JNK1,JNK2,and JNK3 are involved in P-glycoprotein-mediated multidrug resistance of hepatocellular carcinoma cells

BACKGROUND:Multidrug resistance(MDR)is extremely common in hepatocellular carcinoma(HCC)and is a major problem in cancer eradication by limiting the efficacy of chemotherapy.Modulation of c-Jun NH2-terminal kinase(JNK)activation could be a new method to reverse MDR.However,the relationship between JNK activity and MDR in HCC cells is unknown.This study aimed to explore the relationship between MDR and JNK in HCC cell lines with different degrees of MDR.METHODS:A MDR human HCC cell line,SMMC-7721/ ADM,was developed by exposing parental cells to gradually increasing concentrations of adriamycin.The MTT assay was used to determine drug sensitivity.Flow cytometry was used to analyze the cell cycle distribution and to measure the expression levels of P-glycoprotein(P-gp)and MDR-related protein(MRP)-1 in these cells.JNK1,JNK2 and JNK3 mRNA expression levels were quantified by real-time PCR.Expression and phosphorylation of JNK1,JNK2,and JNK3 were analyzed by Western blotting.RESULTS:The MDR of SMMC-7721/ADM cells resistant to 0.05 mg/L adriamycin was mainly attributed to the overexpression of P-gp but not MRP1.In addition,these cells had a significant increase in percentage in the S phase,accompanied by a decrease in percentage in the G0/G1 phase,which is likely associated with a reduced ability for cell proliferation and MDR generation.We found that JNK1,JNK2,and JNK3 activities were negatively correlated with the degree of MDR in HCC cells.CONCLUSION:This study suggests that JNK1,JNK2,and JNK3 activities are negatively correlated with the degree of MDR in HCC cells.

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 10μM and induced the mRNA expression of MRP2 at 50μM 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.

Effect of Histone Deacetylase Inhibition on the Expression of Multidrug Resistance-associated Protein 2 in a Human Placental Trophoblast Cell Line

Background： Placental multidrug resistance-associated protein 2 （MRP2）, encoded by ABCC2 gene in human, plays a significant role in regulating drugs＇ transplacental transfer rates. Studies o11 placental MRP2 regulation could provide more therapeutic targets for individualized and safe pharmacotherapy during pregnancy. Currently, the roles of epigenetic mechanisms in regulating placental drug transporters are still unclear. This study aimed to investigate the effect of histone deacetylases （HDACs） inhibition on MRP2 expression in the placental trophoblast cell line and to explore whether HDAC 1/2/3 are preliminarily involved in this process. Methods： The human choriocarcinoma-derived trophoblast cell line （Bewo cells） was treated with the HDAC inhibitors-trichostatin A （TSA） at different concentration gradients of 0.5, 1.0, 3.0, and 5.0 μmol/L. Cells were harvested after 24 and 48 h treatment. Small interfering RNA （siRNA） specific for HDACI/HDAC2/HDAC3 or control siRNA was transfected into cells. Total HDAC activity was detected by colorimetric assay kits. HDAC 1/2/3/ABCC2 messenger RNA （mRNA） and protein expressions were determined by real-time quantitative polymerase chain reaction and Western-blot analysis, respectively. Immunofluorescence for MRP2 protein expression was visualized and assessed using an immunofluorescence microscopy and ImageJ software, respectively. Results： TSA could inhibit total HDAC activity and HDAC 1/2/3 expression in company with increase ofM RP2 expression in Bewo cells. Reduction of HDAC 1 protein level was noted after 24 h of TSA incubation at 1.0, 3.0, and 5.0 μmol/L （vs. vehicle group, all P 〈 0.001 ）, accompanied with dose-dependent induction of MRP2 expression （P = 0.045 for 1.0 μmol/L, P = 0.001 for 3.0 μmol/L, and P 〈 0.001 for 5.0 μmol/L）, whereas no significant diferences in MRP2 expression were noted after HDAC2/3 silencing. Fluorescent micrograph images of MRP2 protein were expressed on the cell membrane. The fluorescent intensities of MRP2 in the control, HDAC2, and HDAC3 siRNA-transfected cells weir week, and no significant differences were noticed among these three groups （all P 〉 0.05）. However, MRP2 expression was remarkably elevated in H DAC1 siRNA-transfected cells, which displayed an almost 3.19-fold changes in comparison with the control siRNA-transfected cells （P 〈 0.001 ）. Conclusions： HDACs inhibition could up-regulate placental MRP2 expression in ritzy, and HDAC 1 was probably to be involved in this process.

头孢妥仑测定方法的建立及胆汁排泄机制研究

目的:建立高效液相色谱内标法测定大鼠血浆、胆汁、尿液中头孢妥仑含量,阐明多药耐药相关蛋白2(Mrp2)是否与头孢妥仑胆汁排泄有关。方法:用4-二甲氨基安替比林作内标。色谱柱为CapcellpakC18UG120(4.6mm×250mm,5μm),流动相为0.1%醋酸铵-甲醇(67:33,V/V),流速为0.8mL/min,进样量为25μL;用肝灌流法探讨头孢妥仑是否经Mrp2转运。设立对照组(头孢妥仑1μmol/L)和实验组(头孢妥仑1μmol/L加Mrp2抑制剂丙磺舒20μmol/L),在设定时间点于灌流的大鼠肝脏收集出口灌流液和胆汁样品,高效液相色谱法测定样品中头孢妥仑含量,考察实验组和对照组中肝摄取率和胆汁累积排泄率的差异。结果:血浆样品中头孢妥仑在0.1～4μg/mL范围内、胆汁样品中头孢妥仑在0.1～5μg/mL范围内、尿液样品中头孢妥仑在0.1～5μg/mL范围内线性关系良好。回收率为85%～115%,日内、日间RSD均小于13.5%。实验组和对照组相比,肝摄取率变化无统计学意义上的差别;实验组中,肝灌流25min后,头孢妥仑胆汁累积排泄率减少至对照组的40.0%。结论:本方法经济、简单、灵敏、快速,可用于头孢妥仑血浆、胆汁、尿液样品中药物浓度检测和药物代谢动力学研究;头孢妥仑是Mrp2的底物,Mrp2与头孢妥仑的胆汁排泄有关。

Different gap junction-propagated effects on cisplatin transfer result in opposite responses to cisplatinin normal cells versus tumor cells

OBJECTIVE Previous work has shown that gap junction intercel ular communication(GJIC)enhances cisplatin(Pt)toxicity in testicular tumor cells but decreases it in non-tumor testicular cells.In this study,these different GJIC-propagated effects were demonstrated in tumor versus non-tumor cells from other organ tissues(liver and lung).METHODS We use several different mani pulations(no cell contact,pharmacological inhibition,and si RNA suppression)to down-regulate GJIC function.The in vivo results using xenograft tumor models were consistent with those from the above-mentioned cells.To better understand the mechanism(s)involved,we studied the effects of GJIC on Pt accumulation in tumor and non-tumor cells from the liver and lung.RESULTS The intracel ular Pt and DNA-Pt adduct contents clearly increased in non-tumor cells but decreasedin tumor cells when GJIC was downregulated.Further analysis indicated that the opposite effectsof GJIC on Pt accumulation in normal versus tumor cells from the liver were due to its different effects on copper transporter1 and multidrug resistance-associated protein2,membrane transporters attributed to intracellular Pt transfer.CONCLUSION GJIC protects normal organs from cisplatin toxicity while enhancing it in tumor cells via its different effects on intracellular Pt transfer.