resistant to the toxicity of dioxin-like compounds and to have a reduced aryl hydrocarbon receptor (AhR) signaling response. Little is known about the responses of these fish to non-dioxin-like PCBs, which are at extraordinarily high levels in NBH fish. In mammals, some non-dioxin-like PCB congeners act through nuclear receptor 1I2, the pregnane-X-receptor (PXR). To explore this pathway in killifish, a PXR cDNA was sequenced and its molecular phylogenetic relationship to other vertebrate PXRs was determined. Killifish were also collected in 2009 from NBH and SC, and after four months in the laboratory they were injected with a single dose of either the dioxin-like PCB 126 (an AhR agonist) or the non-dioxin-like PCB 153 (a mammalian PXR agonist). Gills and liver were sampled three days after injection and transcript levels of genes encoding PXR, cytochrome P450 3A (CYP3A), P-glycoprotein (Pgp), AhR2 and cytochrome P450 1A (CYP1A) were measured by quantitative PCR. As expected, there was little effect of PCB exposure on mRNA expression of AhR2 or CYP1A in liver and gills of NBH fish. In NBH fish, but not in SC fish, there was increased mRNA expression of hepatic PXR, CYP3A and Pgp upon exposure to either of the two PCB congeners. However, basal PXR and Pgp mRNA levels in liver of NBH fish were significantly lower than in SC fish. A different pattern was seen in gills, where there were no differences in basal mRNA expression of these genes between the two populations. In SC fish, but not in NBH fish, there was increased mRNA expression of branchial PXR and CYP3A upon exposure to PCB126 and of CYP3A upon exposure to PCB153. The results suggest a difference between the two populations in non-AhR transcription factor signaling in liver and gills, and that this could involve killifish PXR. It also implies possible cross-regulatory interactions between that factor (presumably PXR) and AhR2 in liver of these fish.

Abstract 1828: A genomic-transcriptomic-phosphoproteomic study to interrogate lapatinib resistance in an HER2 over expressing SKBR3 human breast cancer cell line model

Abstract Background: Lapatinib is a tyrosine kinase inhibitor that inhibits EGFR and HER2 and is licensed to treat HER2-amplified breast cancer. Understanding mechanisms of acquired resistance is an unmet clinical need. Methods: SKBR3 cells were continuously exposed to increasing concentrations of lapatinib over a 12-month period. The parent cell line (SKBR3) and resulting lapatinib-resistant cell line (SKBR3LR) then underwent a set of studies including: a) western blot analysis for PGP, b) whole exome sequencing, c) whole genome expression microarray, d) quantification of 587 phosphoproteins. All data were integrated using Metacore software. Sensitivity of SKBR3 and SKBR3LR to a panel of drugs based on the findings was carried out by 96-hr sulforhodamine assays. Results: The GI50 values for lapatinib in SKBR3LR and SKBR3 cells were 3582 nM (SD 241.6) and 10.3 nM (SD 1.1). There was no induction of PGP protein or mRNA expression levels in SKBR3LR compared to SKBR3. Somatic mutations in KIT and MEK3 which were not present in SKBR3 were detected in SKBR3LR. There was a reduction in the levels of p-HER2 and p-EGFR in SKBR3LR; however, no differences in the levels of p-HER3, p-HER4, and p-KIT between the cell lines were observed. Analysis of proteins downstream in the RAS-RAF-MEK and PI3K-AKT-m-TOR showed increased levels of p-MEK but no significant changes in p38MAPK and no differences in p-AKT levels, but an increase in p-P70S6K in SKBR3LR compared to SKBR3. The phosphorylation network changes were integrated with transcription factors that showed significant enrichment for binding sites in the genes that were differentially expressed in SKBR3LR. Deregulation of the MAPK signalling network was one of the proposed mechanisms of resistance of SKBR3LR cells to lapatinib. Differential sensitivity to small-molecule agents was used to interrogate the mechanism of lapatinib resistance further. There was no significant differential sensitivity of SKBR3 and SKBR3LR to imatinib, GI50 values 16.2 uM (SD 1.2) and 13.64 uM (SD1.1), respectively. SKBR3LR was more resistant than SKBR3 to an AKT inhibitor AKT1/2i; GI50 values 8 uM (SD 0.45) vs 17.3 uM (SD 1.1) respectively. Interestingly, SKBR3LR was more sensitive to the HSP90 inhibitor, AUY922; GI50 values 3.2 nM (SD 1.1) and 11.2 nM (SD 0.42) in SKBR3LR and SKBR3 cells, respectively. Experiments are ongoing using targeted agents to understand the mechanism of lapatinib resistance and suggest potential therapeutic strategies to overcome these, including novel combinations, particularly the use of HSP90 inhibitors. Conclusions: Integrating genomic-transcriptomic and phosphoproteomic data provides useful insights into the mechanism of resistance to lapatinib. The lapatinib-resistant cell line model was sensitive to the HSP90 inhibitor, AUY922, suggesting HSP90 inhibitors could be clinically effective in this setting. Citation Format: Parames Thavasu, Robert Te Poele, Mark Hughes, Adam Stewart, Paul Workman, Udai Banerji. A genomic-transcriptomic-phosphoproteomic study to interrogate lapatinib resistance in an HER2 over expressing SKBR3 human breast cancer cell line model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1828. doi:10.1158/1538-7445.AM2014-1828

Effect of prostaglandin E2 on multidrug resistance transporters in human placental cells.

Prostaglandin (PG) E2, a major product of cyclooxygenase (COX)-2, acts as an immunomodulator at the maternal-fetal interface during pregnancy. It exerts biologic function through interaction with E-prostanoid (EP) receptors localized to the placenta. The activation of the COX-2/PGE2/EP signal pathway can alter the expression of the ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 [P-glycoprotein (Pgp); gene: ABCB1], and breast cancer resistance protein (BCRP; gene: ABCG2), which function to extrude drugs and xenobiotics from cells. In the placenta, PGE2-mediated changes in ABC transporter expression could impact fetal drug exposure. Furthermore, understanding the signaling cascades involved could lead to strategies for the control of Pgp and BCRP expression levels. We sought to determine the impact of PGE2 signaling mechanisms on Pgp and BCRP in human placental cells. The treatment of placental cells with PGE2 up-regulated BCRP expression and resulted in decreased cellular accumulation of the fluorescent substrate Hoechst 33342. Inhibiting the EP1 and EP3 receptors with specific antagonists attenuated the increase in BCRP. EP receptor signaling results in activation of transcription factors, which can affect BCRP expression. Although PGE2 decreased nuclear factor κ-light chain-enhancer of activated B activation and increased activator protein 1, chemical inhibition of these inflammatory transcription factors did not blunt BCRP up-regulation by PGE2. Though PGE2 decreased Pgp mRNA, Pgp expression and function were not significantly altered. Overall, these findings suggest a possible role for PGE2 in the up-regulation of placental BCRP expression via EP1 and EP3 receptor signaling cascades.

DNA adduct formation and induction of detoxification mechanisms in Dreissena polymorpha exposed to nitro-PAHs.

Derived polycyclic aromatic hydrocarbons (PAHs) such as nitro-PAHs are present in the environment and are known to be much more toxic than PAHs compounds. However, very few studies have analysed their effects on the aquatic environment and none have investigated the freshwater environment. In the present study, we determined whether 1-nitropyrene (1-NP), a model of nitro-PAHs, can induce DNA adducts in gills and digestive glands of the freshwater mussel Dreissena polymorpha. Two concentrations of 1-NP (50 and 500 μM) were tested. In addition, in order to understand the metabolic pathways involved in 1-NP genotoxicity, mRNA expression of genes implicated in biotransformation mechanisms was assessed by quantitative reverse transcription-PCR. Results showed the presence of DNA adducts in both gills and digestive glands, with highest levels obtained after 5 days of exposure to 500 μM. Metallothionein mRNA levels were enhanced in digestive glands exposed to 50 μM. Surprisingly, at the higher concentration (500 μM), aryl hydrocarbon receptor and HSP70 genes were only up-regulated in digestive glands while PgP mRNA levels were increased in both tissues. Results suggested a cytotoxic and genotoxic effect of 1-NP. Mussels seemed to be able to partially detoxify this compound, in view of the low amount of DNA adducts observed after 5 days exposure to 50 μM. For the first time, 1-NP biotransformation and detoxification systems have been characterised in D. polymorpha.

Carboxymethylcellulose-Based and Docetaxel-Loaded Nanoparticles Circumvent P-Glycoprotein-Mediated Multidrug Resistance

Taxanes are a class of anticancer agents with a broad spectrum and have been widely used to treat a variety of cancer. However, its long-term use has been hampered by accumulating toxicity and development of drug resistance. The most extensively reported mechanism of resistance is the overexpression of P-glycoprotein (Pgp). We have developed a PEGylated carboxymethylcellulose conjugate of docetaxel (Cellax), which condenses into ∼120 nm nanoparticles. Here we demonstrated that Cellax therapy did not upregulate Pgp expression in MDA-MB-231 and EMT-6 breast tumor cells, whereas a significant increase in Pgp expression was measured with native docetaxel (DTX) treatment. Treatment with DTX led to 4-7-fold higher Pgp mRNA expression and 2-fold higher Pgp protein expression compared with Cellax treatment in the in vitro and in vivo system, respectively. Cellax also exhibited significantly increased efficacy compared with that of DTX in a taxane-resistant breast tumor model. Against the highly Pgp expressing EMT6/AR1 cells, Cellax exhibited a 6.5 times lower IC50 compared with that of native DTX, and in the in vivo model, Cellax exhibited 90% tumor growth inhibition, while native DTX had no significant antitumor activity.

Expression of P-glycoprotein and CYP3A4 along the porcine oral-gastrointestinal tract: implications on oral mucosal drug delivery

Objectives: To characterize the expression of Pgp and CYP3A4 along the oral-gastrointestinal (GI) tract for understanding the potential roles of CY3A4 and Pgp in oral mucosal drug delivery.Design: Porcine buccal mucosa, sublingual mucosa, esophagus and jejunum, ileum and colon tissues were used for studying the mRNA and protein expression of CYP3A4 and Pgp. mRNA and protein were determined using real-time quantitative polymerase chain reaction (PCR) and western blot, respectively. The expression levels of CYP3A4 and Pgp in different segments of oral-GI tract were compared.Results: Levels of Pgp mRNA were significantly lower (14–40 times lower) in buccal and sublingual mucosa than that in intestine. In contrast, higher levels of CYP3A4 mRNA were observed in the oral mucosa as compared to that in intestine, but the difference was not statistically different. The levels of Pgp protein along the oral-GI tract followed the order: sublingual ∼buccal ∼esophagus < jejunum ∼ileum ∼ colon while the expression of CYP3A4 protein in the oral mucosa was similar to that in intestine.Conclusion: Expression of Pgp in oral mucosa is lower than that in intestine, while the expression of CYP3A4 in oral mucosa is similar to that in intestine. Because of lower Pgp in oral mucosa, oral mucosal drug delivery can be used as an alternative strategy to avoid the coordination of Pgp and CYP3A4 metabolism in drug absorption. However, CYP3A4-dependent metabolism may play a role in oral mucosal drug delivery as in per oral-GI absorption.

Molecular Characterization and Transcription Analysis of P-Glycoprotein Gene from the Salmon Louse Caligus rogercresseyi

Despite the efforts to manage the infestation Caligus rogercresseyi on salmon species, overuse of chemical antiparasites such as avermectins, organophosphates and pyrethroids have increasingly generated drug resistance, impacting on the efficacy of salmon lice control measures. So far, previous reports have evidenced that the ATPbinding cassette transporter P-glycoprotein (Pgp) is a candidate gene implicated in the response of salmon lice to neurotoxins. However, transcription patterns of Pgp in presence of pyrethroids and the expression pattern during the ontogenetic stages are still not complete elucidated. Herein, this study characterizes the Pgp mRNA from C. rogercresseyi (Cr-Pgp) and evaluates the transcription expression in during its lifecycle, and also in adults exposed to the antiparasitic drug deltamethrin (AlphaMaxi£¨). The molecular characterization of Cr-Pgp showed a complete sequence of 4,730 bp, containing a 5’UTR of 56 bp, 3’UTR of 833 bp, and open reading frame (ORF) of 3,840 bp encoding for 1,280 amino acids. Interestingly, eleven SNPs were identified, being two of them nonsynonymous polymorphisms. Cr-Pgp transcription expression was evaluated in conjunction with cytochrome P450 due its wellestablished role as key molecule in drug detoxifications. Herein, Cr-Pgp transcription was mainly associated to adult females than males exposed to deltamethrin, which was also linked with cytochrome P450 expression in adult females at 2 ppb of deltamethrin. This study suggests that Cr-Pgp gene is involved in pyrethroid detoxification and evidences specific expression patterns related to developmental stages, as well as provides novel SNPs that could be associated with resistance/susceptibility to delousing drugs

Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT

Background:Primary Ewing's sarcoma family of tumours (ESFTs) may respond to chemotherapy, although many patients experience subsequent disease recurrence and relapse. The survival of ESFT cells following chemotherapy has been attributed to the development of resistant disease, possibly through the expression of ABC transporter proteins.Methods:MRP-1 and Pgp mRNA and protein expression in primary ESFTs was determined by quantitative reverse-transcriptase PCR (RT-qPCR) and immunohistochemistry, respectively, and alternative splicing of MRP-1 by RT-PCR.Results:We observed MRP-1 protein expression in 92% (43 out of 47) of primary ESFTs, and cell membrane MRP-1 was highly predictive of both overall survival (P<0.0001) and event-free survival (P<0.0001). Alternative splicing of MRP-1 was detected in primary ESFTs, although the pattern of splicing variants was not predictive of patient outcome, with the exception of loss of exon 9 in six patients, which predicted relapse (P=0.041). Pgp protein was detected in 6% (38 out of 44) of primary ESFTs and was not associated with patient survival.Conclusion:For the first time we have established that cell membrane expression of MRP-1 or loss of exon 9 is predictive of outcome but not the number of splicing events or expression of Pgp, and both may be valuable factors for the stratification of patients for more intensive therapy.

Effects of β-cyclodextrin on the intestinal absorption of berberine hydrochloride, a P-glycoprotein substrate

The major objective of this work is to investigate the enhancing effect of β-cyclodextrin on the intestinal absorption of berberine hydrochloride, a P-glycoprotein (Pgp) substrate. The inclusion complexation behavior of BBH with β-CD was investigated by phase-solubility diagram, Fourier transform infrared spectroscopy, differential scanning calorimetry, X-ray powder diffractometry, NMR spectroscopy, and molecular modeling studies. Results indicated that the 1,3-benzodioxole of BBH was included into the cavity of β-CD to form an inclusion complex which exhibited higher dissolution rate than BBH in vitro. The intestinal absorption of the inclusion complex in rats was significantly higher than the free drug due to its solubilizing effect and Pgp modulatory activity. The mechanisms of β-CD on Pgp modulation were demonstrated by modifying the Pgp ATPase activity, the Pgp mRNA level and the Pgp expression.

Abstract 879: Multidrug resistant tumor cells-derived microparticles induce a multifactorial resistance phenotype through regulation of oncogenic-related pathways in sensitive cancer cells.

Abstract Background: Multidrug resistance (MDR) is associated with unsuccessful clinical cancer treatment. Different proteins are enhanced in MDR including the efflux pump P-glycoprotein (Pgp/MDR1) and inhibitor of apoptosis proteins (IAP). Newly, it has been reported that Pgp and microRNAs (miR) associated with it can be transferred through of membrane microparticles (MPs) from MDR cells to sensitive cells. Based on that, we analyzed intercellular transfer of Pgp, IAPs (c-IAP1, survivin and XIAP) and miRs related to Pgp and tumorigenesis, from MDR cells to sensitive cells through MPs. Besides that, we also analyzed NFκB and YB-1 oncogenic pathway in sensitive cells after co-culture with MDR cells-derived MPs - both transcriptional Pgp and/or IAPs regulators. Methods: MDR cells-derived MPs were isolated by ultracentrifugation, identified by flow citometry and analyzed for proteins (Pgp/MDR1 and IAPs) by Western blot, for mRNA (Pgp/MDR1 and IAPs) and for miRs (miR-27a, -451 and -21) by qRT-PCR. Sensitive cancer cells were co-culture with MDR cells for 24 separated by Transwell inserts, with isolated MPs, and culture with conditioned medium from MDR cells; and then analyzed for proteins (Pgp/MDR1, IAPs, IBα and YB-1), immunofluorescence (Pgp, NFκB and YB-1), mRNA (Pgp, IAPs and YB-1), miRs (miR-27a, -451 and -21), and drug resistance profile by annexin-V/PI staining. Results: It was observed that MDR cells-derived MPs carry miR-27a, -451 and -21; Pgp and IAPs mRNA and proteins except for XIAP mRNA. After co-culture, sensitive cells acquired Pgp mRNA and protein and showed clusters of Pgp suggesting the presence of MPs. It was also observed an enhancement of IAPs (mRNA and protein) except for cIAP1; and a reduction of drug-inducible apoptosis index. No changes were observed in Pgp or IAPs in cells cultured with conditioned medium, which show that co-culture inducing proteins enhancement is not related with shedding soluble molecules by MDR cells. In parallel, we observed equivalent Pgp and IAPs enhancement when sensitive cells were co-culture with MDR cells-derived MPs, which show that co-culture inducing Pgp and IAPs changes are direct related with MPs. Also, sensitive cells co-cultured with MDR cells showed NFκB translocated to the nucleus and peri or nuclear YB-1 localization suggesting theirs participation on Pgp and IAPs endogenous expression. In addition, we observed an enhancement on miR-21, - 27a and -451, which also suggest a positive endogenous regulation of Pgp and IAPs. Conclusion: Our data show that sensitive cells acquired multifactorial resistance through intercellular transfer of resistant cells-derived MPs, and suggest that oncogenic pathways could be involved with this phenomenon. These findings contribute to our knowledge for the emergence of MDR in cancer cells and could be helpful for new treatment approaches. Citation Format: Paloma Silva de Souza, André L. de Souza Cruz, Joao Paulo de Biaso Viola, Raquel Ciuvalschi Maia. Multidrug resistant tumor cells-derived microparticles induce a multifactorial resistance phenotype through regulation of oncogenic-related pathways in sensitive cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 879. doi:10.1158/1538-7445.AM2013-879

Keratinocyte growth factor-2 stimulates P-glycoprotein expression and function in intestinal epithelial cells

Intestinal P-glycoprotein (Pgp/multidrug resistance 1), encoded by the ATP-binding cassette B1 gene, is primarily involved in the transepithelial efflux of toxic metabolites and xenobiotics from the mucosa into the gut lumen. Reduced Pgp function and expression has been shown to be associated with intestinal inflammatory disorders. Keratinocyte growth factor-2 (KGF2) has emerged as a potential target for modulation of intestinal inflammation and maintenance of gut mucosal integrity. Whether KGF2 directly regulates Pgp in the human intestine is not known. Therefore, the present studies were undertaken to determine the modulation of Pgp by KGF2 using Caco-2 cells. Short-term treatment of Caco-2 cells with KGF2 (10 ng/ml, 1 h) increased Pgp activity (~2-fold, P < 0.05) as measured by verapamil-sensitive [(3)H]digoxin flux. This increase in Pgp function was associated with an increase in surface Pgp levels. The specific fibroblast growth factor receptor (FGFR) antagonist PD-161570 blocked the KGF2-mediated increase in Pgp activity. Inhibition of the mitogen-activated protein kinase (MAPK) pathway by PD-98059 attenuated the stimulatory effects of KGF2 on Pgp activity. Small-interfering RNA knockdown of Erk1/2 MAPK blocked the increase in surface Pgp levels by KGF2. Long-term treatment with KGF2 (10 ng/ml, 24 h) also significantly increased PgP activity, mRNA, protein expression, and promoter activity. The long-term effects of KGF2 on Pgp promoter activity were also blocked by the FGFR antagonist and mediated by the Erk1/2 MAPK pathway. In conclusion, our findings define the posttranslational and transcriptional mechanisms underlying stimulation of Pgp function and expression by KGF2 that may contribute to the beneficial effects of KGF2 in intestinal inflammatory disorders.

Dual regulation of P-glycoprotein expression by Trichostatin A in cancer cell lines

BackgroundIt has been reported that the histone deacetylase inhibitor (iHDAc) trichostatin A (TSA) induces an increase in MDR1 gene transcription (ABCB1). This result would compromise the use of iHDACs in combination with other cytotoxic agents that are substrates of P-glycoprotein (Pgp). It has also been reported the use of alternative promoters by the ABCB1 gene and the existence of a translational control of Pgp protein. Finally, the ABCB1 gene is located in a genetic locus with the nested gene RUNDC3B in the complementary DNA strand, raising the possibility that RUNDC3B expression could interfere with ABCB1 alternative promoter regulation.MethodsA combination of RT-PCR, real time RT-PCR, Western blot and drug accumulation assays by flow cytometry has been used in this study.ResultsThe iHDACs-induced increase in MDR1 mRNA levels is not followed by a subsequent increase in Pgp protein levels or activity in several pancreatic and colon carcinoma cell lines, suggesting a translational control of Pgp in these cell lines. In addition, the MDR1 mRNA produced in these cell lines is shorter in its 5′ end that the Pgp mRNA produced in cell lines expressing Pgp protein. The different size of the Pgp mRNA is due to the use of alternative promoters. We also demonstrate that these promoters are differentially regulated by TSA. The translational blockade of Pgp mRNA in the pancreatic carcinoma cell lines could be related to alterations in the 5′ end of the MDR1 mRNA in the Pgp protein expressing cell lines. In addition, we demonstrate that the ABCB1 nested gene RUNDC3B expression although upregulated by TSA is independent of the ABCB1 alternative promoter used.ConclusionsThe results show that the increase in MDR1 mRNA expression after iHDACs treatment is clinically irrelevant since this mRNA does not render an active Pgp protein, at least in colon and pancreatic cancer cell lines. Furthermore, we demonstrate that TSA in fact, regulates differentially both ABCB1 promoters, downregulating the upstream promoter that is responsible for active P-glycoprotein expression. These results suggest that iHDACs such as TSA may in fact potentiate the effects of antitumour drugs that are substrates of Pgp. Finally, we also demonstrate that TSA upregulates RUNDC3B mRNA independently of the ABCB1 promoter in use.

Genotoxicity assessment and detoxification induction in Dreissena polymorpha exposed to benzo[a]pyrene

The use of DNA adduct analysis has previously focused on the use of marine organisms for biomonitoring, whereas similar investigations in freshwater organisms are sparse. In that context, we have investigated the relevance of DNA adducts as biomarkers of genotoxicity in the freshwater mussels Dreissena polymorpha. The objective of the present study is to determine the stability of DNA adducts induced by benzo[a]pyrene (B[a]P) in zebra mussels. Mussels were exposed to dissolved B[a]P (10-100 µg/l) for 4 days. Afterwards, mussels were kept in clean water for 28 days and DNA adduct levels were subsequently measured in two different organs, the digestive glands and the gills, using the (32)P-postlabelling technique. In parallel, the expression of genes involved in the detoxification system was assessed by qPCR (catalase, superoxide dismutase, glutathione S transferase, HSP70, aryl hydrocarbon receptor, P glycoprotein). We observed a higher level of DNA adducts in the digestive glands compared to the gills. Moreover, in gills, the level of DNA adduct was dependent on the B[a]P concentration. The levels of adducts tended to decrease in both organs after 28 days in clean water. In addition, an early induction of HSP70, PgP, AHR and SOD mRNA levels was noticed in the gills compared to the digestive glands. CAT and GST gene expression increased from 12h exposure in both organs. A higher gene expression level of those genes was observed in the gills, except for AHR and CAT genes. Data converge towards the fact that DNA adducts hence represent a very promising biomarker of B[a]P contamination and potentially of exposure to polycyclic aromatic hydrocarbons. In addition, for the first time in this study, B[a]P detoxification system was characterised in D. polymorpha.

Development of a new permeability assay using low‐efflux MDCKII cells

Permeability is an important property of drug candidates. The Madin-Darby canine kidney cell line (MDCK) permeability assay is widely used and the primary concern of using MDCK cells is the presence of endogenous transporters of nonhuman origin. The canine P-glycoprotein (Pgp) can interfere with permeability and transporter studies, leading to less reliable data. A new cell line, MDCKII-LE (low efflux), has been developed by selecting a subpopulation of low-efflux cells from MDCKII-WT using an iterative fluorescence-activated cell sorting technique with calcein-AM as a Pgp and efflux substrate. MDCKII-LE cells are a subpopulation of MDCKII cells with over 200-fold lower canine Pgp mRNA level and fivefold lower protein level than MDCKII-WT. MDCKII-LE cells showed less functional efflux activity than MDCKII-WT based on efflux ratios. Notably, MDCKII-MDR1 showed about 1.5-fold decreased expression of endogenous canine Pgp, suggesting that using the net flux ratio might not completely cancel out the background endogenous transporter activities. MDCKII-LE cells offer clear advantages over the MDCKII-WT by providing less efflux transporter background signals and minimizing interference from canine Pgp. The MDCKII-LE apparent permeability values well differentiates compounds from high to medium/low human intestinal absorption and can be used for Biopharmaceutical Classification System. The MDCKII-LE permeability assay (4-in-1 cassette dosing) is high throughput with good precision, reproducibility, robustness, and cost-effective.

Emtricitabine: Inhibitor and substrate of multidrug resistance associated protein

Efflux proteins have been shown to greatly affect the uptake of antiretroviral drugs by cells and to prevent their access to the HIV-1 replication site. The active efflux of these drugs might produce subtherapeutic drug levels and favor resistant viral strains and the emergence of sanctuary sites. This study has been performed to investigate whether emtricitabine (FTC) is a substrate and/or inhibitor of MRP1 in human peripheral blood mononuclear cells (PBMCs, HIV-1 target site). Moreover, we have reported the impact of FTC combined with protease inhibitors (PIs) (ritonavir, atazanavir, lopinavir) on Pgp and MRP1 expression and function, and on PI accumulation. Following a 72-h incubation with antiretroviral regimen, Pgp and MRP1 expression and function were assessed on lymphocytes; and intracellular drug concentrations were measured by LC–MS/MS. FTC concentrations were determined following incubation with or without specific efflux proteins inhibitors. FTC inhibitor properties were measured using 2 different MRP substrates. Quantitative real-time PCR showed that PBMCs express high levels of both Pgp and MRP1 mRNA copy number whereas MRP2 and MRP3 were not detectable. Our findings indicate a decrease in MRP1 function after exposure to FTC. MK571 (specific MRP inhibitor) significantly increases FTC accumulation in PBMCs. FTC increases intracellular calcein and [ 3H]-vincristine accumulation. Emtricitabine has both inhibitor and substrate characteristics with MRP1 in PBMCs in vitro, and does not interact with PI accumulation.

Closing the Gaps: A Full Scan of the Intestinal Expression of P-Glycoprotein, Breast Cancer Resistance Protein, and Multidrug Resistance-Associated Protein 2 in Male and Female Rats

Intestinal ATP binding cassette (ABC) transporters may affect the bioavailability and effectiveness of orally administered drugs. Available studies on regional expression of intestinal efflux transporters were done with selected intestinal segments only and inconsistent with regard to the variability of transporter expression and the course of expression along the intestine. For an evaluation of the consistency between mRNA and protein expression, relative expression levels of P-glycoprotein (Pgp; ABCB1), breast cancer resistance protein (Bcrp; ABCG2), and multidrug resistance-associated protein (Mrp) 2 (ABCC2) were determined using quantitative real-time-polymerase chain reaction and Western blot in rat intestinal segments from duodenum, jejunum, ileum, and colon. In addition, the protein expression of Pgp, Bcrp, and Mrp2 from the entire rat intestine was studied by a complete 3-cm segmentation to evaluate the predictive power of expression analyses from selected intestinal segments. Pgp showed an increase from proximal to distal regions, Bcrp showed an arcuate pattern with highest expression toward the end of small intestine, and Mrp2 decreased along the intestinal axis from proximal to distal parts. No gender specific differences could be observed. Regarding the concordance of mRNA and protein expression, Pgp and Bcrp mRNA samples allow good estimations about the corresponding protein expression (for Pgp limited to the mdr1a isoform), but for Mrp2, pronounced deviation could be observed. All transporters showed considerable intra- and interindividual variability, especially at the protein level, making it problematic to take transporter expressions of small sections exemplary for general assumptions on intestinal abundances.