Pgp Expression Research Articles

Evaluation of the potential effect of Allium sativum, Momordica charantia, Eugenia jambolana, Ocimum sanctum, and Psidium guajava on intestinal p-glycoprotein in rats.

This study was evaluated synergistic effect of a polyherbal formulation (PHF) of Allium sativum L., Eugenia jambolana Lam., Momordica charantia L., Ocimum sanctum Linn., and Psidium guajava L. on p-glycoprotein (Pgp) of intestine. These five herbs were traditionally used for diabetes. These herbs are commonly present in Ayurvedic product as antidiabetics in India. PHF was prepared by five indigenous herbs. Different doses (50, 100 and 200 mg/kg/day) of were orally administered to Sprague-Dawley rats of different groups for multiple weeks except control groups. Alteration in Pgp expression was evaluated by real-time-polymerase chain reaction and western blotting while modulation in activity of Pgp was evaluated using rhodamine 123 (Rh123) as transport substrate by in-situ absorption and everted gut sac method. In PHF, pretreated group received 50, 100 and 200 mg/kg/day for 7 days, mRNA level decreased by 1.75, 2.45 and 2.37-fold, respectively, as compared to control. Similarly, when PHF at dose of 100 mg/kg/day was given consequently for 4 weeks, maximum decrease in Pgp expression level was observed only after 1 week and further increase in the treatment duration did not produce significant decrease compared to the 1st week treatment. Pgp mediated transport of Rh123 was significantly decreased with everted gut sac prepared from PHF pretreated rats (1 week) compared to those prepared from vehicle treated rats. We report that PHF pretreatment downregulated the expression of intestinal Pgp and this downregulated intestinal Pgp would result in decreased functional activity. In addition, this downregulated Pgp expression might affect the bioavailability of antidiabetic Pgp substrate drugs.

Relationships of the expression profiles of the mismatch repair genes with the clinicopathology and the expressions of Pgp, GSTπ, Topo II, and Ki-67 in patients with colorectal cancer

Background: To investigate the immunohistochemical expression profiles of mismatch repair (MMR) genes (MLH1, PMS2, MSH2, and MSH6) and explore their relationships with clinicopathological features and the expressions of P-glycoprotein (Pgp), glutathione-S-transferase (GSTπ), DNA topoisomerase II (Topo II), and cell proliferation antigen Ki-67 in patients with colorectal cancer. Methods: The clinicopathological data of 93 patients with colorectal cancer who had been treated in our hospital from February 2014 to May 2015 were retrospectively analyzed. The expressions of MMR genes (MLH1, PMS2, MSH2, and MSH6), Pgp, GSTπ, Topo II, and Ki-67 in cancer tissues were determined by immunohistochemical staining. Then we used multiple sample rate (or constituent ratio) comparisons (i.e., χ 2 test with R×C table) to analyze the relationships of the expressions of MMR genes (MLH1, PMS2, MSH2, and MSH6) with clinopathology and the expressions of Pgp, GSTπ, Topo II, and Ki-67 in colorectal cancer. Pearson correlation analyses were used to analyze the correlation of the expressions between MMR genes (MLH1, PMS2, MSH2, and MSH6) and Ki-67. Results: The missing expression rate of MMR genes MLH1, PMS2, MSH2, and MSH6 were 14.0%, 17.2%, 10.8%, and 11.8%, respectively. The expression rates of Pgp(−), Pgp (+), Pgp (++), and Pgp (+++) were 3.2%, 25.8%, and 51.6%, 19.3%. The expression rates of GSTπ (−), GSTπ (+), GSTπ (++), and GSTπ (+++) were 3.3%, 16.7%, 56.7%, and 23.3%. Topo II had no negative or level IV expression, and its level I, II and III expressions accounted for 19.3%, 77.4%, and 3.2%. The expression rates of Ki-67 50% (+) accounted for 1.3%, 25%, and 50%, respectively. The missing expressions of MMR genes (MLH1, PMS2, MSH2, and MSH6) showed no significant correlation with gender, age, histological differentiation, and TNM staging (all P>0.05). There was also no significant correlation (P>0.05) between MSH2 missing expression and the lesion location (P>0.05). However, the missing expressions of MLH1, PMS2, and MSH6 showed significant correlation with the lesion location (P 0.05). However, the missing expressions of MLH1, PMS2, MSH2, and MSH6 were significantly correlated with the Ki-67 expression (P 50% (+) were 6.1%, 4.1%, 8.2%, and 6.1%, respectively. MLH1, PMS2, MSH2, and MSH6 missing expressions were negatively correlated with Ki-67 expression (correlation coefficient r=−0.969, r=−0.464, r=−0.143, r=−0.344, respectively; all P Conclusions: The missing expressions of MMR genes (MLH1, PMS2, MSH2, MSH6) are more likely to occur in right colon, followed by left colon and rectum. The missing expressions of these MMR genes are negatively correlated with Ki-67 expression.

Predictive value of microparticle-associated tissue factor activity for permeability glycoprotein-mediated multidrug resistance in cancer.

Multidrug resistance (MDR) protein 1, which is also known as permeability glycoprotein (Pgp), and tissue factor (TF) are recurrently overexpressed on the surface of cancer cells, likely in response to stimuli such as chemotherapy. Microparticles (MPs) released from cancer cells into the bloodstream express tumour markers on their surface that may be useful as predictive biomarkers for evaluating disease progression. The present study measured the level of TF/factor VII (FVII)-dependent coagulation of MPs isolated from the plasma of cancer patients with various tumours, who were undergoing chemotherapy. Furthermore, Pgp expression on the surface of MPs was evaluated by immunohistochemistry. A total of 50 cancer patients, as well as 10 healthy volunteers, were enrolled in the present study. MP-associated TF/FVII-dependent coagulation pathways were evaluated as the effect of an anti-FVII antibody on the time to thrombin generation, as compared with controls treated with saline. The significantly lengthened times of coagulation [obtained in 20/50 samples (36.5 ± 16%) after treatment with anti-FVIIa when compared with controls] suggest the presence of TF activity is associated with circulating MPs. Furthermore, the 20 MP/TF-positive samples were associated with Pgp overexpression on their surface. Conversely, in the remaining samples (n=30), treatment with the anti-FVIIa antibody did not significantly lengthen the time to clotting (<10%), and Pgp overexpression was not detected. In addition, in the control samples from healthy individuals, Pgp expression at the plasma membrane and clotting in the presence of the anti-FVII antibody were not observed, indicating the absence of MPs. The present study demonstrated that MPs in the blood of cancer patients promoted fibrin generation via TF/FVII-dependent pathways, thus suggesting that the evaluation of MP-TF activity may have a predictive value for Pgp-mediated MDR in various cancer types. Although further studies are required, the measurement of plasma MP-associated TF activity as a predictive biomarker may provide novel therapeutic perspectives to improve the prognosis and effectiveness of anti-cancer drugs in patients who are at a high-risk of Pgp-mediated MDR.

Involvement of NRF2 Signaling in Doxorubicin Resistance of Cancer Stem Cell-Enriched Colonospheres.

Cancer stem cells (CSCs) are a subset of tumor cells, which are characterized by resistance against chemotherapy and environmental stress, and are known to cause tumor relapse after therapy. A number of molecular mechanisms underlie the chemoresistance of CSCs, including high expression levels of drug efflux transporters. We investigated the role of the antioxidant transcription factor NF-E2-related factor 2 (NRF2) in chemoresistance development, using a CSC-enriched colonosphere system. HCT116 colonospheres were more resistant to doxorubicin-induced cell death and expressed higher levels of drug efflux transporters such as P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) compared to HCT116 monolayers. Notably, levels of NRF2 and expression of its target genes were substantially elevated in colonospheres, and these increases were linked to doxorubicin resistance. When NRF2 expression was silenced in colonospheres, Pgp and BCRP expression was downregulated, and doxorubicin resistance was diminished. Collectively, these results indicate that NRF2 activation contributes to chemoresistance acquisition in CSC-enriched colonospheres through the upregulation of drug efflux transporters.

Alterations in P-Glycoprotein Expression and Function Between Macrophage Subsets.

Macrophages are an important cellular reservoir in HIV, and exist in two phenotypically dissimilar subsets, the pro-inflammatory M1 phenotype, and the anti-inflammatory M2 phenotype. The role of these two subsets is uncertain. We hypothesized that differences in drug efflux transporters exist between the subsets, which would result in altered intracellular drug concentrations between these cells. U937 monocytic cells were polarized to the M1 or M2 phenotype via treatment with interferon-gamma and LPS, or interleukins 4, 13, and LPS, respectively. PGP function was assessed with Hoechst 33342, and expression via western blotting. Intracellular lopinavir was assessed via LC-MS/MS. Data was confirmed with primary monocyte derived macrophages. We observed significant differences in intracellular concentrations of lopinavir, a PGP substrate, with higher concentrations in M1 cells. PGP function and expression was higher in the M2 macrophages. These results were confirmed with primary monocyte derived macrophages. This data shows that there are previously unreported differences in P-glycoprotein expression between macrophage subsets, and suggests that there may be differences for other transporters. These differences can play a role in intracellular drug concentrations in these cells, and may allow for low-level HIV replication.

Selective GPR55 antagonism reduces chemoresistance in cancer cells

G protein-coupled receptor 55 (GPR55) possesses pro-oncogenic activity and its function can be competitively inhibited with (R,R⿿)-4⿲-methoxy-1-naphthylfenoterol (MNF) through poorly defined signaling pathways. Here, the anti-tumorigenic effect of MNF was investigated in the human pancreatic cancer cell line, PANC-1, by focusing on the expression of known cancer biomarkers and the expression and function of multidrug resistance (MDR) exporters such as P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP). Incubation of PANC1 cells with MNF (1μM) for 24h significantly decreased EGF receptor, pyruvate kinase M2 (PKM2), and β-catenin protein levels and was accompanied by significant reduction in nuclear accumulation of HIF-1α and the phospho-active forms of PKM2 and β-catenin. Inhibition of GPR55 with either MNF or the GPR55 antagonist CID 16020046 lowered the amount of MDR proteins in total cellular extracts while diminishing the nuclear expression of Pgp and BCRP. There was significant nuclear accumulation of doxorubicin in PANC-1 cells treated with MNF and the pre-incubation with MNF increased the cytotoxicity of doxorubicin and gemcitabine in these cells. Potentiation of doxorubicin cytotoxicity by MNF was also observed in MDA-MB-231 breast cancer cells and U87MG glioblastoma cells, which express high levels of GPR55. The data suggest that inhibition of GPR55 activity produces antitumor effects via attenuation of the MEK/ERK and PI3K-AKT pathways leading to a reduction in the expression and function of MDR proteins.

Intercellular transfer of P-glycoprotein in human blood-brain barrier endothelial cells is increased by histone deacetylase inhibitors.

The blood–brain barrier (BBB) controls the entry of compounds into the brain, thereby regulating brain homeostasis. Efflux transporters such as P-glycoprotein (Pgp) significantly contribute to BBB function. Multiple signaling pathways modulate the expression and activity of Pgp in response to xenobiotics and disease. A non-genetic way of intercellular transfer of Pgp occurs in cancer cells, but whether this also occurs in non-cancer cells such as endothelial cells that form the BBB is not known. A human brain endothelial cell line (hCMEC/D3) was used to study whether cell-to-cell Pgp transfer occurs during co-culturing with Pgp-EGFP expressing hCMEC/D3 cells. The Pgp-EGFP fusion protein was transferred from donor to recipient cells by cell-to-cell contact and Pgp-EGFP enriched vesicles, which were exocytosed by donor cells and endocytosed by adherent recipient cells. Flow cytometry experiments with the Pgp substrate eFLUXX-ID Gold demonstrated that the transferred Pgp is functional in the recipient cells. Exposure of the donor cells with inhibitors of histone deacetylases (HDACs) resulted in an enhanced intercellular Pgp transfer. Non-genetic transfer of a resistance phenotype and its regulation by HDACs is a novel mechanism of altering BBB functionality. This mechanism may have important implications for understanding drug-induced alterations in Pgp expression and activity.

Knockout of P-glycoprotein does not alter antiepileptic drug efficacy in the intrahippocampal kainate model of mesial temporal lobe epilepsy in mice

Pharmacoresistance to antiepileptic drugs (AEDs) is a major challenge in epilepsy therapy, affecting at least 30% of patients. Thus, there is considerable interest in the mechanisms responsible for such pharmacoresistance, with particular attention on the specific cellular and molecular factors that lead to reduced drug sensitivity. Current hypotheses of refractory epilepsy include the multidrug transporter hypothesis, which posits that increased expression or function of drug efflux transporters, such as P-glycoprotein (Pgp), in brain capillaries reduces the local concentration of AEDs in epileptic brain regions to subtherapeutic levels. In the present study, this hypothesis was addressed by evaluating the efficacy of six AEDs in wildtype and Pgp deficient Mdr1a/b−/− mice in the intrahippocampal kainate model of mesial temporal lobe epilepsy. In this model, frequent focal electrographic seizures develop after an initial kainate-induced status epilepticus. These seizures are resistant to major AEDs, but the mechanisms of this resistance are unknown. In the present experiments, the focal nonconvulsive seizures were resistant to carbamazepine and phenytoin, whereas high doses of valproate and levetiracetam exerted moderate and phenobarbital and diazepam marked anti-seizure effects. All AEDs suppressed generalized convulsive seizures. No significant differences between wildtype and Pgp-deficient mice were observed in anti-seizure drug efficacies. Also, the individual responder and nonresponder rates in each experiment did not differ between mouse genotypes. This does not argue against the multidrug transporter hypothesis in general, but indicates that Pgp is not involved in the mechanisms explaining that focal electrographic seizures are resistant to some AEDs in the intrahippocampal mouse model of partial epilepsy. This was substantiated by the finding that epileptic wildtype mice do not exhibit increased Pgp expression in this model.

Evaluation of the in vitro expression of ATP binding-cassette (ABC) proteins in an Ixodes ricinus cell line exposed to ivermectin.

BackgroundTicks are among the most important vectors of pathogens causing human and animal disease. Acaricides are used to control tick infestation, although there are increasing reports of resistance. Recently, over-expression of ATP-binding cassette (ABC) transporter proteins (P-glycoproteins, PgP) has been implicated in resistance to the acaricide ivermectin in the ticks Rhipicephalus (Boophilus) microplus and Rhipicephalus sanguineus sensu lato. Ixodid tick cell lines have been used to investigate drug resistance mechanisms. The aim of the present study was to evaluate expression of several PgPs in the Ixodes ricinus-derived cell line IRE/CTVM19 and to determine modulation of expression following treatment with ivermectin.FindingsIRE/CTVM19 cells were treated with different concentrations of ivermectin (0, 11, 22 or 33 μM) and incubated for 10 days. Evaluation of viability and relative expression of ABCB1, ABCB6, ABCB8 and ABCB10 genes were carried out at day 10 post treatment. Cell viability ranged between 84 % and 92 % with no significant differences between untreated and treated cells. qRT-PCR showed that ABC pump expression was not significantly modulated by ivermectin treatment. Expression of the ABCB8 PgP subfamily revealed a biphasic trend, based on the ivermectin concentration. ABCB6 and ABCB10 gene expression was not modulated by ivermectin treatment and ABCB1 expression was not detected.ConclusionsThis is the first report of PgP expression in an I. ricinus-derived tick cell line. Development of an in vitro model for the study of acaricide resistance mechanisms would greatly facilitate screening for drug resistance in ticks.

Influence of different hypoxia types on p-glycoprotein functional activity and expression

The review of literature is devoted to researches in vitro and in vivo in which functioning changes P-glycoprotein (Pgp) under the influence of hypoxia of different types are shown. In the majority of scientific works activation of expression and functional activity of Pgp is shown that authors connect with activation of HIF-1 and Sp1 which are described in more detail. However a number of researches disproves the conclusion about induction of activity of Pgp in the conditions of hypoxia that testifies about difficult mechanisms involved in regulation of Pgp activity, their dependence on a type and duration of hypoxia, redox status and other parameters.

Metalloprobes: Fluorescence imaging of multidrug resistance (MDR1) P-Glycoprotein (Pgp)-mediated functional transport activity in cellulo

Radiolabeled metalloprobes offer sensitive tools for evaluating quantitative accumulation of chemical entities within pooled cell populations. Although beneficial in translational nuclear imaging, this method precludes interrogation of effects resulting from variations at a single cell level, within the same segment of cell population. Compared with radiotracer bioassays, fluorescence imaging offers a cost-efficient technique to assess accumulation of metalloprobes at a single cell level, and determine their intracellular localization under live cell conditions. To evaluate, whether or not radiotracer assay and fluorescence imaging provide complementary information on utility of metalloprobes to assess functional expression of P-glycoprotein (Pgp) on plasma membrane of tumor cells, imaging studies of fluorescent cationic Ga(III)-ENBDMPI (bis(3-ethoxy-2-hydroxy-benzylidene)-N,N′-bis(2,2-dimethyl-3-amino-propyl)ethylenediamine) and its neutral counterpart Zn(II)-ENBDMPI are performed. While the uptake profiles of the cationic metalloprobe are inversely proportional to expression of Pgp in tumor cells, the accumulation profiles of the neutral Zn(II)-ENBDMPI in non-MDR and MDR cells are not significantly impacted. The cationic Ga(III)-ENBDMPI maps with Mito-Tracker Red, thereby confirming localization within mitochondria of non-MDR (Pgp−) cells. Depolarization of both plasmalemmal and mitochondrial potentials decreased retention of the cationic Ga(III)-ENBDMPI within the mitochondria. Additionally, LY335979, an antagonist-induced accumulation of the cationic Ga(III) metalloprobe in MDR (Pgp+) cells indicated specificity of the agent. Compared with traits of Ga(III)-ENBDMPI as a Pgp recognized substrate, Zn(II)-ENBDMPI demonstrated uptake in both MDR and non-MDR cells thus indicating the significance of overall molecular charge in mediating Pgp recognition profiles. Combined data indicate that live cell imaging can offer a cost-effective methodology for monitoring functional Pgp expression.

Abstract P4-09-27: ABC transporter expression: Clues into chemoresistance of triple negative breast cancers

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive histological subtype with high rates of recurrence and metastatic disease. Intrinsic or acquired multidrug resistance facilitated by the over-expression of drug efflux pumps (ABC transporters: BCRP [ABCG2], MRP1 [ABCC1] and PGP [ABCB1]), may contribute to the aggressive nature of this disease. We examined the expression patterns of drug efflux pumps for insight on their potential role in chemoresistance of TNBC. Methods: 1393 TNBC patients molecularly profiled with a commercial assay (Caris Life Sciences) were evaluated retrospectively for expression of BCRP, MRP1 and PGP by immunohistochemistry. Antibodies used: PGP (C494), BCRP (6D171) and MRP1 (33A6). IHC threshold: positive = ≥1+ and ≥10%). This data set also included metachronous paired samples from 71 TNBC patients. JMP was used to ascertain distributional differences. Results: PGP and MRP1 positive expression rates were 8.4% (117/1393) compared to 78.2% (248/317), respectively, displaying an inverse association (p=0.0001). BCRP was over-expressed in 52% (90/173). Co-expression data for all three transporters was available for 153 patients. BCRP/MRP1 co-expression was most abundant, 39% (59/153), followed by MRP1/PGP at 4% (6/153) and BCRP/PGP at 2% (3/153). Furthermore, 12% (18/153) of TNBC exhibited positive expression for all three drug pumps (16/18 or 89% were from patients with metastatic disease) and 14% (22/153) exhibited negative expression for all three drug pumps (15/22 or 68% were from patients with metastatic disease). Interestingly, positive MRP1 status, but not BCRP or PGP, correlated with metastatic disease (p=0.0061). In the paired data analysis, PGP expression was absent in 77% (55/71) and retained lack of expression in subsequently profiled specimens, 3% (2/71) retained positive PGP status, 13% (9/71) lost expression of PGP and 7% (5/71) gained expression of PGP. 36/40 patients that lacked PGP expression and 8/9 patients that "lost" PGP expression, exhibited and retained expression of MRP1 in the initial and subsequently-profiled specimens. Conclusion: Biomarker expression patterns of drug efflux pumps may provide insight to the chemoresistance phenotype observed in TNBC. Expression of MRP1 is favored in TNBC and is correlated with metastatic disease status. Although PGP expression may be absent or lost during TNBC progression, MRP1 (and BCRP) expression are almost always retained. Utilization of chemotherapies that are substrates of PGP, but not MRP1, (based on expression status) may be worthy of future investigation. Citation Format: Feldman R, Abbott B, Reddy S, Gatalica Z, Castro M. ABC transporter expression: Clues into chemoresistance of triple negative breast cancers. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-27.

Evaluation of P-glycoprotein (Pgp) expression in human osteosarcoma by high-throughput tissue microarray.

Survival of osteosarcoma patients is currently limited by the development of metastases and multidrug resistance (MDR). A well-established cause of MDR involves overexpression of P-glycoprotein (Pgp) in tumor cells. However, some discrepancies still exist as to the clinical significance of Pgp in osteosarcoma. We sought to elucidate further whether the Pgp expression correlated with clinical behavior in a series of patients with osteosarcoma via high-throughput tissue microarray (TMA) analysis. Immunohistochemical analysis of Pgp expression in a TMA of 114 specimens with a retrospective review of 70 osteosarcoma patients admitted to the Massachusetts General Hospital (MGH) was performed. High Pgp expression was correlated with metastasis development and poor response to pre-operative chemotherapy in osteosarcoma patients. Eighteen of the fifty-seven patients initially admitted with primary osteosarcoma showed high Pgp expression. Among these 18 patients with high Pgp expression, 13 of 18 (72%) patients eventually developed metastases. There was no significant clinical relevance between Pgp expression and osteosarcoma survival. These results support that high expression of Pgp is important, but cannot be assigned as, an individual predictor in the development of human osteosarcoma. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1606-1612, 2016.

Immunohistochemical expressions of p75, s100, PGP 9.5 and c-KIT in resected bowel of hirchsprung’s disease and correlation with postoperative complication

Immunohistochemical expressions of p75, s100, PGP 9.5 and c-KIT in resected bowel of hirchsprung’s disease and correlation with postoperative complication

Morphological substrate and pathogenetic mechanisms of pelvic pain syndrome in endometriosis. Part II. Peripheral nerve tissue remodeling in the foci of endometriosis

Endometriosis (EM) is morphologically characterized by the development of extrauterine endometrioid heterotopies, the major clinical symptoms of which is chronic pelvic pain, which is a serious problem not only in modern gynecology, but also in public health as a whole. to investigate neurogenic markers in the foci of EM of various sites and histological structure in women with and without pain syndrome. The investigation was performed using the operative material (resected segments of the intestine, bladder, rectovaginal septum, and small pelvic peritoneum) obtained from 52 women with an intraoperative and morphologically verified diagnosis of EM and (Group 1) and without (Group 2) pain syndrome. Immunohistochemical examination was made on paraffin-embedded tissue sections in accordance with the standard protocols, by using the antibodies: 1) anti-PGP 9.5 polyclonal rabbit antibodies; 2) mouse anti-human neurofilament (NF) protein monoclonal antibodies (Clone 2F1); 3) mouse anti-nerve growth factor (NGF) monoclonal antibodies; 4) monoclonal mouse anti-human NGF receptor p75 (NGFRp75) antibodies (Dako, Denmark). Our findings demonstrate differences in the expression of PGP 9.5, NFs, NGF, and NGFRp75 in the foci and adjacent tissue in painful and painless EM irrespective of the locations of heterotopies. The found molecular features are a manifestation of the remodeling of nerve fibers and nerve endings in the foci of EM and PGP9.5, NGF, and NGFRp75 give rise to nerve fiber neoformation and pain syndrome in EM. At the same time, the immunohistochemical phenotype of EM foci does not depend on their site and reflects the presence or absence of pain syndrome.

Immunohistochemical Expression of PgP in Canine Cutaneous and Oral Melanomas: Evidence of a Novel Pattern of Expression and Association with Tumour Location and Behaviour

Immunohistochemical Expression of PgP in Canine Cutaneous and Oral Melanomas: Evidence of a Novel Pattern of Expression and Association with Tumour Location and Behaviour

Membrane Transporters and Pharmacological Implications.

Membrane transporters serve to move chemicals in and out of the cells according to metabolic needs or the presence of toxic compounds. These processes are mediated by facilitated diffusion or active transport through the lipid bilayer that is the cell membrane. There exist two main categories of membrane transporters, the more passive solute carrier transporters (SLCs) and the ATP binding cassette transporters (ABCs). SLCs allow for passage of ions, sugars, lipids, amino acids and other compounds down a gradient, contributing to a cell’s passive permeability for such compounds. ABCs feature a highly conserved nucleotide binding domain (NBD) which contain peptide sequences responsible for ATP hydrolysis such as the Walker A and B motifs. ABCs utilize the energy stored in ATP to transport chemicals against their concentration and/or electrical gradient and consequently alter expected biological conditions. Transporters are now recognized as crucial barriers (e.g., efflux transporters) as well as possible delivery pathways to consider when designing new pharmaceutical agents as many traditional therapeutics are being recognized as transporter substrates [1–3]. Drug resistant tumors and the blood-brain barrier (BBB) for example have been shown to actively express efflux transporters preventing therapeutic agents from reaching clinically relevant intracellular concentrations and/or physiological targets in the brain [4–7]. Consequently, in 2010 the International Transporter Consortium put forth the ‘white paper’ detailing the structure, location, and known substrates of various pharmacologically relevant transporters. This prompted action from American and European regulatory agencies to release guidelines on transporter-drug interaction studies [3]. P-Glycoprotein (MDR1), Breast Cancer Resistance Protein (BCRP), Organic Anion Transporter peptide (OATP), Organic Anion Transporter (OAT), Organic Cation Transporter (OCT), and Multi Drug Resistance Protein (MRP) are mentioned directly in the white paper and have been a focus of the ITC to be included in the in vitro to in vivo extrapolation (IVIVE) which refers to the qualitative or quantitative transposition of in vitro experimental results to predicts a physiological and/or pathological phenomena in vivo. P-glycoproteins (Pgp) and the Cytochrome P450 enzyme CYP3A analogs share a significant number of substrates [8] and are both found in the intestines and liver [9–11]. Together they compose a first pass metabolism barrier for therapeutic agents taken orally [12,13]. Because Pgp effluxes compounds from the intestinal wall back into the lumen of the intestines, researchers may mistake increased mean residence time (MRT) for increased absorption when performing PK/PD studies [14]. Some of these CYP analogs such as CYP3A4, CYP2C9, CYP2C19, CYP2A6 and CYP2E1 are also expressed at the BBB endothelial level of the BBB under pathological conditions (e.g., drug resistant epilepsy). In some cases, the expression of a CYP enzyme (e.g., CYP3A CYP2C19 and CYP2C9) is regulated by the activation of the xenobiotic receptor pregnane X receptor (PXR) which also controls the expression of Pgp and other drug efflux systems [15,16]. With regard to the drug efflux transporters at the BBB level, multidrug resistance (MDR) is a major obstacle to treating patients with cancer and is often the result of overexpression of a 170- to 180-kDa plasma membrane glycoprotein known as P-glycoprotein (Pgp) [17,18] and multidrug resistance-related proteins (MRP1, 190 kDa) [19,20]. Human Pgp is encoded by MDR1 and rodent Pgp by Mdr1a and Mdr1b [21–24]. Pgp and MRPs belong to the superfamily of ATP-binding cassette transporters. Unlike other selective (classical) transport proteins, MDR proteins recognize a wide range of substrates. This wide substrate specificity explains the cross-resistance to several chemically unrelated compounds, the characteristic feature found in the multi-drug resistance phenotype. In addition to their overlapping substrates specificity, each transporter can handle unique compounds. Pgp-MDR1 is a transporter for large amphipathic compounds either uncharged or slightly charged while the MRP family is mostly transporting hydrophobic anionic conjugates with glucuronide, sulfate or glutathione and also extrudes hydrophobic uncharged drugs [19]. Experiments designed to define the structure of Pgp suggest that there is no simple single drug-binding site or pore in Pgp. Amino acid substitutions in, or near, most of the transmembrane segments affect substrate specificity efficiency or transport. In drug refractory patients a synergistic effect between MDRs and CYP enzymes has been recently observed which antagonizes the passage of drugs targeting the brain (e.g., antiepileptics, tumor suppressants, etc.) through a concerted set of mechanisms [15]. The efflux transporters extrude the drug from the brain across the BBB back into the blood circulation while the CYP enzyme metabolize the drug substrate into (at large) inactive derivate(s), thus rendering the drug bioavailability ineffective from a therapeutic standpoint. Other than MDR, altered activity of efflux transporters have been linked to a number of neurological disorders including Alzheimer’s disease [25], Parkinson’s diseases [26] and Creutzfeldt-Jakob disease [27].

Potential anti-MDR agents based on the podophyllotoxin scaffold: synthesis and antiproliferative activity evaluation against chronic myeloid leukemia cells by activating MAPK signaling pathways

Compound 9k exhibited excellent cytotoxicity, induced apoptosis and G2/M cell cycle arrest, downregulated Pgp expression and up-regulated the expression of p-ERK1/2, p-JNK and p-p38 in K562/ADR cells.

Influence of bone marrow stromal cell secreted molecules on pulpal and periodontal healing in replanted immature rat molars.

To investigate the effect of paracrine factors secreted from human bone marrow stromal cell (BMSC-CM) on pulpal and periodontal healing following immediate replantation of maxillary rat first molars. Fifty maxillary rat first molars were elevated and replanted after 2min. The left teeth were replanted without treatment, whereas BMSC-CM was injected into the right socket prior to replantation. Twelve un-operated teeth served as reference teeth. The expression of vascular endothelial growth factor A, alkaline phosphatase, Runt-related transcription factor 2 and osteoclast stimulating factor 1 was studied by real-time reverse transcription polymerase chain reaction at day 3 and 14. The dentin thickness together with Laminin- and PGP 9.5-immunoreactivity were studied after 3, 14 and 90days. Real-Time qRT-PCR data showed up-regulated expression of ALP mRNA in the socket specimens of conditioned medium treated replanted teeth after 3days. No morphological differences were found for the expression of Laminin and PGP 9.5 between control and conditioned medium treated replanted teeth. At day 14, external cervical and surface root resorption was found in one BMSC-CM and one control tooth. At 90days, all control replanted teeth had external cervical and surface root resorptions, whereas only one sample was seen among the conditioned medium treated teeth. At day 90, more extensive dentine formation with narrowing of the pulpal space was observed in the control compared with conditioned medium treated teeth. The present findings showed that BMSC-CM treatment reduced the number of replanted teeth with external root resorption and resulted in a significant reduction in new dentin formation.

The Use of Cyclosporine in Association with Chemotherapy As Induction Treatment in Patients with Acute Myeloid Leukemia (AML) and High Rhodamine Efflux at Diagnosis Results in Higher Complete Hematological Remission Rates, but Does Not Prolong Overall Survival

The overexpression of P-glycoprotein (Pgp) in leukemic blasts of Acute Myeloid Leukemia (AML) is associated with lower complete remission (CR) rates and shorter disease-free (DFS) and overall survival (OS). Cyclosporine A (CSA) is a competitive inhibitor of Pgp capable of reduce the P-gp mediated drug efflux. List et al. reported that CSA addition to treatment with cytarabine and daunorubicine reduced the resistance to induction treatment (IT) and prolonged DFS and OS. The effect was greater in AML patients with higher Pgp expression (Blood 2001; vol 98, n 12: 3212-20). Based on this study, we tested the effect of the association of CSA to IT in cases with high Pgp expression, which was established using the Rhodamine effux test as previously described (Pétriz, J. and García-López, J. Leukemia 1997; vol 11: 1124-30). The cut off value of the mean fluorescence intensity adopted to define high P-gp expression was ≥ 1.10. A total of 21 high P-gp AML patients were randomized in two groups: 1. Daunorubicin (DNR) 60mg/m² in continuous infusion -CI- 3x+Cytarabine 100 mg/m² CI 7x + CSA 16mg/m² CI 3x; (CSA group) and 2. DNR 60mg/m² CI 3x + Cytarabine 100 mg/m² CI 7x (Conventional IT group). Patients who did not achieve Complete Hematological Remission (CHR) in the first cycle of chemotherapy received a second course. The 2 groups did not differ regarding age (mean ± S.D.: 44.5 ± 11.9 vs. 48.5 ± 15.2 years in CSA and conventional IT group, respectively), leukocyte counts at diagnosis (44,200 ± 56,100 vs. 21,540 ± 20,200/µl), frequency of de Novo AML (81.8 vs. 80%) and of Core Binding Factor Leukemia cases (18.2 vs. 10%). The median follow up among survivors was of 6.2 years. The mean serum levels of CSA at 24 hours after infusion was of 1,157 ng/mL (range: 4 - 1,600ng/mL), which was similar to that reported by List et al. The CHR rate after the first cycle of induction was higher in the group using CSA (63.6% vs. 30%; p = 0.09) but the DFS (2.05 years vs. not reached; p= 0.27) and OS (4.08 vs. 21.12 months; p = 0.19) were higher in conventional IT group. These results may reflect the effect of the second course of induction in patients who failed to achieve CHR (CHR after second IT: 0% vs. 33.3%, p=0.34, in CSA and conventional IT group, respectively) and higher relapse rate in CSA group (42.8% vs. 33.3%, p=0.78). Furthermore, there was a lower frequency of induction deaths (18.2% vs. 9%; p = 0.59) and of hyperbilirubinemia (bilirubin > 1,5x the upper limit of normality) in the Conventional IT group (6 vs. 0 patients; p = 0.02). There were no cases of acute cardiac toxicity. The study was interrupted due to the cost of CSA and the lack of significant improvement in the outcomes. In conclusion, despite the higher rate of CR after one cycle of chemotherapy, there was not improvement in long term survival of AML patients with high Pgp expression treated with CSA in combination with anthracyclines and cytarabine. DisclosuresNo relevant conflicts of interest to declare.