顺铂诱导的人NCI-H460细胞获得性耐药与p53基因突变的关系

目的　探讨p53基因突变与肺癌化疗后获得性耐药的关系。方法　采用顺铂(DDP)大剂量(50μmol/L)间歇诱导法,体外诱导具有野生型p53基因的人大细胞肺癌NCIH460细胞株,建立多药耐药细胞系H460/DDP;用免疫细胞化学法检测P糖蛋白(Pgp)、多药耐药相关蛋白(MRP1)、肺相关蛋白(LRP)、拓扑异构酶Ⅱ(TOPOⅡ)、谷胱甘肽转移酶π(GSTπ)及P53蛋白的表达状况;用荧光免疫法测定P53蛋白磷酸化状态;对p53cDNA全长进行扩增及测序,用含野生型p53基因的质粒pShuttleCMVwtp53cDNA转染耐药细胞,测定转染细胞的药物敏感性。结果　新建立的人大细胞肺癌多药耐药株H460/DDP,对DDP及卡铂的耐药指数分别为1021和998,对氟尿嘧啶、多柔比星、表柔比星、甲氨蝶呤、依托泊苷及异长春新碱等有不同程度的交叉耐药。多药耐药株H460/DDP细胞的P53蛋白滞留于细胞浆,在DDP刺激下不能发生磷酸化;LRP表达明显增加(P<005);但其他耐药相关蛋白水平与DDP诱导前相比差异无统计学意义(P>005);耐药株细胞p53基因第277位点后插入一个“t”;耐药株转染pShuttleCMVwtp53cDNA后,其耐药性可发生部分(532%)逆转。结论　铂类化疗药物诱导的NCIH460细胞p53基因突变与其对化疗药物耐药性的产生有密切关系,p53基因替代疗法可能是克服这类获得性耐药的一个有效方     

结肠癌细胞中TS、TP、GST-π、Pgp、MRP1表达对奥沙利铂化疗敏感性的预测价值

目的探讨结肠癌细胞中TS、TP、GST-π、Pgp、MRP1表达对奥沙利铂化疗敏感性的预测价值。方法 Realtime RT-PCR检测不同结肠癌细胞株中TS、TP、GST-π、Pgp、MRP1mRNA的表达,结合奥沙利铂的体外药物敏感实验,分析TS、TP、GST-π、Pgp、MRP1的表达水平与奥沙利铂化疗敏感性的相关性。结果奥沙利铂IC50均值与GST-π(rs=0.609,P=0.001)、Pgp(rs=0.428,P=0.026)、TP(rs=0.394,P=0.042)呈正相关,与TS(rs=0.322,P=0.101)、MRP1(rs=0.090,P=0.655)无显著相关关系。结论检测结肠癌细胞中TP、GST-π、Pgp表达水平对奥沙利铂化疗敏感性具有一定预测价值,对临床医生合理选择结肠癌化疗方案具有指导意义。     

非小细胞肺癌肿瘤药敏与耐药基因MDR1、MRP、GST-π表达的相关性研究

目的研究非小细胞肺癌(NSCLC)多药耐药(MDR1)、谷胱甘肽转移酶(GST-π)、多药耐药相关蛋白(MRP)基因表达和肿瘤药物敏感试验之间的相关性及临床指导意义.方法 48例NSCLC(可手术)进入研究,药敏方法采用磷脂结合蛋白V(Annexin V)联合碘化丙啶(PI)双参数法,耐药基因MDR1、GST-π、MRP采用逆转录聚合酶链反应(RT-PCR)检测.结果抗癌药物检测显示紫杉醇(商品名泰素)、顺铂、去甲长春花碱、丝裂霉素、鬼臼乙叉甙、双氟胞苷、长春碱酰胺、长春新碱的平均抑瘤率分别为(15.7±21.8)%, (20.7±22.2)%,(7.9±16.2)%,(10.3±17.1)%,(9.7±20.1)%,(11.2±13.8)%,(5.6±14.9)%,(4.7 ±8.7)%.耐药基因MDR1、MRP、GST-π阳性率分别为67%(32/48),42%(20/48),48%(23/48).耐药基因MRP、GST-π阳性及阴性表达与病理类型之间未见明显相关;而在MDR1组中鳞癌及腺癌的MDR1阳性表达明显高于阴性表达组(P<0.05).NSCLC各期别中MDR1、MRP、GST-π阳性表达统计学上未见明显差异.在所检测的所有抗癌药物与MDR1耐药基因阳性与阴性表达未见相关性.MRP表达阳性者对去甲长春花碱、长春碱酰胺、长春新碱和丝裂霉素的肿瘤抑制率显著低于阴性表达组(P<0.05).而MRP表达与顺铂、鬼臼乙叉甙、紫杉醇肿瘤药敏未见明显相关.耐药基因GST-π阳性表达者对顺铂、去甲长春花碱、丝裂霉素的抑瘤率显著低于阴性表达组(P<0.05).结论肺癌耐药基因GST-π、MRP表达与部分化疗药物肿瘤药敏存在相关性,耐药基因检测对指导临床化疗药物的选择具有一定的帮助.     

肺癌药敏与耐药基因MRP1、P-gp、GST-π和TopoⅡ表达的相关性研究

目的 研究多药耐药相关蛋白1 （MRP1）、P-糖蛋白（P-gp）、谷胱甘肽S转移酶-π（GST-π）、拓扑异构酶Ⅱ（TopoⅡ）基因表达和肺癌药物敏感试验相关性.方法 药物敏感试验采用MTT比色法,MRP1、P-gp、GST-π及TopoⅡ采用免疫组化检测.结果 MRP1、P-gp、GST-c及TopoⅡ在肺癌中总的阳性率分别为72.5％、67.5％、57.5％、50.0％.在鳞癌和腺癌中,这四种因子其表达无显著性差异（P＞0.05）,但P-gp、GST-π在鳞癌或腺癌中与在小细胞肺癌中表达有明显差异（P＜0.05）.P-gp和GST-π表达与顺铂、吉西他滨、长春瑞滨、紫杉醇耐药性呈正相关（P＜0.05）,与异环磷酰胺的耐药性无相关性（P ＞0.05）;MRP1的表达与顺铂、吉西他滨、长春瑞滨的耐药性均呈正相关（P＜0.05）,其表达与紫杉醇、异环磷酰胺的耐药性无相关性（P＞0.05）;TopoⅡ的表达与顺铂、吉西他滨、紫杉醇、异环磷酰胺的耐药性均呈正相关（P＜0.05）,其表达与长春瑞滨的耐药性无相关性（P＞0.05）.结论 MRP1、P-gp和GST-π的高表达及TopoⅡ的低表达共同介导参与了肺癌耐药的机制,且与检测的化疗药物耐药性有不同程度的相关性.     

耐药相关蛋白在肺癌组织的表达与药物敏感性的关系

目的探讨耐药相关蛋白在肺癌组织中的表达与药物敏感性的关系。方法应用免疫组化法、四唑蓝快速比色法(MTT法)分别检测P-糖蛋白(P-gp)、多药耐药相关蛋白(MRP)、肺耐药蛋白(LRP)、谷胱苷肽s转移酶(GST-π)蛋白在87例肺癌组织中的表达和10种抗癌药的体外药敏试验。结果P-gp、MRP、GST-π阳性表达及高表达者中,腺癌组药物敏感数明显低于鳞癌组(P<0.05),LRP阳性表达者中NSCLC组药物敏感数明显低于LRP阴性表达者(P<0.05)。LRP与MMC、5-FU、VP-16、VCR均呈负相关(P<0.05或0.01)。P-gp与顺铂(CDDP)呈负相关(P<0.05)。GST-π与MTX、HCPT呈正相关(P<0.05行)。结论P-gp、MRP、LRP、GST-π的蛋白表达及共表达与体外药敏试验存在较好相关性,可作为监测肺癌细胞原发性耐药的指标。     

肺癌组织中LRP、GST-π和p53的表达及相关性研究

目的：探讨肺耐药蛋白(LRP)基因、胎盘型谷胱甘肪S转移酶(GST—π)及p53蛋白在肺癌组织中的表达及相关性。方法：应用免疫组化S—P法检测58例术前未经化疗的肺癌组织中LRP、GST—π及p53蛋白的表达情况。结果：LRP、GST—π及p53在58例肺癌组织中的阳性表达率分别为55．17％、60．34％和74．14％，LRP的表达在不同的组织类型中以腺癌最高，达78．57％，它与鳞癌组相比差异有显性(p＜0．05)；男女组相比，女性组表达率达78．95％，差异亦有显性(p＜0.05)，而与肿瘤的大小及淋巴结转移均无关(p＞0．05)；GST—π的表达在淋巴结转移阳性组高达78．57％，与阴性组比较差异有显性(p＜o．05)，而与肿瘤的类型、大小及男女性别无关(p＞0.05)。p53蛋白在淋巴结转移阳性组为89．29％，与阴性组相比差异有显性(p＜0．05)，而p53的表达与肿瘤的类型、大小及性别无明显关系(p＞0．05)；将LRP、GST—π及p53蛋白的表达进行相关性比较发现GST—π及p53蛋白的表达关系密切(p＜0.05)。结论:LRP的表达与肺癌，尤其是肺腺癌的耐药有关，GST—π及P53蛋白的简表达与肺癌的淋巴结转移相关，两的表达关系密切，并可作为肺癌病人预后判断的一个指标。     

耐药相关蛋白在肺癌组织中的表达及意义

目的　探讨耐药相关蛋白在肺癌组织中的表达及意义。方法　应用免疫组化法检测治疗前 90例肺癌组织标本中 P-糖蛋白 (P- gp)、多药耐药相关蛋白 (MRP)、肺耐药蛋白 (L RP)、谷胱苷肽 S转移酶 (GST- π)、DNA拓扑异构酶 (Topo- )蛋白的表达 ;流式细胞术检测肺癌组织增值指数 (PI)、DNA指数 (DI)及细胞周期各时相分布。结果　非小细胞癌 (NSCL C组 ) P- gp、L RP表达明显高于小细胞癌 (SCL C)组 (P <0 .0 5 ) ,MRP+L RP共表达者高于 SCL C组 (P <0 .0 5 ) ;中、高分化癌组 P- gp、L RP表达高于 SCL C组 (P <0 .0 5 ) ,MRP表达高于低分化癌组 (P <0 .0 5 ) ;腺癌组 MRP、L RP、MRP+L RP、MRP+L RP+GST- π、MRP+L RP+P- gp+GST-π表达高于鳞癌组、SCL C组、低分化癌组 (P <0 .0 5 ) ,MRP+GST- π共表达者高于鳞癌组、低分化癌组 (P <0 .0 1)。P- gp与 Topo 蛋白表达呈正相关 (P <0 .0 5 )。MRP与肺癌组织增殖指数 (PI)、G2 / M期细胞比例呈正相关 (P <0 .0 5 ) ,与 G0 / G1 期细胞比例呈负相关 (P <0 .0 5 )。结论　联检肺癌组织中耐药相关基因蛋白的表达有助于判断化疗疗效及预后。 P- gp、L RP、MRP、GST-π可作为判断肺癌细胞原发性耐药的指标。     

肺腺癌患者胸水中肿瘤耐药相关基因蛋白的表达及临床意义

目的检测P-糖蛋白(P-gp)、肺癌耐药蛋白(LRP)、多药耐药相关蛋白(MRP)在肺腺癌及恶性间皮瘤患者胸水沉渣包埋组织中的表达及临床意义。方法采用S-P免疫组化方法,对63例未做放化疗的肺腺癌或恶性间皮瘤患者进行检测,记录其胸水沉渣包埋组织中P-gp、LRP、MRP的表达水平。结果肺腺癌患者胸水沉渣包埋组织中P-gp、LRP、MRP的阳性率均高于非肿瘤性胸水患者(P0.05);恶性间皮瘤患者胸水沉渣包埋组织中P-gp、LRP的阳性率均高于非肿瘤性胸水患者(P0.05),但两者MRP阴性率无显著差异(P0.05)。P-gp、LRP、MRP与肺腺癌患者年龄、组织分化程度及淋巴结转移无密切相关(P0.05)。结论肺腺癌及恶性间皮瘤肿瘤细胞对转运蛋白类耐药基因有较强的耐药性,检测Pgp、LRP、MRP的阳性表达对指导临床化疗方案的实施有重要意义。     

胃癌组织FAT10与突变型p53表达及其相互关系

目的 检测FAT10与突变型p53在胃癌组织中的表达情况及相互间关系.方法 采用免疫组化和逆转录-聚合酶链反应(RT-PCR)方法检测FAT10和突变型p53在62例胃癌患者的胃癌组织、癌旁组织(距癌边缘2～5 cm)及正常胃组织(距癌边缘>5 cm)中的表达水平,统计分析两者表达间的关联性及FAT10与胃癌预后之间的关系.结果 FAT10蛋白和突变型p53蛋白在胃癌组织中的表达阳性率分别为51.61%(32/62)和45.16%(28/62),显著高于癌旁组织[12.90%(8/62)和14.51%(9/62),χ2值分别=21.26和20.69,P值均<0.01]及正常胃组织[6.45%(4/62)和9.68%(6/62),χ2值分别=13.91和19.61,P值均<0.01].胃癌FAT10蛋白及mRNA的表达上升主要与胃癌的淋巴转移及TNM分期密切相关(P值均<0.05).胃癌组织中FAT10表达在蛋白及基因水平均与突变型p53表达呈正相关性(r值分别=0.865和0.761,P值分别<0.05和0.01).FAT10蛋白阳性表达或mRNA表达水平高者,其累计生存时间显著低于FAT10阴性者(P值均<0.05).结论 FAT10与突变型p53在胃癌组织中表达上调且呈正相关性,两者可能共同参与胃癌的形成与发展,FAT10对评价胃癌的预后可能有较好的价值.     

p73基因蛋白表达与老年人肺癌临床病理学特征及预后关系的探讨

目的探讨p73基因蛋白与老年人肺癌发生、发展的关系。方法应用免疫组化SABC法检测了65例老年人肺癌组织中抑癌基因p73基因蛋白的表达,并与癌旁组织和正常肺组织对比,同时结合肺癌的临床病理学特征及预后进行分析。结果老年人肺癌组织中p73基因蛋白阳性表达率(477%)明显高于癌旁组织(92%)和正常肺组织(46%),P<005;不同年龄组、不同类型和不同分化程度肺癌组织中p73基因蛋白的阳性表达率差异无显著性(P>005);Ⅰ期和Ⅱ期老年人肺癌组织中p73基因蛋白阳性表达率(605%)明显高于Ⅲ期和Ⅳ期(227%),P<005;生存3年或以上的老年人肺癌组织中p73基因蛋白阳性表达率(722%)明显高于生存3年以下者(160%),P<005。结论老年人与非老年人肺癌在p73基因蛋白免疫组化染色表达中有共同性,与正常肺组织或癌旁组织相比明显增加,对于进一步探讨p73基因蛋白在肺癌发生发展中的表达及调节有参考价值;p73基因蛋白表达在各型肺癌发生、发展中可能具有普遍意义,可作为判断老年人肺癌生物学行为和预后的一个参考指标。     

非小细胞肺癌多药耐药及其与神经内分泌分化的相关性研究

目的　检测非小细胞肺癌 (NSCLC)多药耐药 (MDR)有关蛋白的表达 ,探讨其与神经内分泌 (NE)分化的关系。方法　采用链霉亲和素 过氧化酶连接 (SP)免疫组化法检测 113例NSCLC中谷胱甘肽S转移酶π(GST π)、多药耐药相关蛋白 (MRP)、肺耐药相关蛋白 (LRP)、神经元特异性烯醇化酶 (NSE)、突触素 (SYN)和嗜铬素A(CgA)的表达。结果　 (1)NSCLC中MRP和LRP的表达与组织学类型有关 (P <0 0 5 )。GST π与MRP、MRP与LRP表达之间呈显著相关 (P <0 0 5 )。 (2 )NSCLC中NSE、SYN、CgA的阳性率分别为 5 3 1%、2 6 6%、6 2 %。至少有 2种NE标记物表达者有 2 4例 (2 1 2 % ) ,其表达与肿瘤的分化程度有关 (P <0 0 5 )。 (3 ) 3种MDR有关蛋白在至少有 2种NE标记物阳性组的表达均较阴性组为低 (P <0 0 5 )。结论　GST π、MRP和LRP的过表达是NSCLC产生原发性多药耐药的重要原因。NE分化可能是影响多药耐药有关蛋白表达的因素之一     

In vitro chemoresistance profile and expression/function of MDR associated proteins in resistant cell lines derived from CCRF-CEM,K562, A549 and MDA MB 231 parental cells

Although cellular experiments have elucidated a number of active principles in the study of the multidrug resistance (MDR) phenomena, most of the drug resistant tumor cells were derived from different parental cell lines. This fact limits generalization of some experimental data and conclusions, and therefore we selected and characterized cell lines resistant to various anti-cancer agents derived from four parental cell lines: CEM (human T-lymphoblastic leukemia), K562 (human myeloid leukemia), A549 (human lung adenocarcinoma) and MDAMB 231 (human breast adenocarcinoma). In total we obtained a set of 42 resistant sublines, which is an excellent tool for the future studies of different aspects of MDR. In this study we report on some basic characteristics of these sublines, namely, cross-resistance to other anti-cancer drugs investigated by in vitro MTT assay, expression of MDR associated proteins (Pgp, MRP1, LRP, GST-pi and Topo IIalpha) as well as the functional activity of Pgp and MRP.     

Relationship Between Chemotherapy Response of Small Cell Lung Cancer and P-glycoprotein or Multidrug Resistance-Related Protein Expression

The resistance of small cell lung cancer (SCLC) to anticancer drugs is a serious clinical problem often encountered during chemotherapy. Therefore, how to prevent this drug resistance need to be investigated. Multidrug resistance 1 (MDR1) gene and multidrug resistance-related protein (MRP) gene, two genes known to be associated with the development of drug resistance, are very common in SCLC. The purpose of this study was to evaluate retrospectively the relationship between chemotherapy responses to MDR1 gene encodes 170 kDa P-glycoprotein (Pgp) expression or MRP gene encodes 190 kDa MRP expression in SCLC. Before chemotherapy, multiple nonconsecutive sections of the bronchoscopy biopsy specimens of SCLC from 50 patients were analyzed immunohistochemically to detect Pgp and MRP expressions. Chemotherapy responses of the 50 patients were evaluated in the third month after completion of treatment by clinical and radiological methods. Of the 23 SCLC patients with poor response to chemotherapy, 11 had positive Pgp and MRP expressions, 2 had positive Pgp but negative MRP expressions, 6 had positive MRP but negative Pgp expressions, and 4 patients had negative Pgp and MRP expressions. All 27 SCLC patients with good response had negative Pgp and MRP expression. Immunohistochemical analyses of Pgp or MRP expression are potential tools for predicting patients' chemotherapy response in SCLC.     

ATP binding cassette transporters and drug resistance in breast cancer

Resistance to chemotherapy is a critical issue in the management of breast cancer patients. The nature of clinical drug resistance is likely to be multifactorial. However, in the last decade considerable attention has been dedicated to the role played by membrane transporter proteins belonging to the ATP binding cassette protein superfamily, and in particular by the MDR1 product P-glycoprotein (Pgp) and the multidrug resistance protein (MRP1). Heterogeneity of results is a common feature of studies evaluating the expression and prognostic role of these proteins, due to both methodological and biological factors. Nonetheless, Pgp and MRP1 are detected in a significant proportion of untreated breast cancers (on average 40 and 50% respectively, by immunohistochemistry), without a clear and consistent association with cancer stage. Exposure to chemotherapy increases the expression of both proteins. In vitro studies on primary cultures of breast cancer cells obtained at surgery consistently show an association between Pgp (protein) or MDR1 (mRNA) expression and resistance to chemotherapy. However, the correlation with clinical drug resistance is not as well defined. A stronger association of Pgp/MDR1 with response rates has been observed when expression or an increase in expression are detected immediately following chemotherapy. Correlations with prognosis appear more evident in studies using immunohistochemistry, in adjuvant and neoadjuvant settings. Evidence of clinical reversal of drug resistance by verapamil suggests a functional role of Pgp in drug resistance, although the significance of the evidence is generally weakened by poor trial designs. Future studies should take into account the multifactorial nature of drug resistance in breast cancer and use standardized approaches with adequate controls. Expression studies should be complemented by well-designed trials of drug-resistance reversal using target-specific chemosensitizing agents, and relating the results to the levels of expression of the target proteins.     

p53-dependent regulation of MDR1 gene expression causes selective resistance to chemotherapeutic agents

Loss of functional p53 paradoxically results in either increased or decreased resistance to chemotherapeutic drugs. The inconsistent relationship between p53 status and drug sensitivity may reflect p53’s selective regulation of genes important to cytotoxic response of chemotherapeutic agents. We reasoned that the discrepant effects of p53 on chemotherapeutic cytotoxicity is due to p53-dependent regulation of the multidrug resistance gene (MDR1) expression in tumors that normally express MDR1. To test the hypothesis that wild-type p53 regulates the endogenous mdr1 gene we stably introduced a trans-dominant negative (TDN) p53 into rodent H35 hepatoma cells that express P-glycoprotein (Pgp) and have wild-type p53. Levels of Pgp and mdr1a mRNA were markedly elevated in cells expressing TDN p53 and were linked to impaired p53 function (both transactivation and transrepression) in these cells. Enhanced mdr1a gene expression in the TDN p53 cells was not secondary to mdr1 gene amplification and Pgp was functional as demonstrated by the decreased uptake of vinblastine. Cytotoxicity assays revealed that the TDN p53 cell lines were selectively insensitive to Pgp substrates. Sensitivity was restored by the Pgp inhibitor reserpine, demonstrating that only drug retention was the basis for loss of drug sensitivity. Similar findings were evident in human LS180 colon carcinoma cells engineered to overexpress TDN p53. Therefore, the p53 inactivation seen in cancers likely leads to selective resistance to chemotherapeutic agents because of up-regulation of MDR1 expression.     

Simultaneous activity of MRP1 and Pgp is correlated with in vitro resistance to daunorubicin and with in vivo resistance in adult acute myeloid leukemia.

In adult acute myeloid leukemia (AML), the weight of the contribution of the combined activity of Pgp and MRP1 to drug resistance is not known. To address this question, we compared the activity of these proteins to the in vitro resistance to daunorubicin (DNR), etoposide, and cytosine arabinoside (Ara-C), using the calcein-AM uptake and the 3-[4, 5-di-methyl-thiazol-2, 5-diphenyl] tetrazolium bromide (MTT) assay in 80 adult AML patients. We found no correlation or only a weak correlation between the in vitro drug resistance to DNR and etoposide and MRP1 or Pgp expression or function when tested separately. However, a strong correlation was observed between the simultaneous activity of MRP1 and Pgp (quantified as the modulation of calcein-AM uptake by cyclosporin A and probenecid) and the LC50 of DNR (r =.77, P <.0001). This emphasized the role of these two proteins, not separately, but together in the resistance to DNR. In contrast, Mvp/LRP expression did not correlate with the LC50 of DNR. A high level of simultaneous activity of Pgp and MRP1 was predictive of a poor treatment outcome (for achievement of CR [P =.008], duration of relapse-free survival [RFS; P =.01], and duration of overall survival [OS; P =.02]). In addition, high LC50 of DNR and high LC50 of etoposide together were also predictive of a poor treatment outcome (for duration of RFS [P =.02] and duration of OS [P =.02]). The unfavorable cytogenetic category was more closely associated with the combined activity of both MRP1 and Pgp (P =.002) than with the activity of Pgp or MRP1 separately. This could explain the poor prognosis and the in vitro resistance to daunorubicin in this group of patients. These data suggest that treatment outcome may be improved when cellular DNR and etoposide resistance can be circumvented or modulated. Modulation of not only Pgp but also MRP1 could be essential to attain this aim in adult AML.