EXPRESSION AND CLINICAL SIGNIFICANCE OF MULTIDRUG RESISTANCE GENE AND MULTIDRUG RESISTANCE-ASSOCIATEDPROTEIN GENE IN ACUTE LEUKEMIA

Objective: To evaluate the expression and clinical significance of multidrug resistance gene (mdr1) and multidrug resistance-associated protein (MRP) gene in acute leukemia. Methods: The expression of mdr1 and MRP assay in 55 patients with acute leukemia (AL) by reverse transcription polymerase chain reaction (RT-PCR). Results: The mdr1 and MRP gene expression levels in the relapsed AL and the blastic plastic phases of CML were significantly higher than those in the newly diagnostic AL and controls. The mdr1 and MRP gene expression levels in the clinical drug-resistant group were significantly higher than those in the non-drug-resistant group. The complete remission (CR) rate in patients with high mdr1 expression (14.3%) was significantly lower than that with low mdr1 expression (57.5%); similarly the CR rate in patients with high MRP level was also lower than that with low MRP level. Using both high expression of mdr1 and MRP gene as the indicator for evaluating multidrug resistance (MDR), the positive predictive value and accuracy increased in comparison with single gene high expression. Conclusion: Elevated level of mdr1 or MRP gene expression might be unfavorable prognostic factors for AL patient and may be used as an important index for predicting drug-resistance and relapse in AL patient. Measuring both mdr1 and MRP gene expression would increase accuracy and sensibility of evaluating MDR in acute leukemia.

EXPRESSION OF MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN (MRP) AND ITS RELATIONSHIP WITH CLINICOPATHOLOGICAL FACTORS IN NON-SMALL CELL LUNG CANCER

Objective: To investigate the relationship between the expression of multidrug resistance-associated protein (MRP) and clinicopathological factors and prognosis. Methods: The expression of MRP in 62 cases with non-small cell lung cancer (NSCLC) was detected using immunohistochemistry method. The expression of MRP in 30 cases of NSCLC and corresponding normal lung tissues were detected using immunohistochemistry and Western Blot. Results: this study of tumor tissues confirmed the plasma membrane and/or cytoplasm locations of MRP. There was apparent difference between normal lung tissues and NSCLC in MRP. The survival analysis of 62 NSCLC showed that the mean survival time of the patients with negative MRP expression was 69.8117.41 months and that of patients with positive MRP expression, 25.384.46 months. Log-rank test suggested that the difference between them was significant (P=0.0156). It was also found that in squamous cell lung cancer the statistically significant difference between the mean survival time of patients with positive MRP expression and those with negative MRP expression (P=0.0153). Multivariate Cox model analysis suggested that the survival time was significantly related to expression of MRP (P=0.035) and lymphatic metastasis (P=0.038). Conclusion: MRP expression in NSCLC is significantly higher compared with normal lung tissues. The mean survival time of patients with negative MRP was relative longer and expression of MRP was an independent factor for prognosis.

Expression and significance of multi-drug resistance-associated protein 3 in different tumor cell lines

Objective To investigate the expression and meaning of MRP3 in different tumor cells. MethodsThe monoclonal antibody against MRP3 was used to identify the expression of MRP3 by flow cytometer in seven tumor cells and human embryo kidney cell lines 293T.And RT-PCR was used to detect the mRNA of MRP3 in eight cell lines. ResultsThe mRNA of MRP3 was expressed in three pancreatic carcinoma cell lines.MRP3 protein was observed in BxPC-3 and AsPC-1 cells. ConclusionMRP3 may express in different tumor in tissue-specific manner.BxPC-3 and AsPC-1 may serve as cellular models for in vitro studies on multidrug resistance of pancreatic carcinoma.

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.

Effects of Hypoxia on Expression of P-gp and Mutltidrug Resistance Protein in Human Lung Adenocarcinoma A549 Cell Line

To study the effects of hypoxia on the expression of P-gp and mutltidrug resistance protein in human lung adenocarcinoma A549 cell line, and to explore the probable mechanism of hypoxia in tumor cell of MDR. The expression of hypoxia inducible factor-1α, P-gp and mutltidrug resistance protein was immunohistochemically detected by culturing human lung adenocarcinoma A549 cell under hypoxia (2 % O_2) for 24 h. After interaction with adriamycin or cisplatin under hypoxia (2 % O_2) for 24 h, the cell survival rate was detected by MTT. Our results showed that the expression of hypoxia inducible factor-1α, P-gp and mutltidrug resistance protein under hypoxia were higher than the expression under normoxia, and correlations between the expression of HIF-1α and P-gp or multidrug resistance-associated protein was observed (P<0.05). The resistance of adriamycin of A549 cell was enhanced under hypoxia. It is concluded that the resistance of tumor chemotherapy is enhanced in hypoxia. The expression of HIF-1α is obviously correlated with the expression of P-gp and mutltidrug resistance protein.

茵栀黄颗粒对雌激素诱导的胆汁瘀积大鼠肝细胞膜多药耐药相关转运体Mrp1~4的调节作用

目的考察茵栀黄颗粒对胆汁瘀积大鼠肝脏转运体多药耐药相关蛋白1~4（Mrp1~4）表达的影响。方法 Wistar雄性大鼠20只,随机分为4组,即正常组、模型组、对照组和茵栀黄组,每组5只。大鼠颈部皮下连续注射苯甲酸雌二醇[EB,5 mg/（kg·d）]造模。Western blot实验考察茵栀黄颗粒对肝脏转运体Mrp1~4的调节作用。结果与对照组相比,茵栀黄组肝细胞膜转运体Mrp1~3的表达均显著增加（P〈0.05）,而Mrp4的表达差异无统计学意义（P〉0.05）。结论茵栀黄颗粒能明显上调胆汁瘀积模型大鼠肝细胞膜转运体Mrp1~3的表达,但对Mrp4的表达无影响。

BMP4增强结直肠癌耐药细胞化疗敏感性的作用及机制

目的研究骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)对结直肠癌耐药细胞化疗敏感性的影响及机制。方法采用5-氟尿嘧啶(5-Fu)处理结直肠癌细胞株LoVo,建立耐药细胞株LoVo/5-Fu;通过CCK-8法检测LoVo/5-Fu细胞和亲本细胞对5-Fu、奥沙利铂或伊立替康的敏感性,并检测不同浓度BMP4对LoVo/5-Fu细胞生长的抑制效应;用5-Fu联合BMP4处理LoVo/5-Fu细胞3d,采用Annexin-Ⅴ/PI法检测5-Fu联合BMP4处理后LoVo/5-Fu细胞的凋亡情况;通过裸鼠体内成瘤实验观察BMP4在体内对LoVo/5-Fu细胞化疗敏感性的影响;采用Western blot检测BMP4处理后LoVo/5-Fu细胞中AKT信号通路活性和Bcl-2的蛋白水平。结果 5-Fu诱导形成的耐药细胞株LoVo/5-Fu可以在含5μg/mL 5-Fu的培养液中稳定生长,并对奥沙利铂和伊立替康有交叉耐药;BMP4可以抑制LoVo/5-Fu细胞的生长,抑制效应呈时间和浓度依赖性;细胞凋亡实验显示BMP4可以促进培养在含5μg/mL 5-Fu培养液中的LoVo/5-Fu细胞发生凋亡;裸鼠体内成瘤实验显示BMP4可以有效增强LoVo/5-Fu细胞在体内的对5-Fu的敏感性,抑制其在裸鼠体内的生长;Western blot检测发现BMP4可以抑制LoVo/5-Fu细胞中AKT的磷酸化,并抑制抗凋亡蛋白Bcl-2的表达。结论 BMP4可以增强结直肠癌耐药细胞对化疗的敏感性;其机制有可能是通过抑制细胞中AKT通路的活性和Bcl-2表达,降低细胞抵抗凋亡的能力。

Hsp90抑制剂WH-4对肝癌细胞株SK-HEP-1增殖、凋亡及耐药基因表达的影响观察

目的探讨Hsp90抑制剂WH-4对肝癌细胞SK-HEP-1增殖、凋亡及耐药基因表达的影响。方法将肝癌细胞SK-HEP-1随机分为A、B、C组,A组分别加入0.625、1.25、2.5、5、10μmol/L WH-4,B组分别加入0.625、1.25、2.5、5、10μmol/L 17-AAG,C组加入等量生理盐水,培养48 h后,采用MTT法测算各组肝癌细胞SK-HEP-1增殖抑制率,并计算IC 50。将肝癌细胞SK-HEP-1随机分为A1、B1、C1组,分别加入2.3μmol/L WH-4、2.6μmol/L 17-AAG及生理盐水,培养48 h后,采用BrdU法观察肝癌细胞SK-HEP-1增殖活性。将肝癌细胞SK-HEP-1随机分为A2、B2、C2组,分别加入2.25μmol/L WH-4、2.80μmol/L 17-AAG、生理盐水,培养48 h后,采用软琼脂平板实验检测肝癌细胞SK-HEP-1克隆形成率,流式细胞术检测肝癌细胞SK-HEP-1凋亡率,采用Western blotting法检测肝癌细胞SK-HEP-1凋亡蛋白Bcl2、Bax、Bcl-xL。将肝癌细胞SK-HEP-1随机分为A3、B3、C3组,分别加入2.30μmol/L WH-4、2.65μmol/L 17-AAG及生理盐水。培养48 h后,采用qPCR法检测耐药基因ABCB1、ABCG2。结果WH-4对肝癌细胞SK-HEP-1有抑制作用,且呈浓度依赖性(R 2=0.647(48h),P均<0.05),IC 50为(2.35±0.25)μmol/L。与C1组相比,A1、B1组SK-HEP-1绿色荧光减弱。A2、B2组克隆形成率相比,P<0.05。与B2组比较,A2组细胞凋亡率升高(t=0.342,P<0.05),Bax蛋白相对表达量升高,B淋巴细胞瘤-2基因及Bcl-xL蛋白相对表达量降低。A3组ABCB1、ABCG2基因相对表达量低于B3组(t分别为-8.88、-13.00,P均<0.05)。结论Hsp90抑制剂WH-4对肝癌细胞SK-HEP-1具有增殖抑制、诱导凋亡作用,同时可降低耐药基因的表达。

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.

人MRP4蛋白结构与功能预测

多药耐药相关蛋白4(multidrug resistance-associated protein 4,MRP4)是一种能够利用ATP水解产生的能量转运多种物质的六重跨膜蛋白。本研究应用Expasy、Jpred4、UniProt-KB、NCBI等公共数据库和在线软件分析其理化性质、蛋白结构、结构域、亚细胞定位、互作蛋白以及功能等信息,为其在多种生理病理过程中的作用与机制研究奠定基础。MRP4蛋白含有1325个氨基酸,相对分子质量约为149526.77,理论等电点为8.41。以α-螺旋为主,占比58.42%。含有2个跨膜结构域和2个ATP结合盒以及8个保守结构域。表观遗传学修饰位点56个,其中磷酸化位点27个,泛素化位点27个,乙酰化位点2个。MRP4蛋白分布于质膜上,具有ATP酶和跨膜转运蛋白活性,参与内外源性物质运输、前列腺素分泌、纤毛组装、信号转导运输等生命过程。本研究系统性梳理并整合了MRP4蛋白结构及功能等信息,为深入研究其在机体生命活动及疾病发生中的作用与机制奠定理论依据。

MRP4在去势抵抗性前列腺癌中的表达及意义

目的探讨多药耐药蛋白4(MRP4)在去势抵抗性前列腺癌(CRPC)中的表达及其作用机制。方法首先采用雄激素递减法建立CRPC细胞株(CR-LNCaP),应用RT-PCR及Western-blotting检测雄激素依赖性前列腺癌细胞株(LNCaP)及CR-LNCaP中MRP4基因的表达变化。然后用慢病毒感染RNA干扰技术(RNAi)沉默CR-LNCaP(KD组)中MRP4表达,并设计未感染病毒的细胞株(CON组)及加阴性对照病毒感染细胞株(NC组)作为对照,应用MTS法、流式细胞术检测RNAi-MRP4后3组细胞的增殖活性及凋亡情况。结果 CR-LNCaP中MRP4 mRNA(0. 89±0. 18 vs. 0. 35±0. 10,P <0. 05)和蛋白(0. 92±0. 28 vs.0. 12±0. 05,P <0. 01)明显高于LNCaP。KD组中MRP4 mRNA(0. 15±0. 01)及蛋白表达(0. 09±0. 02)明显低于CON组(mRNA:0. 95±0. 15,P <0. 01;蛋白:0. 91±0. 11,P <0. 01)及NC组(mRNA:0. 85±0. 11,P <0. 05;蛋白:0. 83±0. 09,P <0. 01)组,并伴随细胞增殖能力下降(P <0. 05)及凋亡数目增加(P <0. 01)。结论雄激素依赖性前列腺癌向CRPC转变过程中伴随有MRP4过表达,下调MRP4表达有抑制肿瘤细胞增殖及促凋亡作用,提示MRP4有可能成为CRPC治疗的新靶点。

慢性阻塞性肺疾病合并肺动脉高压患者血清多药耐药蛋白4水平的变化及临床意义

目的探讨多药耐药蛋白4(MRP4)在慢性阻塞性肺疾病(COPD)合并肺动脉高压(PH)患者血清中的表达及临床意义。方法选取2023年2月13日至2024年2月13日贵州省人民医院住院确诊COPD患者150例,根据超声心动图结果将COPD患者分为COPD组(50例)和COPD合并PH组(COPD+PH组,100例)。另选取本院同期体检健康受试者50例作为对照组;按肺动脉收缩压(PASP),将COPD+PH组分为轻度组[36 mmHg(1 mmHg=0.133 kPa)≤PASP≤50 mmHg]、中度组(51 mmHg≤PASP≤70 mmHg)和重度组(PASP>70 mmHg)。检测MRP4、肺功能、血气分析指标、红细胞体积分布宽度(RDW)、平均血小板体积(MPV)、B型脑钠肽(BNP)及白蛋白水平。Pearson相关性模型进行相关性分析;受试者工作特征(ROC)曲线评估MRP4预测COPD合并PH的价值。结果COPD组MRP4水平低于对照组,COPD+PH组MRP4水平高于对照组和COPD组(均P<0.05)。COPD+PH组白蛋白水平低于COPD组,RDW、MPV、BNP水平均高于COPD组(均P<0.05)。中度组、重度组MRP4、RDW、MPV、BNP水平均高于轻度组,且重度组均高于中度组;白蛋白水平均低于轻度组,且重度组低于中度组(均P<0.05)。COPD+PH组患者MRP4水平与BNP、PASP、RDW、MPV呈正相关(r=0.72、0.85、0.70、0.62,均P<0.01),与白蛋白呈负相关(r=-0.60,P<0.01)。ROC曲线分析结果显示,当MRP4截断值为2.895μg/L时,预测COPD合并PH的曲线下面积为0.917(95%置信区间:0.865~0.970),敏感度为93.00%,特异度为88.00%。结论MRP4在COPD合并PH患者血清中高表达,其可能是潜在的生物标志指标。

NNK对BEP2D细胞耐药基因表达的影响

目的探讨4-甲基亚硝胺-1-3-呲啶基-1-丁酮(NNK)对BEP2D细胞多药耐药相关蛋白(MRP)、肺耐药相关蛋白(LRP)表达的影响。方法应用NNK 500μg/m l干预BEP2D细胞生长,收集连续干预后第10、20、30代NNK-500细胞,行接种裸鼠成瘤试验检验其成瘤性;采用RT-PCR检测BEP2D细胞,以及第10、20、30代NNK-500细胞的MRP、LRP表达量。结果经NNK干预后的第20、30代NNK-500细胞接种裸鼠成瘤,病理切片显示为高分化鳞癌;RT-PCR显示,在BEP2D细胞中MRP、LRP呈低度表达,第10、20代NNK-500细胞的表达量略有增加,第30代NNK-500细胞的表达量明显增加(P<0.01)。结论 NNK对BEP2D细胞有致癌性,可增加BEP2D细胞的MRP、LRP表达,降低化疗敏感性。

尿酸转运蛋白与血清尿酸平衡

尿酸在肝生成到肾和肠道的排泄、重吸收这一过程有多种尿酸转运蛋白参与。现有研究表明葡萄糖转运体9在肝、肾和肠道的尿酸跨膜转运中起着重要作用,参与尿酸的重吸收;三磷酸腺苷结合盒转运蛋白G2主要表达在肾近端小管上皮细胞顶膜,参与尿酸的分泌作用;多药耐药蛋白4表达于肾小管上皮细胞顶端膜,可将尿酸从肾小管上皮细胞转运到肾小管腔内;尿酸盐阴离子转运体1、有机阴离子转运体1和3均属于有机阴离子转运蛋白,是SLC22A家族跨膜转运蛋白的成员,参与肾的尿酸转运,尤其是尿酸的分泌和排泄。本文综述了这些尿酸转运蛋白参与调控血清尿酸平衡相关的研究进展。

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.

Calculus Bovis Sativus up-regulates hepatic protein 2(Mrp2) and Mrp4 in 17α-ethynylestradiol-induced cholestasis via a regulatory effect on ER signaling

OBJECTIVE:To investigate the pathway through which Calculus Bovis Sativus (CBS) up-regulates hepatic multidrug resistance-associated protein 2 (Mrp2) and Mrp4 in 17α-ethynylestradiol (EE)-induced cholestasis.METHODS:Five groups of rats were designed:control group,EE+ICI182780 group,EE group,EE+CBS 50 mg/kg group and EE + CBS 150 mg/kg group.CBS (50 and 150 mg.kg-1· d-1) was orally given to rats by gavage for five consecutive days in coadministration with EE.The levels of cholestasis biomarkers,alanine aminotransferase (ALT),aspartate aminotransferase (AST),alkaline phosphatase (ALP) and total bilirubin (TBIL) were determined by biochemical methods.The bile flow was measured.The histopathology of the liver tissue was evaluated.The expression of Mrp2,Mrp3,Mrp4,estrogen receptor α(ERα) and ERβ was determined by Western blotting.RESULTS:CBS markedly improved EE-induced cholestasis.EE exposure significantly reduced hepatic Mrp2 and Mrp4 expression compared with the control group.EE also dramatically up-regulated the expression of Mrp3.Compared to the EE group,CBS notably up-regulated hepatic Mrp2 and Mrp4 but failed to influence the Mrp3 level significantly.ICI182780,an ER antagonist,showed similar beneficial effects as CBS.Decreased expression of Mrp2 and Mrp4 caused by EE was also restored by IC1182780.Additionally,EE significantly induced hepatic ERα expression,which was reversed by ICI182780 or CBS (150 mg/kg) treatment,suggesting that CB5 exerted a moderate regulatory effect on ER signaling.CONCLUSION:CBS up-regulated hepatic Mrp2 and Mrp4 expression in EE-induced cholestasis,which might be associated with its regulation of ER signaling.

EXPRESSION OF MDRII MRP AND LRP GENES IN GASTRIC CARCINOMA AND THEIR CLINICAL SIGNIFICANCE

Objective: To explore the expression of mdrl,multidrug resistance-associated protein (MRP) and lungresistance protein (LRP) genes in human gastric cancerand their clinical significance. Methods: The mdrlmRNA was assayed by RT-PCR, the MRP and LRPwere detected by flow cytometry. Results: The positiverate of mdrl mRNA was 44.4% (12/27), and the meanMRP and LRP expression were independent uponpatient histologic type, nodal involvement, and TNMstage. The mdrl mRNA expression in patients withserosa invasion was 30.0% (6120), much lower than thatwithout serosa invasion (85.7%). Conclusion: Themultidrug resistance cells are present in primary gastriccarcinomas prior to chemotherapy, and analysis of mdrlgene, MRP, LRP may have guiding significance in thetreatment of gastric carcinoma.

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.

Construction of Multi-ribozyme Expression System and Its Characterization of Cleavage on the MDR1/MRP1 Double Target Substrate in vitro

To improve catalytic activity of ribozyme on its substrate, the multi-ribozyme expression system was designed and constructed from 20 cis-acting hammerhead ribozymes undergoing self-cleavage with 10 trans-acting hammerhead ribozymes inserted alternatively regularly and the plasmid of pGEM-MDR1/MRP1 used to transcribe the MDR1/MRPl（196/210） substrate containing double target sites was also constructed by DNA recombination. Endonuclease digestion analysis and DNA sequencing indicate all the recombinant plasmids were correct. The clea- vage activities were evaluated for the multi-ribozyme expression system on the MDR1/MRP1 substrate in the cell free system. The results demonstrate that the cis-acting hammerhead ribozymes in the multi-ribozyme expression system were able to cleave themselves and the 72 nt of 196Rz and the 71 nt of 210Rz trans-acting hammerhead ribozymes were liberated effectively, and the trans-acting hammerhead ribozymes released were able to act on the MDR1/MRP1 double target RNA substrate and cleave the target RNA at specific sites effectively. The multi- ribozyme expression system of the [Coat＇A196Rz/Coat＇B210Rz]5 is more significantly superior to that of the [Coat＇A 196Rz/Coat＇B210Rz] 1 in cleavage of RNA substrate. The fractions cleaved by [Coat＇A 196Rz/Coat＇B210Rz]5 on the MDR1/MRP1 substrate for 8 h at observed temperatures showed no marked difference. The studies of Mg^2＋ on cleavage efficiency indicate that cleavage reaction is dependent on Mg^2＋ ions concentration. The plot of lg（kobs） vs. lgc（Mg^2＋） displays a linear relationship between 2.5 mmol/L and 20 mmol/L Mg^2＋. It suggests that Mg^2＋ ions play a crucial role in multi-ribozyme cleavage on the substrate.

EFFECTS OF NEOADJUVANT CHEMOTHERAPY ON MDR1 AND MRP GENE EXPRESSION IN PRIMARY BREAST CANCER

Objective: To investigate the effects of neoadjuvant chemotherapy on the expression of drug resistance genes, multidrug resistance-1 (MDR1) and multidrug resistance-associated protein (MRP), in patients with primary breast cancer. Methods: MDR1 and MRP expression were detected by semi-quantitative RT-PCR in 20 patients with primary breast cancer, before and after chemotherapy. Results: Before chemotherapy, MDR1 and MRP expression can be detected in 15 cases (75%) and 18 cases (90%) respectively. After chemotherapy, expression of MDR1 is not significantly different from that before chemotherapy, but expression of MRP is significantly different from that before chemotherapy. Conclusion: Expression of drug resistance gene MRP, but not MDR1, is enhanced in patients with primary breast cancer submitted to neoadjuvant chemotherapy.