MRP1 Expression Research Articles

Lung resistance-related protein (LRP) predicts favorable therapeutic outcome in Acute Myeloid Leukemia

There is conflicting evidence that MDR1, MRP2 and LRP expression is responsible for chemotherapy resistance. We conducted this study to explore their role in AML therapy outcomes. Bone marrow and peripheral blood samples of 90 AML patients, receiving chemotherapy, were analyzed by real time PCR. Gene expression was calculated by the 2−ΔΔCt method. The patients who had a persistent remission were labelled ‘Good Responder’ (GRes) whereas, those with relapse or drug resistance were labelled ‘Poor Responders’ (PRes). Higher LRP expression in bone marrow, but not in peripheral blood, was positively associated with persistent remission (p = 0.001), GRes (p = 0.002), 1-year overall as well as disease-free survival (p = 0.02 and p = 0.007, respectively). Marrow and blood MDR1 and MRP2 expression did not differ significantly between the above groups. Logistic regression analysis showed that only a diagnosis of acute promyelocytic leukemia (APL; M3) or high marrow LRP expression significantly predicted a favorable therapeutic outcome. This is the first report showing that high bone marrow LRP expression predicts significant favorable therapeutic outcome. Peripheral blood LRP expression as well as marrow and blood MDR1 and MRP2 expression have no predictive value in AML patients treated with standard dose cytarabine and daunorubicin 3+7 regimen.

Evaluation of the Mrp2-mediated flavonoid-drug interaction potential of quercetin in rats and in vitro models

Quercetin is a biologically active flavonoid that has been used as a popular health supplement. It is reported that quercetin may cause flavonoid-drug interaction mediated by P-glycoprotein, the most predominant efflux transporter. In this study, we comprehensively evaluated the potential of the pharmacokinetic interaction of quercetin mediated by multidrug resistance-associated protein 2 (MRP2), another major efflux transporter. MRP2-transfected MDCKII cells and LS174T cells were used to evaluate the potential inhibition and induction of MRP2 by quercetin in vitro. To evaluate the induction effect of quercetin on Mrp2 in vivo, Mrp2 mRNA expression in rat liver, kidney, and small intestinal tissues was determined after the oral administration of quercetin (50, 100, or 250 mg/kg) for seven days. Mrp2-mediated interaction potential was also evaluated by the pharmacokinetic study of phenolsulfonphthalein in rats after single or multiple doses of quercetin. Additionally, the effect of quercetin on absorption of docetaxel, a P-glycoprotein and CYP3A4 substrate, was also evaluated. Quercetin inhibited the function of MRP2 at 10 µM and induced the mRNA expression of MRP2 at 50 µM in vitro. Additionally, at 100 mg/kg, quercetin markedly increased Mrp2 expression in the small intestine of rats. However, there was no significant change in phenolsulfonphthalein pharmacokinetics due to single- (50, 100, or 250 mg/kg) or multiple-dose (50, 100, or 250 mg/kg for seven days) quercetin co-administration. By contrast, a significant interaction caused by quercetin (100 mg/kg) was observed in the absorption of docetaxel. The results suggested that although quercetin modulates the function and expression of MRP2 in vitro, it may have a low potential of Mrp2-mediated interaction and present negligible safety concerns related to the interaction.

The Circadian Clock Gene Bmal1 Controls Intestinal Exporter MRP2 and Drug Disposition

The intestinal exporter MRP2 plays an important role in disposition and elimination of a wide range of drugs. Here, we aimed to clarify the impact of circadian clock on intestinal MRP2, and to determine the molecular mechanisms for generation of diurnal MRP2 expression.Methods: The regulatory effects of Bmal1 on intestinal MRP2 expression were assessed using intestine-specific Bmal1 knockout (Bmal1iKO) mice and colon cancer cells. The relative mRNA and protein levels were determined by qPCR and Western blotting, respectively. Everted gut sac, cell viability and in situ intestinal perfusion experiments were performed to evaluate intestinal efflux of the MRP2 substrate methotrexate (MTX). Toxicity and pharmacokinetic experiments were performed with Bmal1iKO mice and control littermates (Bmal1fl/fl mice) after oral gavage of MTX. Transcriptional gene regulation was investigated using luciferase reporter, mobility shift and chromatin immunoprecipitation (ChIP) assays.Results: Bmal1iKO mice were generated by inter-crossing the mice carrying a Bmal1 exon 8 floxed allele (Bmal1fl/fl) with Villin-Cre mice. Intestinal MRP2 expression exhibited a diurnal oscillation in Bmal1fl/fl mice with a zenith value at ZT6. Bmal1 ablation caused reductions in Mrp2 mRNA and protein levels [as well as in transport activity (measured by MTX)], and blunted their diurnal rhythms. Intestinal ablation of Bmal1 abrogated circadian time-dependency of MTX pharmacokinetics and toxicity. Bmal1/BMAL1 regulation of rhythmic Mrp2/MRP2 expression was also confirmed in the colon cancer CT26 and Caco-2 cells. Based on a combination of luciferase reporter, mobility shift and ChIP assays, we found that Dbp activated and E4bp4 repressed Mrp2 transcription via specific binding to a same D-box (-100/-89 bp) element in promoter region. Further, Bmal1 directly activated the transcription of Dbp and Rev-erbα through the E-boxes, whereas it negatively regulated E4bp4 via the transcriptional repressor Rev-erbα. Positive regulation of Mrp2 by Rev-erbα was also observed, and attained through modulation of E4bp4.Conclusion: Bmal1 coordinates temporal expressions of DBP (a MRP2 activator), REV-ERBα (an E4BP4 repressor) and E4BP4 (a MRP2 repressor), generating diurnal MRP2 expression.

Hepatoprotective Effect of S-ademetionine in the treatment of Intrahepatic Cholestasis through Farnesoid X Receptor Mechanism in Rats

S-ademetionine (AdoMet) had been extensively studied in several clinical studies to treat Intrahepatic Cholestasis (IHC). The exact mechanism of action of AdoMet to regulator bile acid is unknown. Farnesoid X Receptor (FXR), a multifunctional nuclear receptor, plays vital roles in metabolism of bile acid. This study aims to investigate the mechanism of action of S-ademetionine in FXR to treat IHC. The IHC Sprague Dawley (SD) rat model was developed by administering Alpha Naphthyl Isothiocyanate (ANIT, 50 mg/kg). FXR excitant GW4064 (3 mg/kg) was used as a control to observe the impact of AdoMet (60 mg/kg) on the changes in serum direct levels in these rats. The hepatic histology between the HE staining contrast groups was performed using the Q-PCR to test FXR and the in situ hybridization was used to test the changes of bile salt export pump (Bsep), multidrug resistance-associated protein 2 (Mrp2), and Na+-taurocholate cotransporting polypeptide (Ntcp). AdoMet significantly reduced the serum direct levels and the hepatic tissue damage. AdoMet can also increase the expression of FXR, Bsep, Mrp2 and Ntcp. These findings confirm that AdoMet could reduce serum direct levels as well as improving hepatic tissue damage by enhancing the expression of FXR, Bsep, Mrp2 and Ntcp. These findings could also explain the great therapeutically effect of AdoMet in intrahepatic cholestasis.

Circadian Clock Gene Bmal1 Regulates Bilirubin Detoxification: A Potential Mechanism of Feedback Control of Hyperbilirubinemia.

Controlling bilirubin to a low level is necessary in physiology because of its severe neurotoxicity. Therefore, it is of great interest to understand the regulatory mechanisms for bilirubin homeostasis. In this study, we uncover a critical role for circadian clock in regulation of bilirubin detoxification and homeostasis.Methods: The mRNA and protein levels of Bmal1 (a core clock gene), metabolic enzymes and transporters were measured by qPCR and Western blotting, respectively. Luciferase reporter, mobility shift and chromatin immunoprecipitation were used to investigate transcriptional gene regulation. Experimental hyperbilirubinemia was induced by injection of bilirubin or phenylhydrazine. Unconjugated bilirubin (UCB) and conjugated bilirubin were assessed by ELISA.Results: We first demonstrated diurnal variations in plasma UCB levels and in main bilirubin-detoxifying genes Ugt1a1 and Mrp2. Of note, the circadian UCB levels were antiphase to the circadian expressions of Ugt1a1 and Mrp2. Bmal1 ablation abrogated the circadian rhythms of UCB and bilirubin-induced hepatotoxicity in mice. Bmal1 ablation also decreased mRNA and protein expressions of both Ugt1a1 and Mrp2 in mouse livers, and blunted their circadian rhythms. A combination of luciferase reporter, mobility shift, and chromatin immunoprecipitation assays revealed that Bmal1 trans-activated Ugt1a1 and Mrp2 through specific binding to the E-boxes in the promoter region. Further, Bmal1 ablation caused a loss of circadian time-dependency in bilirubin clearance and sensitized mice to chemical induced-hyperbilirubinemia. Moreover, bilirubin stimulated Bmal1 expression through antagonism of Rev-erbα, constituting a feedback mechanism in bilirubin detoxification.Conclusion: These data supported a dual role for circadian clock in regulation of bilirubin detoxification, generating circadian variations in bilirubin level via direct transactivation of detoxifying genes Ugt1a1 and Mrp2, and defending the body against hyperbilirubinemia via Rev-erbα antagonism. Thereby, our study provided a potential mechanism for management of bilirubin related diseases.

The combination therapy of HIF1α inhibitor LW6 and cisplatin plays an effective role on anti-tumor function in A549 cells.

Hypoxia-inducible factor 1α (HIF1α) has been demonstrated to be involved in the resistance of various human cancer cells to chemotherapies. However, the correlation between HIF1α and the sensitivity of human non-small cell lung cancer (NSCLC) cells to cisplatin has not been illuminated. The aim of the present study was to investigate the effects of HIF1α on drug resistance in NSCLC cells. A549 cells were incubated in 21% or 0.5% O2 followed by the assessment of the level of HIF1α with qRT-PCR and western blot and ROS level by DCFH-DA assays. Effects of hypoxia or HIF1α inhibitor LW6 on the proliferation and apoptosis of A549 cells were evaluated via CCK-8 and flow cytometry assays. IC50 of A549 cells to cisplatin was determined by MTT assay. The mitochondrial membrane potential (MMP) was measured via JC-1 staining. Moreover, the expression of apoptosis related protein (Bcl-2, Bax) and drug resistance related proteins (MDR1, MRP1) were measured by western blotting. Exposure of A549 cells to 1% O2 significantly up-regulated HIF1α expression, maintained cell viability to cisplatin but decreased the ROS level, which promoted chemoresistance to cisplatin. LW6-treated A549 cells showed an increase in ROS level that blocked the hypoxia induced resistance to cisplatin and in addition, decreased expression of MDR1 and MRP1 in cisplatin-treated cells. This study revealed that hypoxia-improved cisplatin chemoresistance of NSCLC cells by regulated MDR1 and MRP1 expression via HIF1α/ROS pathway is reversed by LW6, suggesting that LW6 may act as effective sensitizer in chemotherapy for NSCLC.

Chemosensitivity and survival of non-small cell lung carcinoma patients receiving neoadjuvant therapy depend on the expression of multidrug efflux transporters

The current study aimed to evaluate the impact of neoadjuvant chemotherapy (NACT) on the expression of three major multidrug resistance efflux transporters, P-glycoprotein (P-gp/ABCB1), multidrug resistance associated protein (MRP1/ABCC1) and breast cancer resistance protein (BCRP/ABCG2) in non-small cell lung carcinoma (NSCLC) patients. Particularly, their role as molecular markers of chemosensitivity and prognosis of NSCLC patients receiving NACT was investigated. To that end, we specifically studied mRNA and protein expression of these three efflux transporters in two independent groups, each consisting of 35 NSCLC patients who did or did not receive platinum-based NACT. Alterations in the expression of MDR efflux transporters were statistically analyzed in relation to NACT status, and their associations were evaluated regarding patients’ survival. The frequency of samples with positive MRP1 expression was significantly decreased in the NACT group, regardless of the applied platinum drugs which are known to induce MRP1 expression. On the other hand, the incidence of BCRP expressing tumor specimens, doubly positive BCRP and P-gp as well as triple positive samples increased in the NACT group. Importantly, patients lacking P-gp expression had more favorable prognosis with NACT than without NACT, whereas the status of MRP1 and BCRP did not influence the patients’ survival in both investigated groups. Collectively, we show that decreased MRP1 and increased BCRP expression after NACT could determine the chemosensitivity of NSCLC following adjuvant therapy, whereas P-gp expression status could be considered a prognostic marker for NSCLC patients who can benefit from NACT treatment.

Cotargeting Plk1 and androgen receptor enhances the therapeutic sensitivity of paclitaxel-resistant prostate cancer.

Backgrounds:Despite the clinical success of taxanes, they still have limitations, such as chemoresistance. To overcome the limitations of paclitaxel, genetic alterations and targeting effects of altered genes were observed in paclitaxel-resistant cancer. Because paclitaxel-resistant cancer shows high levels of Plk1, a promising target in chemotherapy, the effectiveness of Plk1 inhibitors in paclitaxel-resistant cancer cells has been investigated.Methods:Paclitaxel-resistant cancer cells were developed by exposure of stepwise escalating levels of paclitaxel. Genetic alterations were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunoblotting. Using a cell viability assay, combined targeting effects for Plk1 and androgen receptor (AR) were determined. Clinical data were analyzed to understand the relationship between Plk1 and AR in prostate cancer patients.Results:Treatment with Plk1 inhibitors markedly reduced the expression of MDR1, MRP1, and Plk1 in the paclitaxel-resistant cancer. Among Plk1 inhibitors, genistein, recently found as a direct Plk1 inhibitor, tended to be more effective in the paclitaxel-resistant prostate cancer than the parental cancer cells, which was related to the suppression of the AR, as well as inhibition of Plk1 activity. A combination of Plk1 inhibitors and AR antagonist bicalutamide exhibited a synergistic effect in LNCaPTXR, as well as LNCaP cells, by inhibiting Plk1 and AR. Analysis of clinical data provides evidence for the relevance between Plk1 and AR in prostate cancer patients, showing that Plk1 and AR are strong predictors of poor survival rates.Conclusions:We suggest that cotargeting Plk1 and AR would be effective in advanced chemoresistant prostate cancer cells to overcome the limitations associated with paclitaxel.

Protection of Cardiomyocytes against Doxorubicin-Induced Toxicity by Drug Transporter Mediated Efflux

Doxorubicin is an anti-cancer drug used in treating a variety of malignancies. However, its major adverse effect is cardiotoxicity, which is dose dependent and can be either acute or chronic. Doxorubicin causes injury by DNA damage, formation of free reactive oxygen radicals and induction of apoptosis. Our aim is to induce expression of the multiple drug transporter gene (MRP1) in cardiomyocytes derived from human iPS cells (iPSC-CM), to determine whether this will allow cells to effectively remove doxorubicin. We generated a lentivirus vector for inducing expression of MRP1 (LV.MRP1) and validated its function in iPSC-CM by qPCR and western blot. We successfully showed increase of MRP1 mRNA and protein in transduced cells. The activity of the overexpressed MRP1 was also tested, by using an efflux assay to quantify the amount of CDFA dye exported from the cell by the transporter. We demonstrated reduced dye sequestration in cells overexpressing MRP1. We also determined the dose of doxorubicin at which cell viability is significantly reduced. Finally, we demonstrated that iPSC-CM transduced with LV.MRP1 were protected against doxorubicin, and resulted in a reduced level of apoptosis. In conclusion, we have optimised the conditions for gene delivery to human iPSC-CM in vitro. We have also shown that we can successfully over-express MRP1 protein in iPSC-CM, with functional transporter activity leading to protection against doxorubicin-induced toxicity.

The transcription factor ZEB1 promotes chemoresistance in prostate cancer cell lines.

One of the factors promoting tumoral progress is the abnormal activation of the epithelial–mesenchymal transition (EMT) program which has been associated with chemoresistance in tumoral cells. The transcription factor zinc finger E-box-binding homeobox 1 (ZEB1), a key EMT activator, has recently been related to docetaxel resistance, the main chemotherapeutic used in advanced prostate cancer treatment. The mechanisms involved in this protective effect are still unclear. In a previous work, we demonstrated that ZEB1 expression induced an EMT-like phenotype in prostate cancer cell lines. In this work, we used prostate cancer cell lines 22Rv1 and DU145 to study the effect of ZEB1 modulation on docetaxel resistance and its possible mechanisms. The results showed that ZEB1 overexpression conferred to 22Rv1 cell resistance to docetaxel while its silencing made DU145 cells more sensitive to it. Analysis of resistance markers showed no presence of ATP-binding cassette subfamily B member 1 (MDR1) and no changes in breast cancer resistance protein (BCRP) or ATP-binding cassette subfamily C member 10 (MRP7). However, a correlation between ZEB1, multidrug resistance-associated protein 1 (MRP1), and ATP-binding cassette subfamily C member 4 (MRP4) expression was observed. MRP4 inhibition, using MK571, resensitized cells with ZEB1 overexpression to docetaxel treatment. In addition, modulation of ZEB1 and subsequent change in MRP4 expression correlated with a lower apoptotic response to docetaxel, characterized by lower B-cell lymphoma 2 (Bcl2), high BCL2-associated X protein (Bax), and high active caspase 3 expression. The response to docetaxel in our model seems to be mediated mainly by activation of the apoptotic death program. Our results showed that modulation of MRP4 could be a mediator of ZEB1-related resistance to docetaxel in prostate cancer, making it a possible marker for chemotherapy response in patients who do not express MDR1.

Hepatotoxicity induced by psoralen and isopsoralen from Fructus Psoraleae: Wistar rats are more vulnerable than ICR mice

Fructus Psoraleae (FP) causes cholestatic liver injury; however, its main toxic constituents that are responsible for causing hepatotoxicity remained undetermined in previous studies. In the present study, psoralen and isopsoralen, the two main constituents of FP, were administered orally to rats (80 and 40 mg/kg, respectively) and mice (320 and 160 mg/kg, respectively) for 28 days, followed by biochemical and histopathological examinations to evaluate their hepatotoxicity. The results showed that psoralen and isopsoralen could induce the toxic reactions of liver and other organs in rats, while mice were not sensitive to these two compounds. Furthermore, the corresponding results indicated that administration of psoralen and isopsoralen repressed the expression of CYP7A1, BSEP, MRP2 and SULT2A1 and increased the expression of FXR and MRP3 in the rat liver. In summary, the toxic reactions of psoralen and isopsoralen are different in different species. In this study, multiple organ toxicity, such as cholestatic liver injury, occurs in rats, but not in mice. Psoralen and isopsoralen are the two main toxic constituents of FP. In addition, psoralen and isopsoralen cause liver injury, possibly through inhibiting bile acid excretion in the liver, leading to the accumulation of toxin in hepatocytes.

THE MODULATION OF DRUG EFFLUX TRANSPORTER BY CURCUMIN IN MCF7 BREAST CANCER CELLS AFTER REPEATED EXPOSURE OF ENDOXIFEN AND ESTRADIOL

Objective: The aim of the present study was to determine whether curcumin (CM) can prevent drug sensitivity of breast cancer (BC) cells when E andβ-E2 are administered together and whether the underlying mechanism involves modulation of drug efflux transporters.Methods: MCF7 BC cells were treated with the vehicle only, E+β-E2, or E+β-E2+CM repeatedly for 8 weeks. Afterward, the cells were harvested,counted, and isolated for total RNA extraction. Total RNA was then processed into cDNA and further processed for the determination of mRNAexpression patterns of drug efflux transporters (P-glycoprotein, BCRP, and MRP1).Results: Decreased sensitivity of BC cells was shown by the increased cell viability of MCF7 cells after 8 weeks. This condition was accompanied withincreased mRNA expression of P-glycoprotein, BCRP, and MRP1 in cells treated with E+β-E2, as compared with the vehicle only. CM, administered incombination with E+β-E2, resulted in decreased cell viability versus E and β-E2 and also decreased in mRNA expression of P-glycoprotein, BCRP, andMRP1.Conclusion: CM partially reversed the sensitivity loss of BC cells to E in the presence of β-E2 by modulating drug efflux transporters.

Non-steroidal anti-inflammatory drugs increase MRP4 expression in an endometriotic epithelial cell line in a PPARa dependent manner.

Endometriosis is a debilitating disease characterized by chronic inflammation. The transporter multidrug resistance-associated protein 4 (MRP4/ABCC4) is expressed in human endometrial tissue; it is overexpressed in ectopic endometrial tissue, and is modulated by the anti-inflammatory lipid Lipoxin A4 (LXA4). Recently, it was demonstrated that aspirin induces platelet MRP4 over-expression, through genomic modulation in megakaryocytes. Since patients with endometriosis frequently use aspirin or other non-aspirin Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), the aim of this study was to verify whether aspirin and other NSAIDs enhance MRP4 expression in 12Z human endometriotic epithelial cells and whether this was peroxisome proliferator-activated receptor alpha (PPARa) dependent. MRP4 and PPARa expression was analyzed by Q-RT-PCR using TaqMan® Master Mix and TaqMan® Assay Reagents (Life Technologies, Monza, Italy) and Western blot. In 12Z cells, aspirin and other NSAIDs enhanced MRP4 mRNA and protein expression; these treatments also induced PPARa expression. Aspirin and diclofenac-induced increases in MRP4 expression were not observed in cells where PPARa was knocked down using siRNA. NSAIDs-induced MRP4 expression was correlated with augmented PGE2 secretion, indicating functional relevance. MRP4 expression was increased in cells treated with NSAIDs and the nuclear receptor PPARa is involved. Elevated PGE2 levels in cell supernatants correlate with its increased transport by MRP4 after NSAID treatment. More importantly, we provide evidence that in endometriotic epithelial cells aspirin and non-aspirin NSAIDs treatments alter gene expression.

Effects of Red Ginseng Extract on the Pharmacokinetics and Elimination of Methotrexate via Mrp2 Regulation.

We aimed to investigate the effects of red ginseng extract (RGE) on the expression of efflux transporters and to study the pharmacokinetics of representative substrate. For this, rats received single or repeated administration of RGE (1.5 g/kg/day) for 1 and 2 weeks via oral gavage. mRNA and protein levels of multidrug resistance-associated protein2 (Mrp2), bile salt export pump (Bsep), and P-glycoprotein (P-gp) in the rat liver were measured via real-time polymerase chain reaction and Western blot analysis. Ginsenosides concentrations from the rat plasma were also monitored using a liquid chromatography–tandem mass spectrometry (LC–MS/MS) system. Plasma concentrations of ginsenoside Rb1, Rb2, Rc, and Rd following repeated administration of RGE for 1 and 2 weeks were comparable but significantly higher than those after single administration of RGE. These dosing regimens did not induce significant biochemical abnormalities in the liver, kidneys, and lipid homeostasis. In the RGE repeated oral administration groups, the mRNA and protein levels of Mrp2 significantly decreased. Accordingly, we investigated the changes in the pharmacokinetics of methotrexate, a probe substrate for Mrp2, following intravenous administration of 3 mg/kg methotrexate to rats in the RGE 1-week repeated oral administration group, compared to that in the control group. Biliary excretion, but not urinary excretion, of methotrexate decreased in the RGE repeated administration group, compared to that in the control group. Consequently, the plasma concentrations of methotrexate slightly increased in the RGE repeated administration group. In conclusion, repeated administration of RGE for 1 week resulted in a decrease in Mrp2 expression without inducing significant liver or kidney damage. Pharmacokinetic herb–drug interaction between RGE and methotrexate might occur owing to the decrease in the mRNA and protein levels of Mrp2.

Adaptive homeostasis of the vitamin D–vitamin D nuclear receptor axis in 8-methoxypsoralen-induced hepatotoxicity

8-methoxypsoralen (8-MOP) with ultraviolet A radiation therapy (PUVA) is the standard therapy for patients with psoriasis, despite the reported potential risks of 8-MOP-induced cholestatic liver injury in both humans and animals. Usually, patients with chronic cholestasis exhibit lower serum 25-hydroxy vitamin D (25(OH)D) levels. But those patients receiving PUVA for psoriasis showed an increase in serum 25(OH)D levels, probably highlighting that the vitamin D–vitamin D nuclear receptor (VD-VDR) axis play a protective role in 8-MOP-induced hepatotoxicity. The present study confirmed 8-MOP could increase serum 25(OH)D levels in conventional lighting and diet (CLD) and vitamin D deficient (VDD) Sprague-Dawley rats. Potential liver risks were also found in CLD and VDD rats after 8-MOP treatment. We proved that 8-MOP could be a potent ligand for VDR using molecular docking and luciferase report assay. Effect of 8-MOP on VDR subcellular distribution was determined using human liver cell line L02. We found 8-MOP could increase VDR protein expression in the nuclear and cytosol extracts and also total cell extracts in L02. siRNAs for VDR were used to determine the role of VDR in protecting 8-MOP-induced cholestasis and potential cellular mechanisms. The results showed 8-MOP could affect the CYP7A1, SHP and MRP3 expression via VDR, and such effects could be reversed by knockdown of VDR expression, suggesting a vital role of VDR involved in 8-MOP-regulated bile acid synthesis and transportation. In conclusion, these results revealed activation of VD–VDR axis may play a beneficial role in 8-MOP-mediated regulation of bile acid synthesis and transportation.

The effects of crocetin, extracted from saffron, in chemotherapy against the incidence of multiple drug resistance phenotype.

Objective(s):Crocetin, one of the main substances of saffron extract, has anti-cancer effects. Drug resistance proteins (e.g. MRP1 and MRP2) are important reasons for the failure of cancer therapy. We intended to investigate the efficacy of crocetin on MRP1 and MRP2 activity in human ovarian cisplatin-resistant carcinoma cell line (A2780-RCIS).Materials and Methods: The cytotoxic effect of crocetin was evaluated by the MTT assay. The efficacy of crocetin on MRP1 and MRP2 expression at mRNA level was studied by real-time RT-PCR. The effect of crocetin on the activity of MRP transporters was determined by drug efflux assay.Results:Crocetin decreased cell proliferation in the A2780 (IC50: 183±7 µM) and A2780-RCIS (IC50: 316±9 µM). Crocetin decreased the expression level of MRP1 (22±2 %) and MRP2 (48±8 %) in A2780-RCIS and inhibited MRP pumps function directly in A2780 (44±1 %) and A2780-RCIS (88±10 %) and indirectly in A2780 (32±2 %) and A2780-RCIS (48±15 %) respectively. Conclusion:Our findings showed that crocetin could quench drug resistance through modulation of MRP transporters in the drug resistant human ovarian cancer cells.

Preventive effect of Juniperus procera extract on liver injury induced by lithocholic acid

Bile acids are strong cytotoxic endogenous compounds implicated in several diseases in various organs, such as the liver, gallbladder and small and large intestines. Lithocholic acid is one such acid, produced by flora, and causes liver injury, cholestasis, and colon cancer. The present study aimed to examine the preventive effects of Juniperus procera extract on lithocholic acid‑induced liver injury in experimental mice. Forty adult male mice were divided equally into four groups. The negative control group gained free access to food and water. The second group was orally treated with 150 mg/kg of Juniperus procera extract alone, the third group was treated with 1% lithocholic acid alone and the fourth group was co-treated with 150 mg/kg of Juniperus procera extract and 1% lithocholic acid. Blood and hepatic tissues were collected and assayed for biochemical, molecular and histopathological changes. Lithocholic acid toxicity shows a significant increase in the serum levels of the liver function parameters, which were prevented via the Juniperus procera co-administration. Furthermore, lithocholic acid significantly downregulates the mRNA expression of ABCG8, OATP2, SULT2A, CAR, FXR, CYP2B10, MRP2 and UGT1A, and Juniperus procera prevented this effect. Histopathological investigations of the hepatic tissues showed that lithocholic acid exhibited severe hepatotoxicity, with areas of irregularly distributed necrosis with inflammatory infiltration. Juniperus procera co-treated group showed a slight change in the hepatic tissue, diminished necrotic areas, and inflammatory infiltration. In conclusion, this study clarified the preventive effect of Juniperus procera extract administration on hepatotoxicity induced by lithocholic acid exposure in experimental mice.

Octadecyloxyethyl Adefovir Exhibits Potent in vitro and in vivo Cytotoxic Activity and Has Synergistic Effects with Ara-C in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) continues to be a deadly disease, with only 50–70% of patients achieving complete remission and less than 30% of adults having sustained long-term remissions. In order to address these unmet medical needs, we carried out a high-throughput screen of an in-house library of on- and off-patent drugs with the OCI/AML-2 cell line. Through this screen, we discovered adefovir dipi­voxil (adefovir-DP) as being active against human AML. In addition to adefovir-DP, there are second-generation formulations of adefovir, including octadecyloxyethyl adefovir (ODE-adefovir) and hexadecyloxypropyl adefovir (HDP-adefovir), which were designed to overcome the pharmacokinetic problems of the parent compound adefovir. Given the known clinical benefit of nucleoside analogs for the treatment of AML, we undertook studies to evaluate the potential benefit of adefovir-based molecules. In AML cell lines and patient samples, adefovir-DP and ODE-adefovir were highly potent, whereas HDP-adefovir was significantly less active. Interestingly, ODE-adefovir was remarkably less toxic than adefovir-DP towards normal hematopoietic cells. In addition, ODE-adefovir at a dose of 15 mg/kg/day showed potent activity against human AML in a NOD/SCID mouse model, with a reduction of human leukemia in mouse bone marrow of > 40% in all mice tested within 20 days of treatment. Based on its chemical structure, we hypothesized that the cytotoxicity of ODE-adefovir toward AML was through cell cycle arrest and DNA damage. Indeed, ODE-adefovir treatment induced cell cycle arrest in the S phase and increased levels of pH2Ax, indicating the induction of DNA damage. Furthermore, there was an increase in phospho-p53, transactivation of proapoptotic genes and activation of the intrinsic apoptotic pathway. Subsequent investigation unveiled strong synergism between ODE-adefovir and ara-C, making their coadministration of potential clinical benefit. Expression of MRP4, a nucleoside transporter, appeared to influence the response of AML cells to ODE-adefovir, as its inhibition potentiated ODE-adefovir killing. Taken together, our findings indicate that clinical development of ODE-adefovir or related compounds for the treatment of AML is warranted.

The Protective effect of oleanolic acid on acute cholestatic liver injury in rats

Objective The protective effect of oleanolic acid on acute cholestatic liver injury in rats. Methods Thirty male SD rats were randomly divided into 3 groups ( n= 10 per group): sham group, bile duct ligated (BDL) group, and bile duct ligated with oleanolic acid (BDL+OA ) group. After 7 days, liver samples in all rats were collected. Expressions of bile acids pump and nuclear receptors at mRNA and protein levels were detected by RT- qPCR and Western blotting. Results At mRNA level, the expression of Mrp4 and Oatp1 expression in the BDL and BDL+OA groups were increased as compared with that in the sham group. The expression of Mrp4 increased 1.8 times in the BDL group and increased 2.3 times in the BDL+OA group (P<0.05), but the expression of Oatp1 was not statistically significant; AhR was increased 1.7-fold in the BDL group and 2.8 times in the BDL+OA group, Nrf2 was increased 1.5-fold in the BDL group and 2.1 times in the BDL+OA group with statistically significant difference. At the protein level, in the BDL group, Mrp4 expression increased 1.3 times, Oatp1 expression increased 1.5 times, AhR expression increased 1.3 times, Nrf2 expression increased 1.4 fold; in the BDL+OA group, Mrp4 expression increased 1.8 fold, AhR expression increased 1.9 fold, with statistical significance between the two groups. Oatp1 expression increased 1.4-fold in the BDL+OA group as compared with the BDL group showing no statistical significance. Conclusions Oleanolic acid stimulates the hepatic expression of bile acids pump Mrp4 associated with the activation of nuclear receptors AhR and Nrf2 in acute bile duct-ligated rats. Key words: Oleanolic acid; Cholestasis; MRP4; Nuclear receptors

OATPB1/B3 and MRP3 expression in hepatocellular adenoma predicts Gd-EOB-DTPA uptake and correlates with risk of malignancy.

Hepatobiliary phase (HBP) Gd-EOB-DTPA-enhanced magnetic resonance imaging (MRI) has increased the accuracy in differentiating focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA). However, the ability of this technique to distinguish HCA subtypes remains controversial. The aim of this study was to investigate the expression of hepatocyte transporters (OATPB1/B3, MRP2, MRP3) in HCA subtypes, hence to understand their MRI signal intensity on HBP Gd-EOB-DTPA-enhanced MRI. By means of immunohistochemistry (IHC), we scored the expression of OATPB1/B3, MRP2 and MRP3, in resected specimens of FNH (n=40), subtyped HCA (n=58) and HCA with focal malignant transformation (HCA-HCC, n=4). Results were validated on a supplementary set of FNH (n=6), subtyped HCA (n=17) and HCA-HCC (n=1) with Gd-EOB-DTPA MR images. All FNH showed a preserved expression of hepatocytes transporters. Beta-catenin-activated HCA (at highest risk of malignant transformation) and HCA-HCC were characterized by preserved/increased OATPB1/B3 expression (predictor of hyperintensity on HBP), as opposed to other HCA subtypes (P<0.01) that mostly showed OATPB1/B3 absence (predictor of hypointensity on HBP). HCA-HCC showed an additional MRP3 overexpressed profile (P<0.01). On HBP Gd-EOB-DTPA-enhanced MRI, FNH and HCA signal intensity reflected the profile predicted by their specific OATPB1/B3 tissue expression. The hyperintense vs hypointense HBP signal criterion was able to distinguish all higher risk HCA and HCA-HCC (100% accuracy). OATPB1/B3 and MRP3 IHC and signal intensity on HBP Gd-EOB-DTPA-enhanced MRI can help to stratify HCA according to their risk of malignant transformation.