Reduced P-glycoprotein Expression Research Articles

Microvascular blood-brain barrier alterations in isolated brain capillaries of mice over-expressing alpha-synuclein (Thy1-aSyn line 61)

Dysfunction of the blood-brain barrier (BBB) is suggested to play a critical role in the pathological mechanisms of Parkinson's disease (PD). PD-related pathology such as alpha-synuclein accumulation and inflammatory processes potentially affect the integrity of the BBB early in disease progression, which in turn may alter the crosstalk of the central and peripheral immune response. Importantly, BBB dysfunction could also affect drug response in PD. Here we analyzed microvascular changes in isolated brain capillaries and brain sections on a cellular and molecular level during disease progression in an established PD mouse model that overexpresses human wild-type alpha-synuclein (Thy1-aSyn, line 61). BBB alterations observed in Thy1-aSyn mice included reduced vessel density, reduced aquaporin-4 coverage, reduced P-glycoprotein expression, increased low-density lipoprotein receptor-related protein 1 expression, increased pS129-alpha-synuclein deposition, and increased adhesion protein and matrix metalloprotease expression together with alterations in tight junction proteins. Striatal capillaries presented with more dysregulated BBB integrity markers compared to cortical capillaries. These alterations of BBB integrity lead, however, not to an overt IgG leakage in brain parenchyma. Our data reveals intricate alterations in key proteins of BBB function together with histological evidence for altered structure of the brain vasculature. Thy1-aSyn mice represent a useful model to investigate therapeutic targeting of BBB alterations in synucleinopathies.

Increasing Intracellular Levels of Iron with Ferric Ammonium Citrate Leads to Reduced P-glycoprotein Expression in Human Immortalised Brain Microvascular Endothelial Cells.

P-glycoprotein (P-gp) at the blood-brain barrier (BBB) precludes the brain penetration of many xenobiotics and mediates brain-to-blood clearance of β-amyloid, which accumulates in the Alzheimer's disease (AD) brain. Zinc and copper are reported to modulate BBB expression and function of P-gp; however, the impact of exogenous iron, which accumulates in AD, on P-gp dynamics remains unknown. P-gp protein and MDR1 transcript levels were assessed in immortalised human cerebral microvascular endothelial (hCMEC/D3) cells treated with ferric ammonium citrate (FAC; 250μM, 72h), by Western blotting and RT-qPCR, respectively. P-gp function was assessed using rhodamine-123 and [3H]-digoxin accumulation. Intracellular reactive oxygen species (ROS) levels were determined using 2',7'-dichlorofluorescin diacetate and intracellular iron levels quantified using a ferrozine assay. FAC treatment significantly reduced P-gp protein (36%) and MDR1 mRNA (16%) levels, with no significant change in rhodamine-123 or [3H]-digoxin accumulation. While P-gp/MDR1 downregulation was associated with elevated ROS and intracellular iron, MDR1 downregulation was not attenuated with the antioxidant N-acetylcysteine nor the iron chelators desferrioxamine and deferiprone, suggesting the involvement of a ROS-independent mechanism or incomplete iron chelation. These studies demonstrate that iron negatively regulates P-gp expression at the BBB, potentially impacting CNS drug delivery and brain β-amyloid clearance.

Drug Loaded Nanoparticles of Metal-Organic Frameworks with High Colloidal Stability for Anticancer Application.

Nanoscale metal-organic frameworks are considered for drug delivery due to their ultrahigh porosity and intrinsic biodegradability. However, the poor colloidal dispersity of these nanocarriers in aqueous media has limited the further applications. In this study, a novel dextran-coated zeolitic imidazolate framework-8 (ZIF-8/Dex) nanoparticle was synthesized. The hydrated dextran shell, which was linked to frameworks through coordination bonds, improved the dispersity of particle and reduced the side effects caused by rapid disintegration. Furthermore, this nanosystem with pH-sensitive property may serve as a vehicle of doxorubicin hydrochloride with high loading efficiency and controlling release behavior. Interestingly, the drug-loaded nanocomposite (DOX@ZIF-8/Dex) exhibited synergistic anticancer activity against human breast carcinoma multidrug resistant cells, effectively avoided the efflux effect and reduced P-glycoprotein expression. The successful preparation of such therapeutic platforms promotes the clinical application for anti-tumor therapy.

Quantitative analysis of technetium-99m-sestamibi uptake and washout in parathyroid scintigraphy supports dual mechanisms of lesion conspicuity.

Proposed mechanisms of parathyroid localization in 'dual-phase' technetium-99m-sestamibi imaging include increased presence of mitochondria leading to greater uptake and slower washout compared with thyroid tissues owing to reduced expression of P-glycoprotein. Using new techniques of quantitative single-photon emission computed tomography (SPECT)/computed tomography (CT), we have measured MIBI uptake and washout to better understand factors related to conspicuity. We retrospectively reviewed 125 consecutive patients. Early and delayed SPECT/CT images were reconstructed using a previously validated technique. Maximum standardized uptake values of parathyroid adenomas and thyroid tissue were measured, and corresponding washout rates were calculated. Of 53 patients with localization of parathyroid adenoma (42%), median maximum standardized uptake values were higher for parathyroid adenomas than for thyroid tissue on both early (6.43±3.78 vs. 4.43±1.93, P<0.001) and delayed (3.40±3.09 vs. 1.84±1.05, P<0.001) images, being true on a per-patient basis in 41 (77%) and 48 (91%) patients, respectively. Median washout rates were slower from parathyroid adenomas than from thyroid lobes (0.26±0.16 vs. 0.42±0.18 h, P<0.001), being true on a per-patient basis in 43 (81%) patients. Similar findings were observed in a subgroup of 37 patients with surgically-confirmed adenomas. Of the patients where initial parathyroid uptake did not exceed thyroid uptake, all 12 exhibited slower MIBI washout from the parathyroid adenomas than from the thyroid gland. The characteristics of the thyroid gland did not differ in 67 patients without parathyroid localization. Quantitative analysis of 53 patients with localization of parathyroid adenoma revealed both a generally higher initial absolute uptake and slower rate of washout of MIBI in parathyroid adenomas than in thyroid tissue. The findings may support the hypothesis that both mechanisms proposed for parathyroid conspicuity in the dual-phase examination increased mitochondrial binding and slower washout owing to reduced P-glycoprotein expression. The technique of quantitative SPECT/CT represents a powerful tool for measuring tissue uptake to elucidate the contribution of these factors to lesion conspicuity.

Surfactin-based nanoparticles loaded with doxorubicin to overcome multidrug resistance in cancers.

BackgroundMultidrug resistance (MDR) is one of the major obstacles to successful cancer chemotherapy. Developing efficient strategies to reverse MDR remains a major challenge. Surfactin (SUR), a cyclic lipopeptide biosurfactant, has been found to display anticancer activity.MethodsIn this paper, SUR was assembled by solvent-emulsion method to load the anticancer drug doxorubicin (DOX). The cytotoxicity of DOX-loaded SUR nanoparticles (DOX@SUR) against DOX-resistant human breast cancer MCF-7/ADR is measured by MTT assay. The cellular uptake and intracellular retention of DOX@SUR are determined by flow cytometry. The tumor accumulation and anticancer activity of DOX@SUR are evaluated in MCF-7/ADR-bearing nude mice.ResultsDOX@SUR induce stronger cytotoxicity against DOX-resistant human breast cancer MCF-7/ADR cells compared to free DOX. DOX@SUR nanoparticles exhibit enhanced cellular uptake and decreased cellular efflux, which might be associated with reduced P-glycoprotein expression. After internalization into MCF-7/ADR cells by macropinocytosis- and caveolin-mediated endocytosis, DOX@SUR nanoparticles are colocalized with the lysosomes and translocated to the nucleus to exert cytotoxicity. Furthermore, in vivo animal experiment shows that the DOX@ SUR nanoparticles are accumulated more efficiently in tumors than free DOX. Meanwhile, DOX@SUR nanoparticles display stronger tumor inhibition activity and fewer side effects in MCF-7/ADR-bearing nude mice.ConclusionThis study indicates that SUR-based nanocarrier might present a promising platform to reverse MDR in cancer chemotherapy.

DACT1 overexpression inhibits proliferation, enhances apoptosis, and increases daunorubicin chemosensitivity in KG-1α cells.

DACT1 has been shown to participate in the development of many types of tumors; however, its role and precise molecular mechanisms in leukemia are unclear. In this study, we investigated the effect of DACT1 on KG-1α leukemia cells to further understand the mechanisms of DACT1-mediated tumor suppression. We transfected a DACT1 expression plasmid to upregulate DACT1 in KG-1α cells and analyzed the resulting phenotypic changes. The results demonstrated that DACT1 overexpression inhibited KG-1α proliferation, increased apoptosis, and arrested cells in the G0/G1 phase. Mechanistically, DACT1 overexpression inhibited Wnt/β-catenin signaling by reducing nuclear β-catenin levels in KG-1α cells. Furthermore, the viability of KG-1α cells transfected with DACT1 was significantly reduced when treated with daunorubicin. We also found that DACT1 reduced P-glycoprotein expression in KG-1α cells. These findings revealed an inhibitory role for DACT1 in leukemogenesis and provided evidence that DACT1 is an attractive target for the development of novel anti-leukemia therapies.

Downregulation of NPM reverses multidrug resistance in human hepatoma cells via inhibition of P-glycoprotein expression.

Multidrug resistance (MDR) is an important issue in current cancer treatments. In human cancer, drug resistance is primarily associated with the overexpression of multidrug resistance gene 1 (MDR1). Therefore, the human MDR1 gene promoter may be a target for anti‑MDR drug screening. Numerous methods to prevent MDR have been investigated. However, they have been proven to be clinically ineffective. Therefore, the aim of the present study was to investigate whether downregulation of nucleophosmin (NPM) demonstrates any effects on the reversal of MDR in hepatocellular carcinoma (HCC) cells. In the present study, two invitro MDR HCC cell lines, HepG2/Adriamycin (ADM) and SMMC7721/ADM, were established and the level of MDR was measured. The results demonstrated that NPM downregulation markedly reversed the effects of MDR in the model used. In addition, NPM downregulation reduced P-glycoprotein expression, as well as MDR1 expression. These results suggested that downregulation of NPM may be a novel and effective method of reversing the effects of MDR, and may be a potential adjuvant for tumor chemotherapy.

The synthetic compound 2′-hydroxy-2,4,6′-trimethoxychalcone overcomes P-glycoprotein-mediated multi-drug resistance in drug-resistant uterine sarcoma MES-SA/DX5 cells

The antitumor activity of a novel synthetic chalcone derivative, 2′-hydroxy-2,4,6′-trimethoxychalcone (1H3MC), was evaluated using the multi-drug-resistant human uterine sarcoma MES-SA/Dx5 cells. Treatment with 1H3MC reduced P-glycoprotein expression in a time-dependent manner and inhibited MES-SA/Dx5 cell proliferation. Cisplatin alone had no effect on cell viability, but combined treatment with cisplatin and 1H3MC exhibited synergistic cytotoxicity. Furthermore, the combination of cisplatin and 1H3MC synergistically cleaved both caspase-3 and its substrate protein, poly(ADP-ribose) polymerase, which resulted in the fragmentation of genomic DNA, a hallmark of apoptosis. These results suggest that 1H3MC is a promising adjuvant agent for overcoming P-glycoprotein-mediated multi-drug resistance in cancer cells.

Epigenetic Modulation of the Biophysical Properties of Drug-Resistant Cell Lipids to Restore Drug Transport and Endocytic Functions

In our recent studies exploring the biophysical characteristics of resistant cell lipids, and the role they play in drug transport, we demonstrated the difference of drug-resistant breast cancer cells from drug-sensitive cells in lipid composition and biophysical properties, suggesting that cancer cells acquire a drug-resistant phenotype through the alteration of lipid synthesis to inhibit intracellular drug transport to protect from cytotoxic effect. In cancer cells, epigenetic changes (e.g., DNA hypermethylation) are essential to maintain this drug-resistant phenotype. Thus, altered lipid synthesis may be linked to epigenetic mechanisms of drug resistance. We hypothesize that reversing DNA hypermethylation in resistant cells with an epigenetic drug could alter lipid synthesis, changing the cell membrane's biophysical properties to facilitate drug delivery to overcome drug resistance. Herein we show that treating drug-resistant breast cancer cells (MCF-7/ADR) with the epigenetic drug 5-aza-2'-deoxycytidine (decitabine) significantly alters cell lipid composition and biophysical properties, causing the resistant cells to acquire biophysical characteristics similar to those of sensitive cell (MCF-7) lipids. Following decitabine treatment, resistant cells demonstrated increased sphingomyelinase activity, resulting in a decreased sphingomyelin level that influenced lipid domain structures, increased membrane fluidity, and reduced P-glycoprotein expression. Changes in the biophysical characteristics of resistant cell lipids facilitated doxorubicin transport and restored endocytic function for drug delivery with a lipid-encapsulated form of doxorubicin, enhancing the drug efficacy. In conclusion, we have established a new mechanism for efficacy of an epigenetic drug, mediated through changes in lipid composition and biophysical properties, in reversing cancer drug resistance.

Hyperglycemia induced down-regulation of renal P-glycoprotein expression

The purpose of this study is to investigate the regulation of P-glycoprotein expression in the kidney under diabetic condition. Renal P-glycoprotein expression was examined in inbred mice with type 1 or type 2 diabetes by Western blotting. The underlying mechanisms of P-glycoprotein regulation were examined in Madin–Darby canine kidney type II (MDCK-II) cells by Western blotting or qRT-PCR. 3H-digoxin uptake was measured for P-glycoprotein activity in cells under various treatments. The results showed that P-glycoprotein expression was lower in kidneys of diabetic mice than in controls. In MDCK-II cells, treatments with insulin or IL-6 did not cause any change in P-glycoprotein expression, whereas TNF-α tended to increase P-glycoprotein expression at a concentration of 1ng/ml. On the other hand, P-glycoprotein expression was reduced under high glucose conditions (450mg/dl), while superoxide production was increased, and the reduction in P-glycoprotein expression was abolished by N-acetylcysteine (an antioxidant) and staurosporine (a nonselective PKC inhibitor). Treatment with oxidizing agents (H2O2, BSO) or PMA (a PKC activator) reduced P-glycoprotein expression. Antioxidant (N-acetylcysteine or glutathione) co-treatment abolished the H2O2-induced and BSO-induced reduction in P-glycoprotein expression, whereas it did not prevent the effect of PMA. The PMA-induced P-glycoprotein down-regulation was prevented by co-treatment of LY333531 (a PKC-β inhibitor). 3H-digoxin levels were higher in MDCK-II cells with high glucose, PMA or H2O2 treatments. In conclusion, P-glycoprotein expression is lower in kidneys of diabetic mice and in MDCK-II cells under high glucose conditions. Hyperglycemia induced reactive oxygen species and activated PKC in MDCK-II cells, leading to the decrease in P-glycoprotein expression.

MDR1 C3435T Polymorphism Is Highly Correlated with Early Response to Therapy and Outcome in Childhood Acute Lymphoblastic Leukaemia: Malaysia-Singapore 2003 ALL Study.

Early response to therapy as determined by Day 8 prednisolone response (PR) and minimal residual disease (MRD) are highly predictive of risk of relapse in childhood ALL. They are currently employed in risk-stratification in modern chemotherapeutic regimens. Leukemia biological factors like hyperdiploidy, oncogene fusions like TEL-AML1, MLL rearrangements and BCR-ABL are widely used for risk stratification but patient's pharmacogenetic profile has thus far not been utilised for tailoring therapy because of conflicting or equivocal results. We investigated potential candidate genes involved in drug transport and metabolism of steroids, vincristine, L-asparaginase and anthracyclines used in the BFM induction backbone to exploit the plausible pathways of early chemoresistance. We studied 282 children with ALL enrolled in the Malaysia-Singapore 2003 ALL trial. Poor prednisolone response (PPR) is defined as absolute blast count ≥1,000/uL after 7 days of prednisolone and 1 IT MTX. MRD quantification at day 33 induction using IgH and TCR markers based on European MRD Study Group standards. Four potential candidate genes MDR1 3435C>T, GSTT1, GSTM1, NQO1 609C>T were studied using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism. Statistical analysis was carried out using multinomial regression model. We had 241 good prednisolone responders (GPR), and 34 poor prednisolone responders (PPR). There were 112 patients with negative MRD (<10−4) while 129 had positive MRD ≥10−4 (35 had high MRD > 5 × 10−3). Only MDR1 3435TT genotype was strongly associated with poor PR (p=0.011; OR=2.8; 95%CI=1.3–6.4), MRD positive disease ≥10−4 (p=0.011, OR=2.8, 95%CI= 1.3–5.9) and high levels of MRD >5×10−3 as compared to MRD <10−4 (p<0.0001; OR=5.9; 95%CI=2.3–15.1). Interestingly, MDR1 3435CT heterozygosity is significantly associated good early response: with low levels of <MRD10−4 as compared to high MRD levels >5×10−3 (p=0.022; OR=0.4; 95%CI=0.2–0.9). Oral prednisolone and dexamethasone are the most powerful drugs in the treatment of childhood ALL. MDR1 3435C>T although a synonymous SNP, is associated with lower P-glycoprotein because of reduced MDR1 3435T mRNA stability. MDR1 3435 TT homozygote has 4 fold reduced levels of P-glycoprotein in the duodenum and is associated with reduced drug absorption like digoxin. We postulate that TT homozygote; there is reduced absorption of oral steroids in the gut, resulting in low oral bioavailability and poor early response as determined by poor prednisolone response and high MRD. On the other hand, in the CT heterozygote, drug oral bioavailability is not the drug-limiting step. The predominant effect of reduced P-glycoprotein expression in the cells are associated with reduced efflux of drugs, hence higher levels of intracellular drug resulting in good treatment response. MDR1 C3435T polymorphism is highly correlated with early response to therapy in childhood ALL as determined by prednisolone response and MRD levels at the end of induction.

Variable expression of P-glycoprotein in the human placenta and its association with mutations of the multidrug resistance 1 gene (MDR1, ABCB1).

The MDR1 gene product P-glycoprotein in the human placenta is important for protecting the fetus from unintended, harmful drug exposure, but also for limiting the access of therapeutic drugs to the fetus after maternal drug intake. A polymorphism in exon 26 of the MDR1 gene (C3435T) has previously been shown to be associated with reduced P-glycoprotein expression in the small intestine, kidney and lymphocytes. In the present study, we examined systematically whether MDR1 polymorphisms also have an impact on P-glycoprotein expression in the human placenta. MDR1 mRNA and P-glycoprotein were analysed in 73 full-term human placentas of Caucasians, as well as respective MDR1 genotypes/haplotypes, for the C3435T and G2677T/A polymorphisms of mothers and infants. MDR1 mRNA levels were not different between these genotype groups. However, P-glycoprotein expression was significantly lower when both mother and infant were homozygous for the 3435T allele (TT/tt) compared to maternal and fetal homozygotes for the C-allele (0.40 +/- 0.18 a.u. for TT/tt versus 0.66 +/- 0.30 a.u. for CC/cc, P = 0.01). Moreover, placentas from mothers carrying both polymorphisms (3435T and 2677T; TT/TT) also had a significantly lower P-glycoprotein expression (0.31 +/- 0.12 a.u.) compared to placentas of wild-type individuals (CC/GG, 0.71 +/- 0.31 a.u., P = 0.02). Taken together, the MDR1 polymorphisms C3435T and G2677T are associated with altered P-glycoprotein expression in the human placenta, and may have clinical consequences due to genetically determined, variable drug exposure of the fetus.

Morphology and function of canine small intestinal autografts: with particular interest in the influence of ex vivo graft irradiation.

The intestinal transplantation procedure obligates an early functional deficit in intestinal grafts. Graft irradiation has been used to modulate post-transplant immune reactions; however, irradiation may cause further deterioration of the function of the transplanted intestine. Using the canine model, we investigated the influence of the transplant procedures, with and without ex vivo graft irradiation, on early intestinal graft function and histopathology. Outbred hound dogs underwent autointestinal transplantation with (n=8) or without (n=5) 7.5 Gy ex vivo graft irradiation. Mucosal cytochrome P450 and P-glycoprotein, routine immunohistopathology, and intestinal absorptive function were studied. Weight gain was slow after surgery, but was comparable in the irradiated and nonirradiated groups. During the early post-transplant period, both groups showed defects in intestinal absorption, associated with decreased cytochrome P450 3A4 activity and reduced P-glycoprotein expression, regardless of graft irradiation. These changes returned to normal in both groups by day 28. Histopathologically, epithelial apoptosis showed a slight increase 1 hour after transplantation; however, there was no evidence of histopathologic abnormalities including arterial changes associated with irradiation. In addition, the frequency of T and B lymphocytes in the lamina propria were not significantly influenced by the transplant surgery or ex vivo irradiation. Thus, early after transplantation, intestinal function was impaired and effectiveness of orally administered immunosuppressive drugs was significantly altered. Graft irradiation did not induce further defects in intestinal function or cause histopathologic abnormalities.

Expression of P-glycoprotein in the chicken

The multidrug resistance gene product, P-glycoprotein, may act as a defense mechanism against natural and man-made environmental toxins. Like mammals, chickens show high levels of P-glycoprotein expression in the liver, small intestine, and kidney. Expression of P-glycoprotein rapidly increased with age in the liver and kidney reaching a plateau by 2 and 4 days of age, respectively; however, expression of P-glycoprotein in the duodenum did not significantly change with age. Addition of dietary antibiotics (monensin, bacitracin), as models for dietary toxins, altered P-glycoprotein expression. Monensin increased P-glycoprotein expression in the liver and duodenum. Bacitracin reduced P-glycoprotein expression by 45% in the liver, but did not alter expression in the duodenum. Intraperitoneal injection of E. coli lipopolysaccharide, a model for acute inflammation, rapidly increased expression of Pgp protein in the liver (∼2-fold). Expression then declines to pre-induction levels by 24 h. Similar responses were observed in the spleen and kidney but not the duodenum. These results confirm the presence of an avian P-glycoprotein homologue and suggest that dietary constituents regulate the expression of P-glycoprotein. Changes in P-glycoprotein expression may represent an important physiological response to foods containing toxins and an important component of the acute phase immune response.