In vitro function and in situ localization of Multidrug Resistance-associated Protein (MRP)1 (ABCC1) suggest a protective role against methyl mercury-induced oxidative stress in the human placenta.

Methyl mercury (MeHg) is an organic highly toxic compound that is transported efficiently via the human placenta. Our previous data suggest that MeHg is taken up into placental cells by amino acid transporters while mercury export from placental cells mainly involves ATP binding cassette (ABC) transporters. We hypothesized that the ABC transporter multidrug resistance-associated protein (MRP)1 (ABCC1) plays an essential role in mercury export from the human placenta. Transwell transport studies with MRP1-overexpressing Madin-Darby Canine Kidney (MDCK)II cells confirmed the function of MRP1 in polarized mercury efflux. Consistent with this, siRNA-mediated MRP1 gene knockdown in the human placental cell line HTR-8/SVneo resulted in intracellular mercury accumulation, which was associated with reduced cell viability, accompanied by increased cytotoxicity, apoptosis, and oxidative stress as determined via the glutathione (GSH) status. In addition, the many sources claiming different localization of MRP1 in the placenta required a re-evaluation of its localization in placental tissue sections by immunofluorescence microscopy using an MRP1-specific antibody that was validated in-house. Taken together, our results show that (1) MRP1 preferentially mediates apical-to-basolateral mercury transport in epithelial cells, (2) MRP1 regulates the GSH status of placental cells, (3) MRP1 function has a decisive influence on the viability of placental cells exposed to low MeHg concentrations, and (4) the in situ localization of MRP1 corresponds to mercury transport from maternal circulation to the placenta and fetus. We conclude that MRP1 protects placental cells from MeHg-induced oxidative stress by exporting the toxic metal and by maintaining the placental cells' GSH status in equilibrium.

The C-type ATP-Binding Cassette Transporter OsABCC7 Is Involved in the Root-to-Shoot Translocation of Arsenic in Rice.

Rice is a major dietary source of inorganic arsenic (As), a nonthreshold carcinogen. Reducing As accumulation in rice grain is of critical importance for food safety. In the present study, we investigated the role of a member of the rice C-type ATP-binding cassette (ABC) transporter (OsABCC) family, OsABCC7, in arsenite [As(III)] accumulation in rice. Quantitative real-time RT-PCR showed that OsABCC7 was expressed intensively in the roots and the expression was strongly suppressed by As(III) exposure. Transgenic rice plants expressing OsABCC7 Promoter-GUS (ß-glucuronidase) suggest that the gene was predominantly expressed in the xylem parenchyma cells in the stele region of the primary and lateral roots. Transient expression of OsABCC7: GFP fusion protein in Nicotiana benthamiana leaf cells showed that the protein was localized at the plasma membrane. When expressed in Xenopus laevis oocytes, OsABCC7 showed an efflux activity for As(III)-phytochelatin and As(III)-glutathione complexes, but not for As(III). Knockout of OsABCC7 in rice significantly decreased As concentration in the xylem sap and As concentration in the shoots, but had little effect on root As concentration. Taken together, our results indicate that OsABCC7 is involved in the root-to-shoot translocation of As(III).

Dual-Targeting of miR-124-3p and ABCC4 Promotes Sensitivity to Adriamycin in Breast Cancer Cells.

AIMS: Increasing evidence links the abnormal expression of microRNAs and ATP-binding cassette subfamily C member 4 (ABCC4) with tumor development and progression, as well as with chemoresistance. Our aims were to determine the therapeutic potential of targeting both miR-124-3p and ABCC4 in breast cancer cells and to determine if duel targeting increased their sensitivity to chemotherapeutic drugs, in vitro. MATERIALS AND METHODS: The expression of the ABCC4 protein and miR-124-3p were detected, respectively, by immunohistochemical staining and quantitative real-time polymerase chain reaction in breast cancer tumor tissue, MCF-7 and MCF-7-ADR cell lines. Suppression of ABCC4 expression and miR-124-3p overexpression were performed in MCF-7-ADR cell lines. Western blot assays were used to detect expression of ABCC4 and permeability glycoprotein 1/multi-drug resistance protein 1 (P-gp) in cells. Cell Counting Kit-8, flow cytometry, transwell, and scratch assays were conducted to detect cell proliferation, cell cycle, invasion, and migration of cells. RESULTS: We found that ABCC4 protein expression was significantly increased, while the miR-124-3p level was significantly decreased in breast cancer tissue and cell lines. Tumor size and clinical tumor node metastasis stage were significantly correlated with elevated expression of ABCC4 and decreased expression of miR-124-3p. Interestingly, ABCC4 expression was significantly increased in MCF-7-ADR cells, while miR-124-3p level was significantly decreased compared with MCF-7 cells. The inhibition of ABCC4 and miR-124-3p overexpression both led to a significant decrease in cell proliferation, invasion, and migration of MCF-7-ADR cells, and combination of suppression of ABCC4 with miR-124-3p overexpression had a synergistic inhibitory effect. Our results further demonstrated that inhibition of ABCC4 expression and overexpression of miR-124-3p significantly enhanced the sensitivity to adriamycin (ADR) in MCF-7-ADR cells, and that simultaneous dual-targeting of miR-124-3p and ABCC4 had a stronger promotive effect on the sensitivity to ADR in MCF-7-ADR cells. Moreover, western blot analysis showed that miR-124-3p overexpression significantly inhibited P-gp expression in MCF-7-ADR cells. CONCLUSION: Our data demonstrate that the combination of downregulation of ABCC4 with overexpression of miR-124-3p significantly increased sensitivity to ADR in MCF-7-ADR cells. This finding suggests that similar dual targeting may serve as a means to enhance therapies for drug-resistant breast cancers.

5-Oxo-hexahydroquinoline derivatives as modulators of P-gp, MRP1 and BCRP transporters to overcome multidrug resistance in cancer cells.

Multidrug resistance (MDR) in cancer cells is often associated with overexpression of ATP-binding cassette (ABC) transporters, including P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1) and breast cancer resistance protein (BCRP/ABCG2). Modulators of these transporters might be helpful in overcoming MDR. Moreover, exploiting collateral sensitivity (CS) could be another approach for efficient treatment of cancer. Twelve novel 5-oxo-hexahydroquinoline derivatives bearing different aromatic substitutions at C4, while having 2-pyridyl alkyl carboxylate substituents at the C3 were synthesized and evaluated for MDR reversal activity by flow cytometric determination of rhodamine 123, calcein and mitoxantrone accumulations in P-gp, MRP1 and BCRP-overexpressing cell lines, respectively. Furthermore, to confirm the P-gp inhibitory activity, the effect of compounds on the reduction of doxorubicin's IC50 of drug-resistant human uterine sarcoma cell line, MES-SA/DX5, was evaluated. Compounds D6, D5 and D3 (bearing 3-chlorophenyl, 2,3-dichlorophenyl and 4-chlorophenyl substituents at C4 position of 5-oxo-hexahydroquinoline core) were the most potent P-gp, MRP1 and BCRP inhibitors, respectively, causing significant MDR reversal at concentrations of 1-10 µM. Additionally, D4 (containing 3-flourophenyl) was the most effective MRP1-dependent CS inducing agent. Overall, chlorine containing compounds D6, C4 and D3 were capable of significant inhibition of all 3 important efflux pumps in cancer cells. Moreover, D6 also induced CS triggered by reducing glutathione efflux. In conclusion, some of the 5-oxo-hexahydroquinoline derivatives are effective efflux pump inhibitors capable of simultaneously blocking 3 important ABC transporters involved in MDR, and represent promising agents to overcome MDR in cancer cells.

[Role of drug transporters in rational use of drugs at high altitude area].

Pharmacokinetics plays a key role in rational use of medicines. Many factors can affect the drug's pharmacokinetics. Previous studies mainly focused on the impact of hypoxia on hepatic drug metabolizing enzyme, but uncommon on drug transporters. Actually, drug transporter is a key factor for activation of the drugs transport across the cell membrane into the inside of cells, such as multidrug resistance protein (MDR), breast cancer resistance protein (BCRP), multidrug resistance associated protein (MRP), organic cation transporter (OCT), organic anion-transporting polypeptide (OATP), organic anion transporter (OAT), qligopeptide transporter (PEPT), etc. They are widely present in the small intestine villus epithelial cells, renal tubular epithelial cells, hepatocytes and biliary epithelial cells. They play a very important role in drug absorption, distribution, metabolism and excretion. The changes in drug transporters under hypoxia in intestinal could affect the bioavailability of drugs; the changes in drug transporters in organs could affect drug's distribution, subsequent drug's indications and adverse reactions; the changes in drug transporters in liver and kidney could affect the metabolism and excretion rate of drugs, thereby the drug's residence time and half-life.

ABCC6 plays a significant role in the transport of nilotinib and dasatinib, and contributes to TKI resistance in vitro, in both cell lines and primary patient mononuclear cells.

ATP Binding Cassette family efflux proteins ABCB1 and ABCG2 have previously been demonstrated to interact with Tyrosine Kinase Inhibitors (TKIs); however, evidence for the interaction of other potentially relevant drug transporters with TKIs is lacking. Through Taqman transporter array technology we assessed the impact of nilotinib on mRNA expression of ABC transporters, with ABCC6 identified as a transporter of interest. Additionally, increased expression of ABCC6 mRNA was observed during in vitro development of nilotinib resistance in BCR-ABL1-expressing cell lines. K562 cells exposed to gradually increasing concentrations of nilotinib (to 2 µM) expressed up to 57-fold higher levels of ABCC6 mRNA when compared with control cells (p = 0.002). Analogous results were observed in nilotinib resistant K562-Dox cells (up to 33-fold higher levels of ABCC6, p = 0.002). IC50 experiments were conducted on patient mononuclear cells in the absence and presence of three ABCC6 inhibitors: indomethacin, probenecid and pantoprazole. Results demonstrated that all three inhibitors significantly reduced nilotinib IC50 (p<0.001) indicating ABCC6 is likely involved in nilotinib transport. Cell line data confirmed these findings. Similar results were obtained for dasatinib, but not imatinib. Combined, these studies suggest that nilotinib and dasatinib are likely substrates of ABCC6 and to our knowledge, this is the first report of ABCC6 involvement in TKI transport. In addition, ABCC6 overexpression may also contribute to nilotinib and dasatinib resistance in vitro. With nilotinib and dasatinib now front line therapy options in the treatment of CML, concomitant administration of ABCC6 inhibitors may present an attractive option to enhance TKI efficacy.

Suppression of the ATP-binding cassette transporter ABCC4 impairs neuroblastoma tumour growth and sensitises to irinotecan in vivo.

The ATP-binding cassette transporter ABCC4 (multidrug resistance protein 4, MRP4) mRNA level is a strong predictor of poor clinical outcome in neuroblastoma which may relate to its export of endogenous signalling molecules and chemotherapeutic agents. We sought to determine whether ABCC4 contributes to development, growth and drug response in neuroblastoma in vivo. In neuroblastoma patients, high ABCC4 protein levels were associated with reduced overall survival. Inducible knockdown of ABCC4 strongly inhibited the growth of human neuroblastoma cells in vitro and impaired the growth of neuroblastoma xenografts. Loss of Abcc4 in the Th-MYCN transgenic neuroblastoma mouse model did not impact tumour formation; however, Abcc4-null neuroblastomas were strongly sensitised to the ABCC4 substrate drug irinotecan. Our findings demonstrate a role for ABCC4 in neuroblastoma cell proliferation and chemoresistance and provide rationale for a strategy where inhibition of ABCC4 should both attenuate the growth of neuroblastoma and sensitise tumours to ABCC4 chemotherapeutic substrates.

miR-148a-mediated estrogen-induced cholestasis in intrahepatic cholestasis of pregnancy: Role of PXR/MRP3.

Intrahepatic cholestasis of pregnancy (ICP) is an idiopathic liver disease while the biochemical characteristic is the elevated level of total bile acid (TBA). The present study investigated whether miR-148a mediates the induced effect of estrogen on the development of ICP and the proper mechanism: PXR/MRP3 signal pathway. mRNA expression was detected by qPCR, protein expression was detected by western blotting, the concentration of estrogen and TBA were detected by reagent kit respectively. In the cinical research, it was found that miR-148a expression was positive related with the concentration of TBA in the serum of ICP patients. In in vitro research, estradiol (500 nmol/L, 12 h) significantly upregulated miR-148a expression and LV-148a-siRNA inhibited the function of estradiol (500 nmol/L, 48 h) on TBA secretion. In addition, gene silence of miR-148a upregulated PXR expression which was inhibited by estradiol in LO2 cells. Pretreatment of rifampin (10 µmol/L), the agonist of PXR alleviated the TBA secretion induced by estradiol (500 nmol/L, 48 h). miR-148a-siRNA and PXR had a synergistic action on TBA secretion of LO2. Both of miR-148a-siRNA and rifampin (10 µmol/L) inhibited the upregulated effect of estradiol on MRP3 expression. This research has demonstrated that miR-148a may be involved in the induction of estrogen on ICP via PXR signal pathway, and MRP3 may be involved.

Multiple blood-brain barrier transport mechanisms limit bumetanide accumulation, and therapeutic potential, in the mammalian brain.

There is accumulating evidence that bumetanide, which has been used over decades as a potent loop diuretic, also exerts effects on brain disorders, including autism, neonatal seizures, and epilepsy, which are not related to its effects on the kidney but rather mediated by inhibition of the neuronal Na-K-Cl cotransporter isoform NKCC1. However, following systemic administration, brain levels of bumetanide are typically below those needed to inhibit NKCC1, which critically limits its clinical use for treating brain disorders. Recently, active efflux transport at the blood-brain barrier (BBB) has been suggested as a process involved in the low brain:plasma ratio of bumetanide, but it is presently not clear which transporters are involved. Understanding the processes explaining the poor brain penetration of bumetanide is needed for developing strategies to improve the brain delivery of this drug. In the present study, we administered probenecid and more selective inhibitors of active transport carriers at the BBB directly into the brain of mice to minimize the contribution of peripheral effects on the brain penetration of bumetanide. Furthermore, in vitro experiments with mouse organic anion transporter 3 (Oat3)-overexpressing Chinese hamster ovary cells were performed to study the interaction of bumetanide, bumetanide derivatives, and several known inhibitors of Oats on Oat3-mediated transport. The in vivo experiments demonstrated that the uptake and efflux of bumetanide at the BBB is much more complex than previously thought. It seems that both restricted passive diffusion and active efflux transport, mediated by Oat3 but also organic anion-transporting polypeptide (Oatp) Oatp1a4 and multidrug resistance protein 4 explain the extremely low brain concentrations that are achieved after systemic administration of bumetanide, limiting the use of this drug for targeting abnormal expression of neuronal NKCC1 in brain diseases.

Functional Rescue of ABCC6 Deficiency by 4-Phenylbutyrate Therapy Reduces Dystrophic Calcification in Abcc6-/- Mice.

Soft-tissue calcification is associated with aging, common conditions such as diabetes or hypercholesterolemia, and with certain genetic disorders. ABCC6 is an efflux transporter primarily expressed in liver facilitating the release of adenosine triphosphate from hepatocytes. Within the liver vasculature, adenosine triphosphate is converted into pyrophosphate, a major inhibitor of ectopic calcification. ABCC6 mutations thus lead to reduced plasma pyrophosphate levels, resulting in the calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Most mutations in ABCC6 are missense, and many preserve transport activity but are retained intracellularly. We have previously shown that the chemical chaperone 4-phenylbutyrate (4-PBA) promotes the maturation of ABCC6 mutants to the plasma membrane. In a humanized mouse model of pseudoxanthoma elasticum, we investigated whether 4-PBA treatments could rescue the calcification inhibition potential of selected ABCC6 mutants. We used the dystrophic cardiac calcification phenotype of Abcc6-/- mice as an indicator of ABCC6 function to quantify the effect of 4-PBA on human ABCC6 mutants transiently expressed in the liver. We showed that 4-PBA administrations restored the physiological function of ABCC6 mutants, resulting in enhanced calcification inhibition. This study identifies 4-PBA treatment as a promising strategy for allele-specific therapy of ABCC6-associated calcification disorders.

Triple negative breast cancer development can be selectively suppressed by sustaining an elevated level of cellular cyclic AMP through simultaneously blocking its efflux and decomposition.

Triple negative breast cancer (TNBC) has the highest mortality among all breast cancer types and lack of targeted therapy is a key factor contributing to its high mortality rate. In this study, we show that 8-bromo-cAMP, a cyclic adenosine monophosphate (cAMP) analog at high concentration (> 1 mM) selectively suppresses TNBC cell growth. However, commonly-used cAMP-elevating agents such as adenylyl cyclase activator forskolin and pan phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) are ineffective. Inability of cAMP elevating agents to inhibit TNBC cell growth is due to rapid diminution of cellular cAMP through efflux and decomposition. By performing bioinformatics analyses with publically available gene expression datasets from breast cancer patients/established breast cancer cell lines and further validating using specific inhibitors/siRNAs, we reveal that multidrug resistance-associated protein 1/4 (MRP1/4) mediate rapid cAMP efflux while members PDE4 subfamily facilitate cAMP decomposition. When cAMP clearance is prevented by specific inhibitors, forskolin blocks TNBC's in vitro cell growth by arresting cell cycle at G1/S phase. Importantly, cocktail of forskolin, MRP inhibitor probenecid and PDE4 inhibitor rolipram suppresses TNBC in vivo tumor development. This study suggests that a TNBC-targeted therapeutic strategy can be developed by sustaining an elevated level of cAMP through simultaneously blocking its efflux and decomposition.

A Nitric Oxide Storage and Transport System That Protects Activated Macrophages from Endogenous Nitric Oxide Cytotoxicity.

Nitric oxide (NO) is integral to macrophage cytotoxicity against tumors due to its ability to induce iron release from cancer cells. However, the mechanism for how activated macrophages protect themselves from endogenous NO remains unknown. We previously demonstrated by using tumor cells that glutathione S-transferase P1 (GSTP1) sequesters NO as dinitrosyl-dithiol iron complexes (DNICs) and inhibits NO-mediated iron release from cells via the transporter multidrug resistance protein 1 (MRP1/ABCC1). These prior studies also showed that MRP1 and GSTP1 protect tumor cells against NO cytotoxicity, which parallels their roles in defending cancer cells from cytotoxic drugs. Considering this, and because GSTP1 and MRP1 are up-regulated during macrophage activation, this investigation examined whether this NO storage/transport system protects macrophages against endogenous NO cytotoxicity in two well characterized macrophage cell types (J774 and RAW 264.7). MRP1 expression markedly increased upon macrophage activation, and the role of MRP1 in NO-induced 59Fe release was demonstrated by Mrp1 siRNA and the MRP1 inhibitor, MK571, which inhibited NO-mediated iron efflux. Furthermore, Mrp1 silencing increased DNIC accumulation in macrophages, indicating a role for MRP1 in transporting DNICs out of cells. In addition, macrophage 59Fe release was enhanced by silencing Gstp1, suggesting GSTP1 was responsible for DNIC binding/storage. Viability studies demonstrated that GSTP1 and MRP1 protect activated macrophages from NO cytotoxicity. This was confirmed by silencing nuclear factor-erythroid 2-related factor 2 (Nrf2), which decreased MRP1 and GSTP1 expression, concomitant with reduced 59Fe release and macrophage survival. Together, these results demonstrate a mechanism by which macrophages protect themselves against NO cytotoxicity.

Silencing of histone deacetylase 2 suppresses malignancy for proliferation, migration, and invasion of glioblastoma cells and enhances temozolomide sensitivity.

Histone deacetylases (HDACs) can regulate the progression of various cancers, while their roles in glioblastoma multiforme (GBM) are not well known. Our present study investigated the expression of class I HDACs (HDAC1, 2, 3, 8) in GBM U87, A172, U251, and LN229 cells and compared their levels with that in primary normal human astrocytes (NHA) cells. It showed that HDAC2 expression is significantly up-regulated in GBM cells. Silencing of HDAC2 via its specific siRNAs can suppress the in vitro proliferation, migration, and invasion of GBM U87 and A172 cells. Furthermore, silencing of HDAC2 can increase the sensitivity of GBM cells to temozolomide (TMZ), a standard-of-care during clinical GBM treatment. This might be due to that si-HDAC can significantly down-regulate the mRNA and protein expression of MRP1, while has no effect on ABCB1 and ABCG2. Schisandrin B (Sch B), a specific inhibitor of MRP1, can further increase the TMZ sensitivity in HDAC2-knocked down GBM cells. Collectively, our data revealed that targeted HDAC2 can suppress the malignancy of GBM cells and increase their sensitivity of TMZ via down-regulation of MRP1. It suggested that HDAC2 might be a potential target for GBM therapy and improvement in TMZ therapy efficiency.

Mechanisms of tumor cell resistance to the current targeted-therapy agents.

Resistance to chemotherapy agents is a major challenge infront of cancer patient treatment and researchers. It is known that several factors, such as multidrug resistance proteins and ATP-binding cassette families, are cell membrane transporters that can efflux several substrates such as chemotherapy agents from the cell cytoplasm. To reduce the adverse effects of chemotherapy agents, various targeted-based cancer therapy (TBCT) agents have been developed. TBCT has revolutionized cancer treatment, and several agents have shown more specific effects on tumor cells than chemotherapies. Small molecule inhibitors and monoclonal antibodies are specific agents that mostly target tumor cells but have low side effects on normal cells. Although these agents have been very useful for cancer treatment, however, the presence of natural and acquired resistance has blunted the advantages of targeted therapies. Therefore, development of new options might be necessary. A better understanding of tumor cell resistance mechanisms to current treatment agents may provide an appropriate platform for developing and improving new treatment modalities. Therefore, in this review, different mechanisms of tumor cell resistance to chemotherapy drugs and current targeted therapies have been described.

ATP-binding cassette sub-family C member 4 (ABCC4) is overexpressed in human NK/T-cell lymphoma and regulates chemotherapy sensitivity: Potential as a functional therapeutic target.

Nasal-type natural killer/T-cell (NK/T-cell) lymphomas are subtypes of non-Hodgkin's lymphoma (NHL), which are typically more clinically aggressive. There is, however relatively little understanding of nasal-type NK/T-cell lymphoma molecular pathogenesis. Thus, in this study we applied RNA sequencing to systematically screen for altered gene expression in human NK/T-cell lymphoma cell lines YTS and SNK-6 versus normal NK cells. We found that ATP-binding cassette sub-family C Member 4 (ABCC4) levels were significantly upregulated both in human NK/T-cell lymphoma YTS and SNK-6 cells, as compared with normal NK cells. These expression levels were further confirmed by real-time PCR. Protein levels of ABCC4 were also significantly higher in YTS and SNK-6 cells as compared with normal NK cells. Clinically relevant, ABCC4 expression levels were significantly higher in human NK/T-cell lymphoma tissues as compared with control nasal mucosa tissues, confirmed by immunohistochemical staining. In addition, we explored the biological function of such ABCC4 upregulation. Overexpression of ABCC4 by lentivirus transfection induced chemotherapy resistance to epirubicin (EPI) and cisplatin (DDP) in YTS cells. In contrast, knockdown of ABCC4 expression by shRNA contributed to chemotherapy sensitivity by both EPI and DDP. Furthermore, overexpression of ABCC4 inhibited, while downregulation of ABCC4 increased, YTS cell apoptosis following treatment by EPI or DDP. Therefore, the present study identified ABCC4 to be overexpressed in human NK/T-cell lymphoma cells, to regulate chemotherapy sensitivity to EPI and DDP, and possibly to be a functional therapeutic target. These findings may provide a basic rationale for new approaches in the effort to develop anti-tumor therapeutics for NK/T-cell lymphoma.

Neochamaejasmin B increases the bioavailability of chamaechromone coexisting in Stellera chamaejasme L. via inhibition of MRP2 and BCRP.

Chamaechromone and neochamaejasmin B (NCB) are the most abundant components in the dried roots of the toxic perennial herb Stellera chamaejasme L. and have pharmacological activities. The objective of this study was to investigate the transport mechanism of these two components in vivo and in vitro. The transport and cellular accumulation studies in Madin-Darby canine kidney (MDCK) cells overexpressing human multidrug resistance protein 2 (MRP2) or P-gp and LLC-PK1 cells overexpressing human breast cancer resistance protein (BCRP) were performed. The results showed that chamaechromone was a good substrate of MRP2 and BCRP but not a substrate of P-gp. NCB was found to be a MRP2 inhibitor in transfected cells and significantly enhanced the cellular accumulation of chamaechromone in MDCK cells overexpressing MRP2. Similar results were obtained in LLC-PK1-BCRP cells. In addition, the influence of NCB on the bioavailability of chamaechromone following their co-administration was also determined in rats. The results showed that the area under the plasma concentration-time curve and maximal plasma concentration of chamaechromone in rats were increased by 48.9% and 81.9%, respectively. The mechanism of improving the oral bioavailability of chamaechromone was attributable to the inhibition of the BCRP and MRP2-mediated efflux of chamaechromone by NCB.

Impaired platelet activation and cAMP homeostasis in MRP4-deficient mice.

Molecules that reduce the level of cyclic adenosine 5'-monophosphate (cAMP) in the platelet cytosol, such as adenosine 5'-diphosphate (ADP) secreted from dense granules, trigger platelet activation. Therefore, any change in the distribution and/or availability of cyclic nucleotides or ADP may interfere with platelet reactivity. In this study, we evaluated the role of multidrug resistance protein 4 (MRP4, or ABCC4), a nucleotide transporter, in platelet functions in vivo and in vitro by investigating MRP4-deficient mice. MRP4 deletion resulted in a slight increase in platelet count but had no impact on platelet ultrastructure. In MRP4-deficient mice, the arterial occlusion was delayed and the tail bleeding time was prolonged. In a model of platelet depletion and transfusion mimicking a platelet-specific knockout, mice injected with MRP4(-/-) platelets also showed a significant increase in blood loss compared with mice injected with wild-type platelets. Defective thrombus formation and platelet activation were confirmed in vitro by studying platelet adhesion to collagen in flow conditions, integrin αIIbß3 activation, washed platelet secretion, and aggregation induced by low concentrations of proteinase-activated receptor 4-activating peptide, U46619, or ADP. We found no role of MRP4 in ADP dense-granule storage, but MRP4 redistributed cAMP from the cytosol to dense granules, as confirmed by increased vasodilator-stimulated phosphoprotein phosphorylation in MRP4-deficient platelets. These data suggest that MRP4 promotes platelet aggregation by modulating the cAMP-protein kinase A signaling pathway, suggesting that MRP4 might serve as a target for novel antiplatelet agents.

Natural product modulators to overcome multidrug resistance in cancer.

Multidrug resistance (MDR) is a condition that makes cells simultaneously unresponsive to different drugs, unrelated to their chemical structure and mechanism of action. MDR caused by the presence and overexpression of ABC transporters makes obstacles in cancer treatment and lower the effectiveness of chemotherapy. Natural products are investigated by many researchers as MDR modulators for their low toxicity and potent, selective behavior. When coadministered, MDR modulators compete with cytotoxic agents for binding to the active site of the membrane transporters and reduce drug efflux. Natural product-based drugs are important in struggling against drug resistance during cancer therapy. This review is focused on the potential mechanisms against drug resistance, the development of inhibitors for ABC drug transporters, natural product modulators, and nanoparticle drug delivery.

Overexpression of cyclic adenosine monophosphate effluent protein MRP4 induces an altered response to ß-adrenergic stimulation in the senescent rat heart.

BACKGROUND: In the senescent heart, the positive inotropic response to ß-adrenoceptor stimulation is reduced, partly by dysregulation of ß1- and ß3-adrenoceptors. The multidrug resistance protein 4 (MRP4) takes part in the control of intracellular cyclic adenosine monophosphate concentration by controlling its efflux but the role of MRP4 in the ß-adrenergic dysfunction of the senescent heart remains unknown. METHODS: The ß-adrenergic responses to isoproterenol were investigated in vivo (stress echocardiography) and in vitro (isolated cardiomyocyte by Ionoptix with sarcomere shortening and calcium transient) in young (3 months old) and senescent (24 months old) rats pretreated or not with MK571, a specific MRP4 inhibitor. MRP4 was quantified in left ventricular homogenates by Western blotting. Data are mean ± SD expressed as percent of baseline value. RESULTS: The positive inotropic effect of isoproterenol was reduced in senescent rats in vivo (left ventricular shortening fraction 120 ± 16% vs. 158 ± 20%, P < 0.001, n = 16 rats) and in vitro (sarcomere shortening 129 ± 37% vs. 148 ± 35%, P = 0.004, n = 41 or 43 cells) as compared to young rats. MRP4 expression increased 3.6-fold in senescent compared to young rat myocardium (P = 0.012, n = 8 rats per group). In senescent rats, inhibition of MRP4 by MK571 restored the positive inotropic effect of isoproterenol in vivo (143 ± 11%, n = 8 rats). In vitro in senescent cardiomyocytes pretreated with MK571, both sarcomere shortening (161 ± 45% vs. 129 ± 37%, P = 0.007, n = 41 cells per group) and calcium transient amplitude (132 ± 25% vs. 113 ± 27%, P = 0.007) increased significantly. CONCLUSION: MRP4 overexpression contributes to the reduction of the positive inotropic response to ß-adrenoceptor stimulation in the senescent heart.

A Drosophila ABC transporter regulates lifespan.

MRP4 (multidrug resistance-associated protein 4) is a member of the MRP/ABCC subfamily of ATP-binding cassette (ABC) transporters that are essential for many cellular processes requiring the transport of substrates across cell membranes. Although MRP4 has been implicated as a detoxification protein by transport of structurally diverse endogenous and xenobiotic compounds, including antivirus and anticancer drugs, that usually induce oxidative stress in cells, its in vivo biological function remains unknown. In this study, we investigate the biological functions of a Drosophila homolog of human MRP4, dMRP4. We show that dMRP4 expression is elevated in response to oxidative stress (paraquat, hydrogen peroxide and hyperoxia) in Drosophila. Flies lacking dMRP4 have a shortened lifespan under both oxidative and normal conditions. Overexpression of dMRP4, on the other hand, is sufficient to increase oxidative stress resistance and extend lifespan. By genetic manipulations, we demonstrate that dMRP4 is required for JNK (c-Jun NH2-terminal kinase) activation during paraquat challenge and for basal transcription of some JNK target genes under normal condition. We show that impaired JNK signaling is an important cause for major defects associated with dMRP4 mutations, suggesting that dMRP4 regulates lifespan by modulating the expression of a set of genes related to both oxidative resistance and aging, at least in part, through JNK signaling.