Multidrug Resistance Protein Inhibitor Research Articles

MRP1-Dependent Extracellular Release of Glutathione Induces Cardiomyocyte Ferroptosis After Ischemia-Reperfusion.

The membrane components of cardiomyocytes are rich in polyunsaturated fatty acids, which are easily oxidized. Thus, an efficient glutathione-based lipid redox system is essential for maintaining cellular functions. However, the relationship between disruption of the redox system during ischemia-reperfusion (IR), oxidized lipid production, and consequent cell death (ferroptosis) remains unclear. We investigated the mechanisms underlying the disruption of the glutathione-mediated reduction system related to ferroptosis during IR and developed intervention strategies to suppress ferroptosis. In vivo fluctuations of both intra- and extracellular metabolite levels during IR were explored via microdialysis and tissue metabolome analysis. Oxidized phosphatidylcholines were assessed using liquid chromatography high-resolution mass spectrometry. The areas at risk following IR were assessed using triphenyl-tetrazolium chloride/Evans blue stain. Metabolomic analysis combined with microdialysis revealed a significant release of glutathione from the ischemic region into extracellular spaces during ischemia and after reperfusion. The release of glutathione into extracellular spaces and a concomitant decrease in intracellular glutathione concentrations were also observed during anoxia-reperfusion in an in vitro cardiomyocyte model. This extracellular glutathione release was prevented by chemical inhibition or genetic suppression of glutathione transporters, mainly MRP1 (multidrug resistance protein 1). Treatment with MRP1 inhibitor reduced the intracellular reactive oxygen species levels and lipid peroxidation, thereby inhibiting cell death. Subsequent in vivo evaluation of endogenously oxidized phospholipids following IR demonstrated the involvement of ferroptosis, as levels of multiple oxidized phosphatidylcholines were significantly elevated in the ischemic region 12 hours after reperfusion. Inhibition of the MRP1 transporter also alleviated intracellular glutathione depletion in vivo and significantly reduced the generation of oxidized phosphatidylcholines. Administration of MRP1 inhibitors significantly attenuated infarct size after IR injury. Glutathione was released continuously during IR, primarily in an MRP1-dependent manner, and induced ferroptosis. Suppression of glutathione release attenuated ferroptosis and reduced myocardial infarct size following IR.

Torsemide increases arsenic concentrations by inhibition of multidrug resistance protein 4 in arsenic trioxide treated acute promyelocytic leukemia patients

Torsemide is commonly used to relieve edema during the treatment of acute promyelocytic leukemia (APL) with arsenic trioxide (ATO). We explored the effect of torsemide on the plasma concentrations of inorganic arsenic (iAs), monomethylarsonic acid (MMAV) and dimethyarsinic acid (DMAV) in APL patients treated with ATO and clarified its molecular mechanism in rats and cells. The study included 146 APL patients treated with ATO. 60（41.1 %) of these 146 patients were co-administered with torsemide. The treatment of torsemide increased plasma concentrations of iAs (P < 0.05) and DMAV (P < 0.05) in APL patients. The single co-administration of ATO and torsemide in rats significantly increased the plasma concentrations and AUC(0−t) of iAs (P < 0.05) and MMAV (P < 0.05), decreased the urinary excretion rates and the urine concentrations of iAs (P < 0.05) and DMAV (P < 0.05), and enhanced iAs (P < 0.05) and MMAV (P < 0.05) concentrations in the kidneys of rats. In addition, torsemide decreased the expression of multidrug resistance protein 4 (MRP4) in rat kidneys after 7 days of continuous co-administration (P < 0.05). We also treated MRP4-overexpressing HEK293T cells with ATO and different concentrations of torsemide. Torsemide markedly increased the concentrations of iAs, MMAV and DMAV by inhibiting MRP4 compared with ATO alone (P < 0.05). In conclusion, torsemide increased the plasma concentrations of arsenic metabolites in APL patients treated with ATO by inhibiting the transporter MRP4 in a dose-dependent manner.

Inhibition of CD73 expression or A2AR blockade reduces MRP1 expression and increases the sensitivity of cervical cancer cells to cisplatin.

Recently, a link between the biological activity of CD73 in solid tumors and multidrug resistance protein (MRP) has been proposed. Cisplatin (CP) is the most widely used anticancer agent to treat advanced and recurrent cervical cancer (CC). However, multidrug resistance protein-1 (MRP1) is overexpressed in approximately 85% of these tumors and has been strongly associated with cisplatin resistance (CPR). In this study, we examine the involvement of CD73 and the interaction of adenosine (ADO) with its receptors (ARs) in MRP1 expression in CC cells. We found that ADO positively modulates MRP1 expression in CC cells in a dose-dependent manner. The inhibition of CD73 expression with a CD73-targeted siRNA and A2AR blockade with the selective antagonist ZM241385 significantly decreased MRP1 expression and the extrusive capacity of CC cells, making them significantly more sensitive to CP treatment than cancer cells treated with MK-751, a specific MRP1 inhibitor. These results suggest CD73 inhibition or blocking ADO signaling through A2AR could be strategies to reverse CPR in patients with advanced or recurrent CC, which is characterized by very low response rates to CP (10%-20%).

Co-administration of MDR1 and BCRP or EGFR/PI3K inhibitors overcomes lenvatinib resistance in hepatocellular carcinoma.

Lenvatinib is the first-line treatment for hepatocellular carcinoma (HCC), the most common type of primary liver cancer; however, some patients become refractory to lenvatinib. The underlying mechanism of lenvatinib resistance (LR) in patients with advanced HCC remains unclear. We focused on exploring the potential mechanism of LR and novel treatments of lenvatinib-resistant HCC. In particular, we established a Huh7 LR cell line and performed in vitro, bioinformatic, and biochemical assays. Additionally, we used a Huh7-LR cell-derived xenograft mouse model to confirm the results in vivo. Following LR induction, multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) transporters were markedly upregulated, and the epidermal growth factor receptor (EGFR), MEK/ERK, and PI3K/AKT pathways were activated. In vitro, the co-administration of elacridar, a dual MDR1 and BCRP inhibitor, with lenvatinib inhibited proliferation and induced apoptosis of LR cells. These effects might be due to inhibiting cancer stem-like cells (CSCs) properties, by decreasing colony formation and downregulating CD133, EpCAM, SOX-9, and c-Myc expression. Moreover, the co-administration of gefitinib, an EGFR inhibitor, with lenvatinib retarded proliferation and induced apoptosis of LR cells. These similar effects might be caused by the inhibition of EGFR-mediated MEK/ERK and PI3K/AKT pathway activation. In vivo, co-administration of lenvatinib with elacridar or gefitinib suppressed tumour growth and angiogenesis. Therefore, inhibiting MDR1 and BCRP transporters or targeting the EGFR/PI3K pathway might overcome LR in HCC. Notably, lenvatinib should be used to treat HCC after LR induction owing to its role in inhibiting tumour proliferation and angiogenesis. Our findings could help develop novel and effective treatment strategies for HCC.

Design and synthesis of versatile GSTP1-specific fluorogenic substrates for the highly sensitive detection of GSTP1 activity in living cells

ABSTRACT: Pi-class glutathione S-transferase (GSTP1) is a detoxification enzyme that is highly expressed in various types of cancer cells and is a promising target for cancer imaging and therapy. Ps-TAc, an acetylated derivative of the GSTP1-specific fluorogenic substrate Ps-TG, is attracting attention as an effective GSTP1 fluorescent probe, and has been successfully used to visualize intracellular GSTP1 activity. Ps-TAc is a prodrug type fluorescent probe in which the phenolic hydroxyl group of Ps-TG is acetylated and thus is susceptible to nonspecific hydrolysis, potentially compromising its ability to detect GSTP1 activity. Here, we describe the development of a highly selective fluorogenic GSTP1 substrate that is membrane permeable and does not involve esterification and show its application to live-cell imaging and FACS analysis. We designed and synthesized several compounds with benzylsulfone substituents instead of the mesyl group of Ps-TG and tested their fluorescence activation by GSTP1 catalysis in vitro and in cellulo. Of the test compounds, Ps-TG3 was the most suitable for the visualization of intracellular GSTP1 activity because the signal from living cells increased significantly when MK-571, an inhibitor of multidrug resistance proteins (MRPs), was simultaneously loaded. The results obtained by co-loading Ps-TG3 and MK571 into GSTP1-nonexpressing cells suggest that Ps-TG3 can be a substrate for MRPs. The usefulness of Ps-TG3 was demonstrated by fluorescence imaging of several cancer cell cultures and FACS analysis of lymphoma cells. The results presented here suggest that Ps-TG3, in combination with MK571, is useful for visualizing and detecting intracellular GSTP1 activity in cancer cells that highly express GSTP1.

Inorganic mercury induces liver oxidative stress injury in quails by inhibiting Akt/Nrf2 signal pathway

Mercury is widely used in human life and production. Excessive mercury can accumulate in livestock and poultry through air, drinking water, and food, which causes a serious threat to natural environment and human food safety. However, the exact mechanism of hepatotoxicity caused by mercuric chloride (HgCl2) is still unclear. The purpose of this study is to explore the mechanism of HgCl2 poisoning in the liver. Based on the above background, 28 quails were randomly and equally divided into 4 groups to establish a hepatotoxicity model by oral administration of HgCl2 once daily (0 mg/kg, 2 mg/kg, 4 mg/kg, and 8 mg/kg, respectively). The experiment period was 84 d. The assay results of blood routine and biochemical indexes showed liver injury in quails. Further research found that mRNA and protein levels of nuclear factor-erythroid related factor 2 (Nrf2) and its key promoter protein kinase B (Akt) were significantly decreased, suggesting HgCl2 treatment led to liver oxidative stress injury in a dose-dependent manner by inhibiting the antioxidant signal pathway Akt/Nrf2. Besides, the increase of nuclear factor-κB and tumor necrosis factor-alpha levels as well as the activation of tumor protein 53 indicated the occurrence of inflammation and apoptosis. Moreover, the inhibition of multidrug resistance protein 2 (Mrp2) which participates in HgCl2-mediated toxicant metabolism resulted in toxicity accumulation. Thus, a vicious circle was formed to further aggravate the liver injury. In conclusion, our results demonstrated that HgCl2 leads to liver inflammation, apoptosis, and detoxification dysfunction by inhibiting the antioxidant pathway Akt/Nrf2. Among them, Akt and Mrp2 may be effective therapeutic targets for the treatment of HgCl2-triggered hepatotoxicity.

Abstract 1404: Loss of adenomatous polyposis coli induces DOX resistance through upregulation of ABC transporters

Abstract Chemoresistance is a leading cause of breast cancer related deaths. Therefore, understanding the molecular basis for chemoresistance is essential for novel therapeutic advancement improving patient outcome. The Adenomatous Polyposis Coli (APC) tumor suppressor is lost in up to 70% of sporadic breast cancer; however, little is known about how APC loss contributes to chemoresistance. Using mammary tumor cells isolated from the ApcMin/+ mouse crossed to the Polyoma middle T antigen (PyMT) transgenic model, we were the first to show that APC loss decreased doxorubicin (DOX) induced apoptosis. Therefore, we examined the mechanisms contributing to DOX resistance with APC loss to identify combination therapy options. We previously made the novel observation that APC loss in MMTV-PyMT;ApcMin/+ cells activated signal transducer and activator of transcription 3 (STAT3) thereby increasing the expression of the drug efflux pump, multidrug resistance protein 1 (MDR1). ATP-binding cassette (ABC) transporters, such as MDR1, are one of the most well-known contributors to drug resistance. These ABC transporters are capable of exporting drugs from the cell preventing apoptosis. Decreased intracellular DOX accumulation was observed in APC-deficient cells suggesting enhanced drug efflux. To investigate this decreased intracellular DOX accumulation, we measured the expression and activity of the drug export pumps, MDR1 and multidrug resistance protein 1 (MRP1). APC-deficient cells also have increased expression of MRP1. We used a calcein incorporation assay and demonstrated that APC loss increased MDR1 activity, which was restored by an MDR1 inhibitor. In addition, MDR1 inhibition sensitized the MMTV-PyMT;ApcMin/+ cells to DOX-mediated apoptosis. MRP1 inhibition through genetic manipulation will be used to assess MRP1 activity using the calcein AM assay and its contribution to DOX resistance. MDR1 and MRP1 inhibition will also be used to restore intracellular DOX accumulation. Future studies including evaluating human tumor protein lysates will for APC, MDR1, and MRP1 expression. Correlation studies will be used to determine whether loss of APC correlates with either high expression of MDR1 or MRP1. Taken together, APC loss mediates DOX resistance via exporting DOX demonstrating the potential use of combination therapy to overcome chemoresistance. Citation Format: Casey D. Stefanski, Jenifer R. Prosperi, Lauren Milac. Loss of adenomatous polyposis coli induces DOX resistance through upregulation of ABC transporters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1404.

Abrogating Treatment Resistance By Mir-451 in FLT3-ITD-Positive Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a fatal malignant hematologic disorder. Although many patients with AML initially respond to standard chemotherapy and achieve complete remission (CR), the majority of patients relapse due to the development and clonal expansion of chemoresistant subclones. Albeit being a heterogenous disease, individual mutations are still scarcely considered in the current treatment regimen.Approximately 30% of AML patients exhibit gain-of-function mutations in the FMS-like tyrosine kinase 3 receptor (FLT3-receptor) gene. Here, an internal tandem duplication (ITD) in the juxtamembrane region of the receptor is among the most frequently observed mutations. This usually entails a constitutive activation of FLT3 receptor downstream signaling pathways, leading to differential gene expression patterns and aberrant cell growth. Clinically, FLT3-ITD mutations confer adverse prognosis. Thus, many promising FLT3 signaling inhibitor-based therapies are currently utilized to improve patient survival. However, the phenomenon of treatment resistant subclones persists and constantly calls for new angles of treatment.MicroRNAs, a group of small non-coding RNAs, regulate gene expression on a posttranscriptional level by a complementary binding to 3'UTRs of protein-coding mRNAs. They have been shown to influence healthy and malignant hematopoiesis and to predict treatment response in cancer. In addition, several candidates have been connected to treatment resistance or sensitivity in AML.Within this study, we hypothesized that microRNAs are biological important players in the adverse treatment response and the development of chemoresistance in FLT3-ITD associated AML.Initially, we were interested in the microRNA expression pattern in FLT3-ITD associated AML. Therefore, we performed a next generation sequencing screen in bone marrow mononuclear cells from healthy donors, AML patients with FLT3-wildtype and AML patients with FLT3-ITD mutation. Here, we identified miR-451 to be significantly lower expressed in AML compared to healthy individuals. Furthermore, it was nearly absent in FLT3-ITD associated AML. In addition, we compared our results to the AML patient cohort of “The Cancer Genome Atlas” (TCGA) and found a similar miR-451 expression distribution. In agreement with this, miR-451 expression increased during differentiation of primary human and mouse hematopoietic cells. Moreover, transduction of U937 as well as 32D cells with FLT3-ITD led to a decrease in miR-451 expression.Downregulation of miR-451 has been shown for various cancerous diseases to correlate with an increased cancer persistence as well as recurrence, as one established target of miR-451 is multi drug resistance protein 1 (MDR1). MDR1 is an ATP dependent efflux pump that mediates chemoresistance by transporting drugs out of the cell. Today, only limited data are available on the miR-451-MDR1 axis in FLT3-ITD associated AML. Thus, we hypothesized that low miR-451 expression and high MDR1 activity is associated with treatment resistance.Therefore, we showed an inverse correlation of miR-451 expression and MDR1 protein levels in various leukemic cells lines. Furthermore, we observed that a repression of FLT3-ITD signaling by both Quizartinib and Gilteritinib causes a miR-451 expression increment and a subsequent reduction of MDR1 protein levels. By luciferase activity assay, we could demonstrate a direct binding of miR-451 to the 3'UTR of MDR1. In order to investigate whether miR-451 alone is able to abrogate treatment resistance, we overexpressed miR-451 in both normal MV4;11 and treatment resistant MV4;11 cells (MV4;11-R). Subsequently, MDR1 protein levels were decreased and cells exhibited an enhanced sensitivity towards both FLT3-ITD inhibitor and chemotherapeutic agent-based treatment. Moreover, exogenous miR-451 was able to restore drug sensitivity in MV4;11-R cells comparable to that of normal MV4;11 cells. As proof of principle, MDR1 inhibitor Tariquidar mimicked miR-451-mediated effects and sensitized MV4;11-R cells to FLT3-ITD inhibitor Quizartinib.In conclusion, we identified a novel mechanism by which FLT3-ITD is able to alter treatment response and confer a poor prognosis. Hence, not only inhibiting FLT3-ITD but mimicking the effects of negatively regulated downstream targets such as miR-451 presents as promising new avenue for FLT3-ITD AML treatment. DisclosuresThiede:Bayer: Consultancy, Speakers Bureau; Roche: Consultancy; Novartis: Consultancy, Speakers Bureau; Agendix: Employment.

Cilostazol: a Review of Basic Mechanisms and Clinical Uses.

Primarily used in the treatment of intermittent claudication, cilostazol is a 2-oxyquinolone derivative that works through the inhibition of phosphodiesterase III and related increases in cyclic adenosine monophosphate (cAMP) levels. However, cilostazol has been implicated in a number of other basic pathways including the inhibition of adenosine reuptake, the inhibition of multidrug resistance protein 4, among others. It has been observed to exhibit antiplatelet, antiproliferative, vasodilatory, and ischemic-reperfusion protective properties. As such, cilostazol has been investigated for clinical use in a variety of settings including intermittent claudication, as an adjunctive for reduction of restenosis after coronary and peripheral endovascular interventions, and in the prevention of secondary stroke, although its widespread implementation for indications other than intermittent claudication has been limited by relatively modest effect sizes and lack of studies in western populations. In this review, we highlight the pleiotropic effects of cilostazol and the evidence for its clinical use.

Clinical MDR1 inhibitors enhance Smac-mimetic bioavailability to kill murine LSCs and improve survival in AML models

AbstractThe specific targeting of inhibitor of apoptosis (IAP) proteins by Smac-mimetic (SM) drugs, such as birinapant, has been tested in clinical trials of acute myeloid leukemia (AML) and certain solid cancers. Despite their promising safety profile, SMs have had variable and limited success. Using a library of more than 5700 bioactive compounds, we screened for approaches that could sensitize AML cells to birinapant and identified multidrug resistance protein 1 inhibitors (MDR1i) as a class of clinically approved drugs that can enhance the efficacy of SM therapy. Genetic or pharmacological inhibition of MDR1 increased intracellular levels of birinapant and sensitized AML cells from leukemia murine models, human leukemia cell lines, and primary AML samples to killing by birinapant. The combination of clinical MDR1 and IAP inhibitors was well tolerated in vivo and more effective against leukemic cells, compared with normal hematopoietic progenitors. Importantly, birinapant combined with third-generation MDR1i effectively killed murine leukemic stem cells (LSCs) and prolonged survival of AML-burdened mice, suggesting a therapeutic opportunity for AML. This study identified a drug combination strategy that, by efficiently killing LSCs, may have the potential to improve outcomes in patients with AML.

Combinatorial Treatment of Birinapant and Zosuquidar Enhances Effective Control of HBV Replication In Vivo.

Chronic hepatitis B virus (HBV) infection remains a global health threat and affects hundreds of millions worldwide. Small molecule compounds that mimic natural antagonists of inhibitor of apoptosis (IAP) proteins, known as Smac-mimetics (second mitochondria-derived activator of caspases-mimetics), can promote the death of HBV-replicating liver cells and promote clearance of infection in preclinical models of HBV infection. The Smac-mimetic birinapant is a substrate of the multidrug resistance protein 1 (MDR1) efflux pump, and therefore inhibitors of MDR1 increase intracellular concentration of birinapant in MDR1 expressing cells. Liver cells are known to express MDR1 and other drug pump proteins. In this study, we investigated whether combining the clinical drugs, birinapant and the MDR1 inhibitor zosuquidar, increases the efficacy of birinapant in killing HBV expressing liver cells. We showed that this combination treatment is well tolerated and, compared to birinapant single agent, was more efficient at inducing death of HBV-positive liver cells and improving HBV-DNA and HBV surface antigen (HBsAg) control kinetics in an immunocompetent mouse model of HBV infection. Thus, this study identifies a novel and safe combinatorial treatment strategy to potentiate substantial reduction of HBV replication using an IAP antagonist.

MRP4 over-expression has a role on both reducing nitric oxide-dependent antiplatelet effect and enhancing ADP induced platelet activation

The impact of inhibition of multidrug resistance protein 4 (MRP4) on nitric oxide (NO) resistance and on ADP-induced platelet aggregation is unknown. The aim of this investigation was to verify whether platelet NO resistance correlates with MRP4 expression and evaluate whether this can be reduced by in vitro MRP4 inhibition mediated by cilostazol. Moreover, we assessed if inhibition of MRP4-mediated transport reduces ADP-induced platelet reactivity. The inhibitory effect of sodium nitroprusside (SNP), a NO-donor that enhances cyclic guanosine monophosphate (cGMP) cytosolic concentration, was assessed in platelets obtained from aspirin treated patients and in a control population. The inhibitory effect of SNP was evaluated by ADP-induced aggregation in SNP-treated platelets. The impact of MRP4 on ADP-induced platelet aggregation was performed in high on aspirin residual platelet reactivity (HARPR) patients and compared to healthy volunteers (HV), and a control cohort (CTR). In aspirin-treated patients with high levels of MRP4, reduced SNP inhibition was found compared to those with low levels of MRP4. MRP4 inhibition by cilostazol significantly reduced ADP-induced platelet aggregation in HARPR population, and to a lesser extent in HV and CTR populations. In conclusion, cilostazol can mitigate the hyper-reactive platelet phenotype of HARPR patients by reducing residual ADP-induced platelet aggregation and increasing NO-dependent endothelial antiplatelet effects.

Characterization of the intestinal absorption of morroniside from Cornus officinalis Sieb. et Zucc via a Caco-2 cell monolayer model.

Morroniside is a biologically active polyphenol found in Cornus officinalis Sieb. et Zucc (CO) that exhibits a broad spectrum of pharmacological activities, such as protecting nerves, and preventing diabetic liver damage and renal damage. However, little data are available regarding the mechanism of its intestinal absorption. Here, an in vitro human intestinal epithelial cell model of cultured Caco-2 cells was applied to study the absorption and transport of morroniside. The effects of donor concentration, pH and inhibitors were investigated. The bidirectional permeability of morroniside from the apical (AP) to the basolateral (BL) side and in the reverse direction was studied. When administered at three tested concentrations (5, 25 and 100 μM), the apparent permeability coefficient (Papp) values in the AP-to-BL direction ranged from 1.59 × 10−6 to 2.66 × 10−6 cm/s. In the reverse direction, BL-to-AP, the value was ranged from 2.67 × 10−6 to 4.10 × 10−6 cm/s. The data indicated that morroniside transport was pH-dependent. The permeability of morroniside was affected by treatment with various inhibitors, such as multidrug resistance protein inhibitors MK571 and indomethacin, as well as the breast cancer resistance protein inhibitor apigenin. The mechanisms of the intestinal absorption of morroniside may involve multiple transport pathways, such as the passive diffusion and efflux protein-mediated active transport especially involving multidrug resistance protein 2 and breast cancer resistance protein. After the addition of CO, the Papp values in the AP-to-BL direction increased significantly, therefore, it can be assumed that some ingredients in the CO promote morroniside absorption in the small intestine.

MK-571, a Cysteinyl Leukotriene Receptor 1 Antagonist, Inhibits Hepatitis C Virus Replication.

The quinoline MK-571 is the most commonly used inhibitor of multidrug resistance protein-1 (MRP-1) but was originally developed as a cysteinyl leukotriene receptor 1 (CysLTR1) antagonist. While studying the modulatory effect of MRP-1 on anti-hepatitis C virus (HCV) direct-acting antiviral (DAA) efficiency, we observed an unexpected anti-HCV effect of compound MK-571 alone. This anti-HCV activity was characterized in Huh7.5 cells stably harboring a subgenomic genotype 1b replicon. A dose-dependent decrease of HCV RNA levels was observed upon MK-571 administration, with a 50% effective concentration (EC50 ± standard deviation) of 9 ± 0.3 μM and a maximum HCV RNA level reduction of approximatively 1 log10 MK-571 also reduced the replication of the HCV full-length J6/JFH1 model in a dose-dependent manner. However, probenecid and apigenin homodimer (APN), two specific inhibitors of MRP-1, had no effect on HCV replication. In contrast, the CysLTR1 antagonist SR2640 increased HCV-subgenomic replicon (SGR) RNA levels in a dose-dependent manner, with a maximum increase of 10-fold. In addition, a combination of natural CysLTR1 agonist (LTD4) or antagonists (zafirlukast, cinalukast, and SR2640) with MK-571 completely reversed its antiviral effect, suggesting its anti-HCV activity is related to CysLTR1 rather to MRP-1 inhibition. In conclusion, we showed that MK-571 inhibits HCV replication in hepatoma cell cultures by acting as a CysLTR1 receptor antagonist, thus unraveling a new host-virus interaction in the HCV life cycle.

Aloe-emodin induces hepatotoxicity by the inhibition of multidrug resistance protein 2

BackgroundAloe-emodin (AE) is among the primary bioactive anthraquinones present in traditional Chinese medicinal plants such as Rheum palmatum L. Multidrug resistance protein 2 (ABCC2/ MRP2) is an important efflux transporter of substances associated with cellular oxidative stress. However, the effects of traditional Chinese medicine on this protein remain unclear. PurposeThe aim of this research is to study the role of ABCC2 in AE-induced hepatotoxicity. MethodsThe expression of ABCC2 protein and mRNA levels were analyzed by Western-Blotting and qRT-PCR, respectively. The intracellular oxidative stress caused by AE was evaluated by quantifying the levels of intracellular reactive oxygen species, malondialdehyde, glutathione reduced and oxidized glutathione. The levels of adenosine triphosphate, mitochondrial membrane potential and mitochondrial DNA were explored to evaluate the effects of AE on mitochondrial function. The effects of AE on cell apoptosis and cell cycle were detected by flow cytometry. To further clarify the key role of ABCC2 in AE induced cytotoxicity, we used pCI-neo-ABCC2 plasmid to over express ABCC2 protein, and small interfering RNA was used to knockdown ABCC2 in HepG2 cells. Additionally, we investigated the impact of AE on ABCC2 degradation pathway and the hepatotoxic effects of AE in mice. ResultsAE was found to inhibit ABCC2 transport activity, downregulate ABCC2 expression and altered intracellular redox balance. Induction of oxidative stress resulted in depletion of intracellular glutathione reduced, mitochondria dysfunction and activation of apoptosis. ABCC2 overexpression significantly reduced AE-induced intracellular oxidative stress and cell death, which was enhanced by ABCC2 knockdown. Furthermore, AE was observed to promote ABCC2 degradation through induction of autophagy and hepatotoxicity was induced in mice by promoting ABCC2 degradation. ConclusionsThe inhibition of ABCC2 is a novel effect of AE that triggers oxidative stress and apoptosis. These findings are helpful in understanding the toxicological effects of AE-containing medicinal plants.

Association of ABC Transporter With Resistance to FK866, a NAMPT Inhibitor, in Human Colorectal Cancer Cells.

Nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the NAD+ biosynthetic pathway, is a drug target of potent anticancer candidates, including FK866 and other reported NAMPT inhibitors. However, it is known that NAMPT point-mutations render resistance to specific NAMPT inhibitors in several cancer cells. We investigated the resistance mechanisms of NAMPT inhibitor FK866 in human colorectal cancer (CRC) cells. We used CRC human cell line HCT116 to determine the expression profiles of FK866-sensitive parental HCT116 cells and FK866-resistant HCT116 (HCT116RFK866) cells by DNA microarray analysis. The levels of multidrug resistance protein 1 (MDR1) were assessed via western blot. In addition, we analyzed the sensitivity of FK866 in parental HCT116 cells and HCT116RFK866 cells by co-treatment with MDR1 inhibitor verapamil. Our results revealed an association between ATP-binding cassette (ABC) transporter gene ABCB1 and resistance to NAMPT inhibitor FK866 in both HCT116RFK866 cells and parental HCT116 cells. The expression of ABCB1, which encodes MDR1, was lower in HCT116RFK866 cells than in parental HCT116 cells. Furthermore, the protein level of MDR1/ATP-binding cassette sub-family B member 1 (ABCB1) was 0.5-fold lower in HCT116RFK866 cells than in parental HCT116 cells. Additionally, HCT116RFK866 cells showed improved sensitivity to FK866 when co-treated with verapamil, an ABCB1 inhibitor. Interestingly, the efficacy of FK866 in parental HCT116 cells was the same for the treatment with FK866 alone or in combination with verapamil. The change in expression of ABCB1 plays a key role in CRC drug resistance to NAMPT inhibitor FK866. This suggests that the MDR1/ABCB1 mechanism may regulate the resistance of anticancer NAMPT inhibitor FK866.

Expanded Physiologically-Based Pharmacokinetic Model of Rifampicin for Predicting Interactions With Drugs and an Endogenous Biomarker via Complex Mechanisms Including Organic Anion Transporting Polypeptide 1B Induction.

As rifampicin can cause the induction and inhibition of multiple metabolizing enzymes and transporters, it has been challenging to accurately predict the complex drug–drug interactions (DDIs). We previously constructed a physiologically‐based pharmacokinetic (PBPK) model of rifampicin accounting for the components for the induction of cytochrome P450 (CYP) 3A/CYP2C9 and the inhibition of organic anion transporting polypeptide 1B (OATP1B). This study aimed to expand and verify the PBPK model for rifampicin by incorporating additional components for the induction of OATP1B and CYP2C8 and the inhibition of multidrug resistance protein 2. The established PBPK model was capable of accurately predicting complex rifampicin‐induced alterations in the profiles of glibenclamide, repaglinide, and coproporphyrin I (an endogenous biomarker of OATP1B activities) with various dosing regimens. Our comprehensive rifampicin PBPK model may enable quantitative prediction of DDIs across diverse potential victim drugs and endogenous biomarkers handled by multiple metabolizing enzymes and transporters.

TARGETING MULTIDRUG RESISTANCE PROTEIN 1 (MDR1) POTENTIATES SMAC-MIMETIC THERAPY TO KILL LEUKEMIC STEM CELLS AND OVERCOME RESISTANCE IN ACUTE MYELOID LEUKEMIA

Acute myeloid leukemia (AML) is an aggressive disease with a current 5-year survival rate of only ∼25%, therefore development of effective treatments towards resistant AML is urgently needed. Potential mechanisms of therapy resistance include overexpression of members of the inhibitor of apoptosis protein (IAP) family. Natural IAP antagonists such as second-mitochondria-derived activator of caspases (Smac) exist, leading to the pharmaceutical development of Smac-mimetics (SMs). The clinical SM birinapant has been trialed in AML and solid cancers, as well as in hepatitis B virus (HBV), with variable and limited success. Using a high-throughput strategy, we screened 5,700 bioactive compounds and identified clinical drugs that overcome birinapant resistance through inhibition of multidrug resistance protein 1 (MDR1). We show that 3rd generation MDR1 inhibitors synergise with birinapant and other SMs to kill AMLs. We use mass spectrometry to show inhibition of MDR1 in SM resistant leukemias increased intracellular levels of birinapant, indicating birinapant is a novel substrate of MDR1. Genetic deletion of MDR1 confirmed combination synergy is mediated specifically by MDR1. Furthermore, we observed a strong correlation between MDR1 expression/activity and sensitivity to birinapant-based therapies in AML cells. Strikingly, combination treatment sensitised leukemic stem cells (LSCs) to birinapant killing and was well-tolerated and effective in treating leukemia in vivo. Moreover, MDR1 inhibitors potentiated birinapant-mediated killing in patient leukemia and ovarian cancer cells, and in HBV infected liver cells in vivo. This study identifies MDR1 as a biomarker of SM therapy and gives clear rationale for MDR1 screening, leading to informed SM clinical trials and a personalised therapy for AML patients.

ARE THE BCL2 FAMILY INHIBITORS SUBSTRATES FOR THE MULTIDRUG RESISTANCE EFFLUX PUMP MDR1?

The BH3 mimetic compounds, a novel class of targeted anti-cancer agents, are highly efficacious for the treatment of certain haematological malignancies. The BCL2 inhibitor, venetoclax, is the most advanced and has been approved for treating patients with high-risk chronic lymphocytic leukemia (CLL) in many countries. While highly effective, clinical resistance is emerging, and we anticipate that resistance to BH3 mimetic therapy will be a problem in the future. Unlike CLL, the BCL2 relative MCL1 is the key survival factor in multiple myeloma (MM) and clinical trials with inhibitors selective for MCL1 have started in patients with this disease. To anticipate what might drive resistance to MCL1 inhibition, we have undertaken studies using model systems in the laboratory. By culturing a highly sensitive MM cell line, AMO1, in gradually escalating doses of a MCL1 inhibitor, we were able to generate resistant pools of these cells. Interestingly, we found marked amplification of the multidrug resistance protein 1 (MDR1) gene in 3/4 generated resistant pools. We conducted a number of studies which validated MDR1 amplification as a potential mechanism of resistance to this class of MCL1 inhibitors. Firstly, ectopic overexpression of MDR1 in multiple hematopoietic cell lines conferred resistance. Secondly, this resistance is reversed by co-treatment with an advanced MDR1 inhibitor. Finally, we could restore sensitivity to this MCL1 inhibitor when MDR1 function was blocked in the resistant AMO1 pools with MDR1 amplification. Overall, our data provide strong evidence that MDR1 amplification could adversely impact this MCL1 inhibitor activity. We are also testing if other BH3 mimetics are substrates of MDR1. These studies are important because MDR1 amplification is a common mechanism that underpins resistance to multiple anti-cancer agents, both conventional and targeted.

Abstract 3478: APC loss mediates doxorubicin response in breast cancer via changes in intracellular drug concentration and DNA repair pathways

Abstract Chemoresistance is one of the leading causes of breast cancer related deaths. Understanding the molecular basis for chemoresistance is essential for novel therapeutic advancement to improve patient outcome. The Adenomatous Polyposis Coli (APC) tumor suppressor is either mutated or hypermethylated in up to 70% of sporadic breast cancer; however, little is known about how APC loss contributes to chemoresistance. Using mammary tumor cells isolated from the ApcMin/+ mouse crossed to the Polyoma middle T antigen (PyMT) transgenic model, we demonstrated that APC loss decreased doxorubicin (DOX) induced apoptosis. DOX, a commonly used chemotherapeutic in breast cancer, inhibits topoisomerase IIa, resulting in double stranded DNA breaks, resulting in cell cycle arrest to allow repair or apoptosis. We made the novel observation that APC loss in MMTV-PyMT;ApcMin/+ cells activated signal transducer and activator of transcription 3 (STAT3) thereby increasing the expression of the drug efflux pump, multidrug resistance protein 1 (MDR1). Therefore, we hypothesized that APC loss prevents doxorubicin-mediated cell death through: 1) reduced intracellular DOX and 2) alterations in DNA damage and repair. To investigate the intracellular accumulation of DOX, we first used calcein incorporation assay and demonstrated that APC loss increased MDR1 activity, which was restored by an MDR1 inhibitor. In addition, MDR1 inhibition sensitized the MMTV-PyMT;ApcMin/+ cells to DOX-mediated apoptosis. To investigate the effect of APC loss on DNA damage repair pathways, we initially monitored damage recognition pathways after 24-hour DOX treatment. The MMTV-PyMT;ApcMin/+ cells exhibited decreased γH2AX and ataxia-telangiectasia mutated (ATM) phosphorylation following DOX treatment compared to controls, suggesting decreased DNA damage either through decreased intracellular DOX or enhanced DNA repair. Decreased phosphorylation of Chk1 and Chk2 was also observed in DOX-treated MMTV-PyMT;ApcMin/+ cells. Preliminary data to investigate if this decreased DNA damage was due to increased DNA damage repair, via monitoring γH2AX expression throughout treatment and recovery from DOX, suggest enhanced DNA repair in MMTV-PyMT;ApcMin/+ cells. Using the ATM inhibitor, we observed an increase in DOX sensitivity in MMTV-PyMT;ApcMin/+ cells. Alternatively, ATR inhibition did not affect DOX-mediated apoptosis. We will further investigate the efficiency of repair pathways through reporter plasmids and radiation induced damage to separate the drug transport alterations. Finally, we will test whether combination therapy of doxorubicin with an MDR1 inhibitor will reduce tumor burden in vivo. Taken together, APC loss mediates DOX resistance via reducing intracellular DOX and increasing DNA damage repair demonstrating the potential use of combination therapy to overcome chemoresistance. Citation Format: Casey Stefanski, Kaitlyn Keffler, Stephanie McClintock, Lauren Milac, Jenifer Prosperi. APC loss mediates doxorubicin response in breast cancer via changes in intracellular drug concentration and DNA repair pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3478.