MRP1 mRNA Research Articles

Drug-resistance in doxorubicin-resistant FL5.12 hematopoietic cells: elevated MDR1, drug efflux and side-population positive and decreased BCL2-family member expression.

Chemotherapeutic drug treatment can result in the emergence of drug-resistant cells. By culturing an interleukin-3 (IL-3)-dependent cell line, FL5.12 cells in the presence of the chemotherapeutic drug doxorubicin, we isolated FL/Doxo cells which are multi-drug resistant. Increased levels of drug efflux were detected in FL/Doxo cells which could be inhibited by the MDR1 inhibitor verapamil but not by the MRP1 inhibitor MK571. The effects of TP53 and MEK1 were examined by infection of FL/Doxo cells with retroviruses encoding either a dominant negative TP-53 gene (FL/Doxo+ TP53 (DN) or a constitutively-activated MEK-1 gene (FL/Doxo + MEK1 (CA). Elevated MDR1 but not MRP1 mRNA transcripts were detected by quantitative RT-PCR in the drug-resistant cells while transcripts encoding anti-apoptotic genes such as: BCL2, BCLXL and MCL1 were observed at higher levels in the drug-sensitive FL5.12 cells. The percentage of cells that were side-population positive was increased in the drug-resistant cells compared to the parental line. Drug-resistance and side-positive population cells have been associated with cancer stem cells (CSC). Our studies suggest mechanisms which could allow the targeting of these molecules to prevent drug-resistance.

Effect of Levetiracetam add-on therapy on expression of multidrug resistant-associated protein 1 and 2 in peripheral blood mononuclear cells of children with intractable epilepsy

Objective To explore the changes of multidrug resistant-associated protein (MRP)1 expression and MRP2 expression in the peripheral blood mononuclear cells in intractable epileptic children after accepting the Levetiracetam (LEV) add-on therapy. Methods From June 2012 to September 2015, 68 intractable epileptic children who had been diagnosed at the Outpatient Department of the Xuzhou Children′s Hospital were randomly selected and given the LEV add-on therapy.The expressions of MRP1 and MRP2 in their peripheral blood mononuclear cells were detected by real-time fluorescence quantitative polymerase chain reaction and Western blot before the LEV add-on therapy and 6 months after the LEV add-on therapy. Results (1) Compared to the expressions of MRP1 mRNA and MRP2 mRNA in the peripheral blood mononuclear of intractable epileptic children before therapy, the expressions of MRP1 mRNA (0.23±0.02) and MRP2 mRNA (0.17±0.03) after therapy were obviously down-regulated with significant differences(tMRP1=32.77, P<0.01; tMRP2=26.73, P<0.01). (2)The relative expressions of protein of MRP1 and MRP2 in the peripheral blood mononuclear of intractable epileptic children before therapy were 0.59±0.03, 0.53±0.02 respectively, and they were 0.39±0.03, 0.38±0.02 after therapy.Compared with those before therapy, the protein expressions of MRP1 and MRP2 were down-regulated, and the differences were significant (tMRP1=11.55, P<0.01; tMRP2=9.87, P<0.01). Conclusions The expressions of MRP1 and MRP2 mRNA and protein in the peripheral blood mononuclear cells in some intractable epileptic children can be down-regulated if patients receive the LEV add-on therapy, and LEV may control the seizures of intractable epileptic children by down-regulating the expressions of MRP1 and MRP2, then reduce the occurrence and development of drug-resistant epilepsy. Key words: Intractable epilepsy; Levetiracetam; Multidrug resistant-associated protein; Child

The phytoestrogens daidzein and equol inhibit the drug transporter BCRP/ABCG2 in breast cancer cells: potential chemosensitizing effect.

The soy isoflavone genistein has been described to up-regulate breast cancer resistance protein (BCRP) and, thus, enhance chemoresistance in breast cancer cells. The aim of this work was to assess the effect of long- and short-term incubation with daidzein, the second most abundant soy isoflavone and its metabolite equol on the expression and activity of P-glycoprotein, multidrug resistance-associated proteins 1 and 2 (MRP1 and MRP2) and BCRP in breast cancer cells. MCF-7 and MDA-MB-231 cells were treated with phytoestrogen concentrations within the range achieved in individuals with a high isoflavone intake. Transporter expression was evaluated at protein and mRNA level through western blot and qRT-PCR, respectively. Transporter activity was determined using doxorubicin, mitoxantrone and carboxy-dichlorofluorescein as substrates. Daidzein (5µM) up-regulated MRP2- and down-regulated MRP1 protein expressions in MCF-7 and MDA-MB-231 cells, respectively. Both effects were ER-dependent, as determined using the antagonist ICI 182,780. The decrease in MRP1 mRNA in MDA-MB-231 cells indicates a transcriptional mechanism. On the contrary, MRP2 induction in MCF-7 cells takes place post-transcriptionally. Whereas changes in the transporter expression had a minor effect on the transporter activity, acute incubation with daidzein, R-equol and S-equol led to a strong inhibition of BCRP activity and an increase in the IC50 of BCRP substrates. In contrast to previous reports for genistein, daidzein and equol do not provoke a major up-regulation of the transporter expression but instead an inhibition of BCRP activity and sensitization to BCRP substrates.

Ecotoxicological properties of ketoprofen and the S(+)-enantiomer (dexketoprofen): Bioassays in freshwater model species and biomarkers in fish PLHC-1 cell line.

The increased use of nonsteroidal anti-inflammatory drugs (NSAIDs) has resulted in their ubiquitous presence in the environment. The toxicological properties of these 2 widely prescribed NSAIDs, namely racemic ketoprofen and its enantiomer S(+)-ketoprofen (dexketoprofen), were evaluated, firstly, by acute and chronic toxicity tests using 3 representative model organisms (Vibrio fischeri, Pseudokirchneriella subcapitata, and Ceriodaphnia dubia) and, secondly, by evaluating the responses of biotransformation systems and multidrug resistance-associated proteins (MRP1/MRP2) using the Poeciliopsis lucida hepatocellular carcinoma 1 (PLHC-1) fish hepatic cell line. Toxicity data from both acute and chronic dexketoprofen exposure indicated higher sensitivity through inhibition of bioluminescence and algal growth and through increased mortality/immobilization compared to racemic ketoprofen exposure. The growth inhibition test showed that racemic ketoprofen and dexketoprofen exhibited different effect concentration values (240.2 and 65.6 μg/L, respectively). Furthermore, racemic ketoprofen and dexketoprofen did not exert cytotoxic effects in PLHC-1 cells and produced compound-, time-, and concentration-specific differential effects on cytochrome P450 1A (CYP1A) and glutathione S-transferase levels. For CYP1A, the effects of racemic ketoprofen and dexketoprofen differed at the transcriptional and catalytic levels. Exposure to racemic ketoprofen and dexketoprofen modulated MRP1 and MRP2 mRNA levels, and these effects were also dependent on compound, exposure time, and concentration of the individual drug. The present study revealed for the first time the interactions between these NSAIDs and key detoxification systems and different sensitivity to the racemic mixture compared to its enantiomer. Environ Toxicol Chem 2018;37:201-212. © 2017 SETAC.

Xenobiotic Nuclear Receptors Pregnane X Receptor and Constitutive Androstane Receptor Regulate Antiretroviral Drug Efflux Transporters at the Blood-Testis Barrier

Poor antiretroviral drug (ARV) penetration in the testes could be due, in part, to the presence of ATP-binding cassette (ABC) membrane-associated drug efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins (MRPs) expressed at the blood-testis barrier (BTB). The functional expression of these transporters is known to be regulated by ligand-activated nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in various tissues. This study aimed to investigate in vitro and ex vivo the role of PXR and CAR in the regulation of ABC transporters at the BTB. Both PXR and CAR proteins were expressed in human testicular tissue and in mouse TM4 Sertoli cells (an in vitro cell line model of the BTB). In addition, we demonstrated an upregulation of P-gp, Bcrp, and Mrp4 mRNA and protein expression, after exposure to PXR or CAR ligands in TM4 cells. Small interfering RNA downregulation of PXR or CAR attenuated the expression of these transporters, suggesting the direct involvement of these nuclear receptors in regulating P-gp, Bcrp, and Mrp4 in this system. In an ex vivo study using freshly isolated mouse seminiferous tubules, we found that exposure to PXR or CAR ligands, including ARVs, significantly increased P-gp expression and function. Together, our data suggest that ABC transporters could be regulated at the BTB during chronic treatment with ARVs that can serve as ligands for PXR and CAR, which could in turn further limit testicular ARV concentrations.

Study on mechanism of hepatotoxicity of Ploygoni Multiflori Caulis based on function inhibition of bilirubin-associated transporters in idiosyncratic rat

To explore the possible mechanism of liver injury, the effects of Ploygoni Multiflori Caulis and its extractive on the function of bilirubin-associated transporters were investigated in normal (N) and idiosyncratic (LPS) rats (M). The normal and LPS rats were respectively administrated powder of Ploygoni Multiflori Caulis, its extractive and same volume of 0.5% CMC-Na solution for 7 d. BSP, a substrate of the transporters of Oatp1a1 and Oatp1b2 was selected, and its pharmacokinetic parameters of intravenous injection were determined to examined the activity these transporters. Meanwhile the mRNA expressions of transporters were detected. Compared with N-blank control group, besides M-powder group, the Cmax has no significantly different from other groups, t1/2, AUC0-t and AUC0-∞ were significantly increased, and CL were significantly decreased. However, compared with N- blank control group, AST and ALT decreased significantly. The expression of Oatp1a1, Oatp1b2 and MRP2 mRNA was significantly decreased (P<0.05), but there was no act synergistically when Ploygoni Multiflori Caulis and extractive were combined with LPS. The function of Oatp1a1, Oatp1b2 and MRP2 in rats were significantly inhibited by Ploygoni Multiflori Caulis and extractive, which may be an important cause of hepatotoxicity.

BCRP mRNA and FLT3‐ITD are independent poor risk factors in adult patients with acute myeloid leukemia and intermediate or normal karyotype

FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is aberration associated with poor prognosis in AML. We have analyzed the expression of MDR-1, MRP-1, and BCRPmRNA in relation to FLT3-ITD in 100 AML adult patients with normal and intermediate karyotype. The RQ-PCR method was performed to assess the expression of MDR-1, MRP-1, and BCRPmRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl's rule. According to univariate analysis, the following pretreatment variables negatively influenced disease-free survival (DFS): WBC count ≥25×109 /L (P=.037), MRP-1 mRNA ≥1.6818 (P=.028), BCRPmRNA ≥1.1892 (P=.004), FLT3-ITD (P=.005) and overall survival (OS): WBC count ≥25×109 /L (P=.031), MRP-1 mRNA ≥1.6818 (P=.01), BCRPmRNA ≥1.1892 (P=.01), FLT3-ITD (P=.001). When all prognostic variables were pooled into a multivariate model, we found that WBC count ≥25×109 /L (P=.026) and BCRPmRNA ≥1.1892 (P=.011). We observed trend in negative influence of FLT3-ITD on DFS (P=.057). BCRPmRNA ≥1.1892 (P=.035) and FLT3-ITD (P=.006) negatively, independently influenced the OS. The high expressions of BCRPmRNA calculated with Pfaffl's rule and FLT3-ITD are independent poor risk factors in adult patients with AML and intermediate or normal karyotype.

Circadian Clock Is Involved in Regulation of Hepatobiliary Transport Mediated by Multidrug Resistance-Associated Protein 2

There has been a growing interest in circadian regulation of the expression and function of drug transporters. In this study, we investigated circadian rhythm in the expression and function of multidrug resistance-associated protein 2 (Mrp2) in mouse liver and involvement of circadian clock in their regulations by using the circadian clock genes (period 1 and period 2) knockout mice. The mRNA and protein expression of Mrp2, P-glycoprotein, and breast cancer resistance protein was measured in the mouse liver at different times of the day. Circadian variation of hepatobiliary excretion of phenolsulfonphthalein, a model substrate of Mrp2, was also investigated in mice. Circadian oscillation of Mrp2 protein expression was clearly observed in the mouse liver with levels down at the light phase and up at the dark phase. The cumulative biliary excretion and biliary clearance of phenolsulfonphthalein from the liver to the bile was 2.37- and 1.74-fold greater in mice administered during the dark phase than in those administered during the light phase, respectively. The circadian oscillation in mRNA expression of Mrp2 disappeared in period 1 and period 2 double knockout mice. These results suggest that the expression and function of Mrp2 show the circadian rhythm, controlled by circadian clock genes.

Pharmacokinetic changes of norfloxacin based on expression of MRP2 after acute exposure to high altitude at 4300m.

This study was to investigate the influence of physiological changes and the expression of MRP2 efflux transporter on the pharmacokinetics of norfloxacin after acute exposure to high altitude 4300m. The rats were randomly divided into high altitude group and plain group. Blood gas and biochemical analysis showed that the physiological parameters significantly changed at high altitude. The mRNA and protein expression of MRP2 in high altitude group were higher than plain group in rat small intestine and kidney, while was reduced in rat liver. The AUC, Ka and Cmax of norfloxacin were significantly reduced in high altitude group (p<0.05). However, the MRT, CL, t1/2 and Vd were significantly increased (p<0.05). These results indicate that physiological indicators and expression levels of drug transporters MRP2 are changed in responded to high altitude, to severely affect norfloxacin pharmacokinetics. These changes may provide basis and new ideas to adjust the dosage and administration, so as to promote rational drug use in the high altitude.

Differential regulation of intestinal efflux transporters by pregnancy in mice

1. In the intestines, the nuclear receptors farnesoid X receptor (Fxr) and pregnane X receptor (Pxr) regulate the transcription of metabolizing enzymes and transporters that dictate the absorption of nutrients and xenobiotics.2. Here, we sought to determine whether Fxr and Pxr signaling pathways are disrupted in response to high-circulating concentrations of steroid hormones late in pregnancy leading to altered transporter expression. To test this, ileum were collected from virgin and pregnant C57BL/6 mice on gestation days 14, 17 and 19.3. Ileum from pregnant mice exhibited suppression of Fgf15 and Cyp3a11 mRNAs, which are the prototypical target genes for Fxr and Pxr, respectively. An overall reduction in the expression of apical efflux transporters, including Mdr1, Mrp2 and Bcrp, was observed in pregnant mice. To assess the ability of steroid hormones to alter intestinal nuclear receptor signaling, transporter mRNA expression was quantified in human intestinal LS174T adenocarcinoma cells. In vitro data demonstrated that progestins reduced CYP3A4, MDR1 and MRP2 mRNA expression by 30–40%.4. These data suggest that progesterone may act as a mediator to negatively regulate efflux transporter expression in the mouse ileum during pregnancy possibly by reducing PXR/Pxr signaling. This may affect drug absorption and disposition during pregnancy.

Ontogeny, aging, and gender-related changes in hepatic multidrug resistant protein genes in rats

Multidrug resistance proteins (Mrps) are efflux transporters playing important roles in endogenous substances and xenobiotics transport out of the liver. Children, elderly, gender and physio-pathological conditions could influence their expression and result in changes in drug disposition. AimThis study was aimed to examine the development-, aging-, and sex-dependent changes in Mrp1–4 and ATP-binding cassette sub-family G member 2 (Abcg2) gene expressions in livers of rats. Main methodsThe livers from male and female SD rats at development (−2, 1,7,14,21,28,35, and 60d) and aging (28, 60, 180 and 540d) were collected and total RNA was isolated, purified and subjected to real-time RT-PCR analysis. Key findingsResults showed that expression of Mrp1 was low, while Abcg2 and Mrp2 were the high in the liver. Mrp1 expression decreased with maturity but remained constant to 540d, while Mrp3 and 4 increased with liver development, reached the peak with maturity at 35–60days of age, and slightly reduced with aging. Mrp2 and Abcg2 were high at 7days of age and maintained at relative high levels till maturity, while Abcg2 was reduced during aging. Females had higher Mrp3 and Abcg2 mRNA expression than male rats, while male rats had higher Mrp2 and Mrp4 mRNA expression. SignificanceThe expression of hepatic Mrp1–4 and Abcg2 mRNA during development, aging in male and female rats was characterized, which could be fundamental to our understanding of age- and sex-associated variations in drug disposition in children, elderly, and women.

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.

Parenteral nutrition dysregulates bile salt homeostasis in a rat model of parenteral nutrition-associated liver disease

Parenteral nutrition (PN), a lifesaving therapy in patients with intestinal failure, has been associated with hepatobiliary complications including steatosis, cholestasis and fibrosis, collectively known as parenteral nutrition-associated liver disease (PNALD). To date, the pathogenesis of PNALD is poorly understood and therapeutic options are limited. Impaired bile salt homeostasis has been proposed to contribute PNALD. The objective of this study was to establish a PNALD model in rats and to evaluate the effects of continuous parenteral nutrition (PN) on bile salt homeostasis. Rats received either PN via the jugular vein or received normal diet for 3, 7 or 14 days. Serum biochemistry, hepatic triglycerides, circulating bile salts and C4, IL-6 and TNF-alpha, and lipogenic and bile salt homeostatic gene expression in liver and ileum were assessed. PN increased hepatic triglycerides already after 3 days of administration, and resulted in conjugated bilirubin elevation after 7 or more days. This indicates PN-induced steatosis and impaired canalicular secretion of bilirubin, the latter which is in line with reduced hepatic expression of Mrp2 mRNA. There was no histological evidence for liver inflammation after PN administration, and circulating levels of pro-inflammatory cytokines IL-6 and TNF-α, were comparable in all groups. Hepatic expression of Fxr mRNA was decreased after 7 days of PN, without apparent effect on expression of Fxr targets Bsep and Shp. Nonetheless, Cyp7a1 expression was reduced after 7 days of PN, indicative for lowered bile salt synthesis. Circulating levels of C4 (marker of bile salt synthesis) were also decreased after 3, 7 and 14 days of PN. Levels of circulating bile salts were not affected by PN. This study showed that PN in rats caused early mild steatosis and cholestasis, while hepatic and systemic inflammation were not present. The onset of these abnormalities was associated with alterations in bile salt synthesis and transport. This animal model serves as an experimental model to further investigate the pathogenesis of PNALD inflicted by steatosis and cholestasis.

Crocin suppresses multidrug resistance in MRP overexpressing ovarian cancer cell line.

BackgroundCrocin, one of the main constituents of saffron extract, has numerous biological effects such as anti-cancer effects. Multidrug resistance-associated proteins 1 and 2 (MRP1 and MRP2) are important elements in the failure of cancer chemotherapy. In this study we aimed to evaluate the effects of crocin on MRP1 and MRP2 expression and function in human ovarian cancer cell line A2780 and its cisplatin-resistant derivative A2780/RCIS cells.MethodsThe cytotoxicity of crocin was assessed by the MTT assay. The effects of crocin on the MRP1 and MRP2 mRNA expression and function were assessed by real-time RT-PCR and MTT assays, respectively.ResultsOur study indicated that crocin reduced cell proliferation in a dose-dependent manner in which the reduction in proliferation rate was more noticeable in the A2780 cell line compared to A2780/RCIS. Crocin reduced MRP1 and MRP2 gene expression at the mRNA level in A2780/RCIS cells. It increased doxorubicin cytotoxicity on the resistant A2780/RCIS cells in comparison with the drug-sensitive A2780 cells.ConclusionTotally, these results indicated that crocin could suppress drug resistance via down regulation of MRP transporters in the human ovarian cancer resistant cell line.

Functional Integrity of the Chimeric (Humanized) Mouse Liver: Enzyme Zonation, Physiologic Spaces, and Hepatic Enzymes and Transporters

Chimeric mouse liver models are useful in vivo tools for human drug metabolism studies; however, liver integrity and the microcirculation remain largely uninvestigated. Hence, we conducted liver perfusion studies to examine these attributes in FRGN [Fah(-/-), Rag2(-/-), and Il2rg(-/-), NOD strain] livers (control) and chimeric livers repopulated with mouse (mFRGN) or human (hFRGN) hepatocytes. In single-pass perfusion studies (2.5 ml/min), outflow dilution profiles of noneliminated reference indicators ((51)Cr-RBC, (125)I-albumin, (14)C-sucrose, and (3)H-water) revealed preservation of flow-limited distribution and reduced water and albumin spaces in hFRGN livers compared with FRGN livers, a view supported microscopically by tightly packed sinusoids. With prograde and retrograde perfusion of harmol (50 µM) in FRGN livers, an anterior sulfation (Sult1a1) over the posterior distribution of glucuronidation (Ugt1a1) activity was preserved, evidenced by the 42% lower sulfation-to-glucuronidation ratio (HS/HG) and 14% higher harmol extraction ratio (E) upon switching from prograde to retrograde flow. By contrast, zonation was lost in mFRGN and hFRGN livers, with HS/HG and E for both flows remaining unchanged. Remnant mouse genes persisted in hFRGN livers (10%-300% those of FRGN). When hFRGN livers were compared with human liver tissue, higher UGT1A1 and MRP2, lower MRP3, and unchanged SULT1A1 and MRP4 mRNA expression were observed. Total Sult1a1/SULT1A1 protein expression in hFRGN livers was higher than that of FRGN livers, consistent with higher harmol sulfate formation. The composite data on humanized livers suggest a loss of zonation, lack of complete liver humanization, and persistence of murine hepatocyte activities leading to higher sulfation.

Effects of Glycyrrhetinic Acid on GSH Synthesis Induced by Realgar in the Mouse Hippocampus: Involvement of System [Formula: see text], System [Formula: see text], MRP-1, and Nrf2.

Realgar, a type of mineral drug-containing arsenic, exhibits neurotoxicity. Brain glutathione (GSH) is crucial to protect the nervous system and to resist arsenic toxicity. Therefore, the main aim of this study was to explore the neurotoxic mechanisms of realgar and the protective effects of glycyrrhetinic acid (GA) by observing the effects of GA on the hippocampal GSH biosynthetic pathway after exposure to realgar. Institute of Cancer Research (ICR) mice were randomly divided into five groups: a control group, a GA control group, a realgar alone group, a low-dose GA intervention group, and a high-dose GA intervention group. Cognitive ability was tested using an object recognition task (ORT). The ultrastructures of the hippocampal neurons and synapses were observed. mRNA and protein levels of EAAT1, EAAT2, EAAT3, xCT, Nrf2, HO-1, γ-GCS (GCLC, GCLM), and MRP-1 were measured, as was the cellular localization of EAAT3, xCT, MRP-1, and Nrf2. The levels of GSH in the hippocampus, the levels of glutamate (Glu) and cysteine (Cys) in the extracellular fluid of hippocampal CA1 region, and the levels of active sulfur in the brain were also investigated. The results indicate that realgar lowered hippocampal GSH levels, resulting in ultrastructural changes in hippocampal neurons and synapses and deficiencies in cognitive ability, ultimately inducing neurotoxicity. GA could trigger the expression of Nrf2, HO-1, EAAT1, EAAT2, EAAT3, xCT, MRP-1, GCLC, and GCLM. Additionally, the expression of γ-GT and the supply levels of Glu and Cys increased, ultimately causing a significant increase in hippocampal GSH to alleviate realgar-induced neurotoxicity. In conclusion, the findings from our study indicate that GA can antagonize decreased brain GSH levels induced by realgar and can lessen the neurotoxicity of realgar.

Timosaponin A-III reverses multi-drug resistance in human chronic myelogenous leukemia K562/ADM cells via downregulation of MDR1 and MRP1 expression by inhibiting PI3K/Akt signaling pathway.

One of the major causes of failure in chemotherapy for patients with human chronic myelogenous leukemia (CML) is the acquisition of multidrug resistance (MDR). MDR is often associated with the overexpression of drug efflux transporters of the ATP-binding cassette (ABC) protein family. Timosaponin A-III (TAIII), a saponin isolated from the rhizome of Anemarrhena asphodeloides, has previously demonstrated the ability to suppress certain human tumor processes and the potential to be developed as an anticancer agent. Nevertheless, the ability of TAIII to reverse MDR has not yet been explored. In this study, the adriamycin (ADM) resistance reversal effect of TAIII in human CML K562/ADM cells and the underlying mechanism was investigated. The Cell Counting Kit-8 (CCK-8) assay showed that TAIII had a reversal effect on the drug resistance of K562/ADM cells. Flow cytometry assay showed increased intracellular accumulation of ADM after cells were pretreated with TAIII, and the changes in the accumulation of rhodamine-123 (Rho-123) and 5(6)-carboxyfluorescein diacetate (CFDA) dye in K562/ADM cells were determined to be similar to the changes of intracellular accumulation of ADM. After pretreatment of cells with TAIII, the decreasing expression of P-gp and MRP1 mRNA was examined by reverse transcription polymerase chain reaction (RT-PCR). Western blotting showed TAIII inhibiting P-gp and MRP1 expression depended on the PI3K/Akt signaling pathway by decreasing the activity of p-Akt. Moreover, wortmannin an inhibitor of PI3K/Akt signaling pathway has a strong inhibitory effect on the expression of p-Akt, P-gp and MRP1. Besides, the combined treatment with TAIII did not have an affect on wortmannin downregulation of p-Akt, P-gp and MRP1. Taken together, our findings demonstrate, for the first time, that TAIII induced MDR reversal through inhibition of P-gp and MRP1 expression and function with regained adriamycin sensitivity which might mainly correlate to the regulation of PI3K/Akt signaling pathway.

MiR-145 sensitizes gallbladder cancer to cisplatin by regulating multidrug resistance associated protein 1.

Gallbladder cancer (GBC) is the most common malignancy in biliary tract with poor prognosis. Due to its high chemoresistance, systemic chemotherapies have had limited success in treating GBC patients. MicroRNAs (miRNAs) are emerging novel regulators of chemoresistance, which modulate the expression of drug resistance-related genes. In this study, we investigated the association between miR-145 expression and cisplatin sensitivity by both in vivo and in vitro analysis. Quantitative PCR (q-PCR) analysis indicated an increased miR-145 expression in GBC tissues. In addition, studies on GBC cell lines suggested an increased cisplatin efficacy with miR-145 overexpression, whereas decreasing miR-145 expression reduced cisplatin sensitivity. Further, we found that miR-145 accelerated MRP1 mRNA degradation by directly targeting its 3'-UTR and therefore caused increased cisplatin toxicity in GBC cells. Moreover, lower miR-145 and higher MRP1 expression levels predicted poor prognosis in GBC patients who received chemotherapy. Collectively, our findings established a rationale for using miR-145 expression as a biomarker to identify cisplatin-resistant GBC patients and propose that treatment strategies increasing the expression of miR-145 could be a new therapeutic approach for GBC patients.

Geniposidic acid protected against ANIT-induced hepatotoxity and acute intrahepatic cholestasis, due to Fxr-mediated regulation of Bsep and Mrp2

Ethnopharmacological relevanceGeniposidic acid (GPA) is the main constituent of Gardenia jasminoides Ellis (Rubiaceae), which has long been used to treat inflammation, jaundice and hepatic disorders. The cholagogic effect of Gardenia jasminoides Ellis (Rubiaceae) and GPA have been widely reported, but the underlying occurrence mechanism remains unclear. Aim of the studyThis investigation was designed to evaluate the hepatoprotection effect and potential mechanisms of GPA derived from Gardenia jasminoides Ellis (Rubiaceae) on fighting against α-naphthylisothiocyanate (ANIT) caused liver injury with acute intrahepatic cholestasis. Materials and methodsSprague-Dawley (SD) rats were intragastrically (i.g.) administered with the GPA (100, 50 and 25mg/kg B.W. every 24h) for seven consecutive days, and then they were treated with ANIT (i.g. 65mg/kg once in the 5th day) which induced liver injury with acute intrahepatic cholestasis. Serum and bile biochemical analysis, bile flow rate and liver histopathology were measured to evaluate the protective effect of GPA fight against ANIT treatment. The protein and mRNA expression levels of farnesoid X receptor (Fxr), bile-salt export pump (Bsep), multidrug resistance associated protein2 (Mrp2), were evaluated to study the effect of liver protection about GPA against ANIT induced hepatotoxicity and underlying mechanisms. ResultsSome abnormalities were observed on ANIT treated rats including weight loss, reduced food intake and hair turned yellow. Obtained results demonstrated that at dose 100 and 50mg/kg B.W. (P<0.01) and 25mg/kg B.W. (P<0.05) of GPA pretreated dramatically prevented ANIT induced decreased in bile flow rate. Compared with ANIT treated group, the results of bile biochemical parameters about total bile acid (TBA) was increased by GPA at groups with any dose (P<0.01), glutathione (GSH) was increased significantly at high dose (P<0.01) and medium dose (P<0.05), total bilirubin (TB) was increased at high and medium dose (P<0.05), direct bilirubin (DB) was only increased at high dose (P<0.01). Serum levels of glutamic-Oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), γ-glutamyltranspeptidase (γ-GT), TB, DB and TBA in comparison with ANIT treated group (P<0.01) were reduced by GPA (between 100 and 50mg/kg B.W.) pretreatment. Histopathology of the liver tissue showed that pathological damages and hepatic portal area filled with bile were relieved after GPA pretreatment compared with ANIT treated group. The protein and mRNA expression of Fxr, Bsep and Mrp2 were decreased in ANIT treated group. On the contrary, the protein and mRNA of Fxr, Bsep and Mrp2 were up regulated significantly pretreatment by GPA at dose of high and medium groups. On protein level of Bsep and Mrp2 the result shown no statistical difference in GPA (25mg/kg B.W.), but it was not same shown in mRNA level. ConclusionThe results of this investigation have demonstrated that the GPA exerts a dose dependent hepatoprotection effect on ANIT induced liver damage with acute intrahepatic cholestasis in rats, which may due to Fxr mediated regulation of bile transporters like Bsep and Mrp2.

Yinzhihuang attenuates ANIT-induced intrahepatic cholestasis in rats through upregulation of Mrp2 and Bsep expressions.

The treatment of intrahepatic cholestasis has been limited, and development of an effective drug is needed. Clinical studies have shown that Yinzhihuang (YZH), a traditional Chinese decoction, enhances bilirubin clearance. The goal of this study was to determine the protective effect of YZH on experimental intrahepatic cholestasis in young rats and to explore its underlying molecular mechanisms. Intrahepatic cholestasis in rats was induced by α-naphthylisothiocyanate (ANIT) on days 1 and 8. The rats received YZH, ursodeoxycholic acid (UDCA), or vehicle for 9 d and were killed on either day 3 or day 10. Serum biomarkers, liver histology, and the distribution of protein and mRNA expression of Mrp2 and Bsep were analyzed. YZH treatment resulted in decreased levels of serum biomarkers except γ-glutamyl transpeptidase, attenuated liver histological injuries, increased protein expressions of Mrp2 and Bsep, and upregulated expressions of Mrp2 and Bsep mRNAs. The effects of YZH on serum biomarkers (aminotransferase, alanine aminotransferase, and direct bilirubin), liver histology, and Mrp2 mRNA expressions were significantly greater and earlier than those of UDCA. Our results suggest that YZH has protective effect against ANIT-induced intrahepatic cholestasis in rats, through upregulation of Mrp2 and Bsep expressions.