MRP、GST-π、TopoⅡα和LRP在胃癌组织中的表达及意义

背景与目的:多药耐药相关蛋白(multidrugresistance-associatedprotein,MRP)、肺耐药蛋白(lungresistanceprotein,LRP)、谷胱甘肽转移酶(glutathione-S-transferase-π,GST-π)和拓扑异构酶Ⅱα(topoisomeraseⅡα,TopoⅡα)均在多药耐药中发挥重要作用。联合检测它们在胃癌组织中的表达,目前国内外报道极少。本研究旨在探讨联合检测MRP、GST-π、TopoⅡα、LRP在胃癌组织中的表达及意义。方法:应用免疫组化SP法检测MRP、GST-π、TopoⅡα、LRP在90例胃癌标本中的表达,采用χ2检验和Fisher精确检验分析它们表达的意义。结果:(1)MRP、GST-π、TopoⅡα、LRP在胃癌组织中的阳性表达率分别为88.9%、91.1%、74.4%和87.7%,均高于在正常胃粘膜组织中的表达(P<0.05)。(2)MRP、GST-π、LRP在高、中分化腺癌中的表达均高于低分化腺癌,而TopoⅡα在高、中分化腺癌中的表达低于低分化腺癌(P<0.05);此四者的表达情况在不同浸润程度及有/无淋巴结转移的胃癌组织之间均无统计学差异(P>0.05)。(3)MRP、GST-π、TopoⅡα、LRP两两间均无相关性。结论:MRP、GST-π、TopoⅡα和LRP均在胃癌原发性多药耐药中起重要作用,它们在胃癌组织中的表达与肿瘤分化程度有关,而与肿瘤浸润程度及是否有淋巴结转移无关。     

Increased LRP mRNA expression is associated with the MDR phenotype in intrinsically resistant human cancer cell lines

Multidrug resistance (MDR) in human cancer cells is multifactorial. Previously, we reported on the association between expression of P-glycoprotein (Pgp), the multidrug resistance-associated protein (MRP), and the lung resistance protein (LRP) with the MDR phenotype in the NCI panel of 60 human cancer cell lines used for in vitro anticancer drug screening. Eight cell lines from this panel, manifesting widely divergent levels of in vitro drug resistance were chosen to investigate the role of MRP and LRP expression at the molecular level. LRP mRNA levels, as determined by ribonuclease protection assay, varied significantly among the 8 cell lines, and correlated closely with in vitro drug resistance to both MDR and non-MDR related drugs. LRP mRNA expression was determined to be a stronger correlate of drug sensitivity than protein expression. In contrast, MRP mRNA levels were not significantly correlated with drug sensitivity. The rates of newly transcribed LRP or MRP mRNA did not correlate with mRNA levels, indicating that mRNA stability or other features of processing may be important in regulation of LRP and MRP mRNA levels. Using Southern blot analysis, LRP gene amplification was shown not to be associated with LRP overexpression. These data suggest that LRP expression may be an important determinant of the MDR phenotype in cell lines intrinsically resistant to cancer chemotherapeutic agents. Int. J. Cancer 72:1021–1026, 1997. © 1997 Wiley-Liss, Inc.     

Expression analysis of multidrug resistance associated genes in neuroblastomas.

In a series of 40 neuroblastomas we analyzed the relative mRNA levels of the MDR associated genes encoding MDR1/P-glycoprotein (MDR1), multidrug resistance associated protein (MRP), lung cancer resistance related protein (LRP) and topoisomerase IIalpha (TOPO IIalpha) by cDNA-PCR. Cyclin A (CYCA) was included to examine cellular proliferation activity. MYCN gene expression was analyzed as it was recently shown to be associated with enhanced MRP gene expression in neuroblastomas. We found that tumors with MYCN gene amplification exhibit significantly increased MYCN and MRP gene expression levels. Tumors with an allelic loss of the chromosomal 1p region showed significant (P<0.05) lower MDR1 gene expression (MDR1: 50+/-29, n=4) than tumors without (MDR1: 117+/-81, P<0.05, n=36). Moreover, significant positive correlations were found for MYCN/TOPO IIalpha (P<0.0001), MYCN/CYCA (P<0.05), TOPO IIalpha/CYCA (P<0.01), MRP/CYCA (P<0.0001) and MRP/LRP (P<0.05). Our results give evidence that MDR in neuroblastomas might be caused by multiple resistance factors and that a higher proliferation rate of neuroblastoma cells possibly based on altered MYCN gene expression is associated with enhanced MRP, CYCA and TOPO IIalpha gene expression.     

LRP,MRP,MDR1基因在非小细胞肺癌中的表达及其临床意义

目的 探讨肺耐药蛋白（ｌｕｎｇ ｒｅｓｉｓｔａｎｃｅ ｐｒｏｔｅｉｎ,ＬＲＰ）,多药耐药蛋白（ｍｕｌｔｉｄｒｕｇ ｒｅｓｉｓｔａｎｃｅａｓｓｏｃｉａｔｅｄ ｐｒｏｔｅｉｎ,ＭＲＰ）,和多药耐药基因（ｍｕｌｔｉｄｒｕｇ ｒｅｓｉｓｔａｎｅ,ＭＤＲ１）ｍＲＮＡ在非小细胞肺癌（ｎｏｎ－ｓｍａｌｌ ｃｅｌｌ ｃａｎｃｅｒ,ＮＳＣＬＣ）中的共表达及临床意义。方法 ＲＴ－ＰＣＲ检测ＮＳＣＬＣ冰冻组织中上述耐药     

Multiple gene expression analysis reveals distinct differences between G2 and G3 stage breast cancers, and correlations of PKC eta with MDR1, MRP and LRP gene expression.

A possible link between protein kinase C (PKC) and P-glycoprotein (P-gp)-mediated-multidrug resistance (MDR) was assumed from studies on MDR cell lines selected in vitro. The functional relevance of PKC for the MDR phenotype remains unclear, and the involvement of a particular PKC isozyme in clinically occurring drug resistance is not known. Recently, we have demonstrated significant correlations between the expression levels of the PKC eta isozyme and the MDR1 or MRP (multidrug resistance-associated protein) genes in blasts from patients with acute myelogenous leukaemia (AML) and in ascites cell aspirates from ovarian cancer patients. To extend these findings to further types of human tumours we analysed specimens from 64 patients with primary breast cancer for their individual expression levels of several MDR-associated genes (MDR1, MRP, LRP (lung cancer resistance-related protein), topoisomerase (Topo) II alpha/IIbeta, cyclin A and the PKC isozyme genes (alpha, beta1, beta2, eta, theta, and mu) by a cDNA-PCR approach. We found significantly enhanced mean values for MRP, LRP and PKC eta gene expression, but significantly decreased Topo II alpha and cyclin A gene expression levels in G2 tumours compared with G3. Remarkably, significant positive correlations between the MDR1, MRP or LRP gene expression levels and PKC eta were determined: MDR1/PKC eta (rs = +0.6451, P < 0.0001) n = 62; MRP/PKC eta (rs = +0.5454, P < 0.0001) n = 63; LRP/PKC eta (rs = +0.5436, P < 0.0001) n = 62; MRP/LRP (rs = +0.7703, P < 0.0001) and n = 62, MDR1/MRP (rs = +0.5042, P < 0.0001) n = 62. Our findings point to the occurrence of a multifactorial MDR in the clinics and to PKC eta as a possible key regulatory factor for up-regulation of a series of MDR-associated genes in different types of tumours.     

非小细胞肺癌MDR1—mRNA,MRP—mRNA及LRP—mRNA表达的研究

目的：观察肺癌组织及癌旁肺组织MDR1－mRNA,MRP-mRNA及LRP－mRNA的表达,方法：采用RT－PCR法检测30例肺癌患者组织和正常肺组织中MDR1－mRNA,MRP-mRNA和LRP－mRNA的表达情况。结果：MDR1－mRNA在肺癌组织及癌旁肺组织的表达阳性率分别为40％及16．67％（P＝0．045）,其表面与细胞分化程度,发期及病理类型无明显关系,MRP－mRNA在肺癌组织及癌旁肺组织的表达分别为43＝．44％及26．67％,低分化者MRP的表达率明显高于中高分化者（P＝0．0004）,其表达与肿瘤类型,临床分期及癌细胞的分化程度无明显关系。30例肺癌组织中MDR1－mRNA,MRP-mRNA,LRP-mRNA共同表达者7例（23．33％）,三者均无表达者10例（33．33％）,三者的一致性达56．67％,结论：MDR1－mRNA,MRP-mRNA,LRP-mRNA在肺癌的耐药中可能起重要作用。     

Chemosensitivity of prostate cancer cell lines and expression of multidrug resistance-related proteins

The aim of this study was to obtain insight into the role of the multidrug resistance (MDR) phenomenon in hormone-independent progressive prostate cancer. Using immunocytochemistry and Western blotting we determined the expression of P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP), glutathione-S-transferase-π (GST-π), Bcl-2, Bax, topoisomerase (Topo) I, IIα and IIβ in the human prostate cancer cell lines PC3, TSU-Pr1, DU145 and LNCaP derivatives LNCaP-R, LNCaP-LNO and LNCaP-FGC. Proliferative activity was assessed by immunocytochemistry. MTT assays were used to determine the sensitivity to etoposide, doxorubicin and vinblastin. Pgp was not expressed in any of the cell lines. MRP was variably expressed. GST-π was expressed in TSU-Pr1, PC3 and DU145. The expression of Bcl-2 was restricted to TSU-Pr1, whereas Bax was found in all cell lines. Topo IIα was expressed at the highest level in the rapidly proliferating cell lines TSU-Pr1 and DU145. Topo I and IIβ were equally expressed. Resistance profiles varied among the cell lines, with TSU-Pr1 being the most sensitive and LNCaP-LNO relatively resistant. Multiple MDR proteins were expressed in prostate cancer cell lines and may well influence response to chemotherapy. Future functional studies, using chemo-selected MDR models, may further help to determine the mechanism or combination of mechanisms underlying the resistance of prostate cancer to chemotherapy.     

Expression of multidrug resistance proteins,P-gp,MRP1 and LRP,in soft tissue sarcomas analysed according to their histological type and grade

The biological behaviour of different histological types and grades of soft tissue sarcomas (STS) varies. This might result in a differing sensitivity to cytotoxic drugs. Cross-resistance to functionally and structurally distinct natural-product drugs, known as multidrug resistance (MDR), is associated with the overexpression of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and lung resistance-related protein (LRP). The purpose of this study was to evaluate the expression of P-gp, MRP1 and LRP in STS according to their histological type and grade. In 141 chemotherapy-naive STS patients, the expression of the three MDR proteins was detected by immunohistochemistry. Nine histological types were documented. These were 19% grade 1, 34% grade 2 and 47% grade 3 tumours. Expression of P-gp and LRP was observed more frequently than the expression of MRP1 (P<0.0001). P-gp expression was most pronounced in malignant fibrous histiocytoma (MFH), but was low in leiomyosarcomas. MRP1 was expressed in most malignant peripheral nerve sheath tumours (MPNST). LRP was strongly expressed in MFH and unspecified sarcomas, but was low in liposarcomas. MRP1 and LRP expression was significantly more common in grades 2 and 3 compared with grade 1 tumours. P-gp expression was correlated with MRP1, especially in grade 3 STS. In conclusion, P-gp, MRP1 and LRP are expressed in the majority of STS, but this expression varies according to the histological type. MRP1 and LRP, but not P-gp expression, were found to be correlated to tumour grade. MDR might contribute to the observed differences in clinical behaviour within the heterogeneous group of STS.     

Induction of P-glycoprotein expression on the plasma membrane of human melanoma cells

Melanoma cells exhibit, both in vivo and in vitro, intrinsic drug resistance to various chemotherapeutic agents. Cultured human melanoma cells (M14) intrinsically express significant amounts of multidrug resistance-related protein (MRP1) and P-glycoprotein (P-gp) in the Golgi apparatus, but do not express these drug transporters on the plasma membrane. A panel of multidrug resistant (MDR) melanoma cell lines (M14Dx), showing different degrees of resistance to doxorubicin (DOX), were isolated. In M14Dx lines, the appearance of surface P-gp, but not of MRP1 or lung resistance related protein (LRP), occurred in cells grown in the presence of DOX concentrations higher than 60 nM. Furthermore, P-gp levels appeared to be dose-dependent. Flow cytometry, laser scanning confocal microscopy and cytotoxicity studies demonstrated that the activity of the drug extrusion system was related to both surface P-gp expression and resistance to DOX. In conclusion, P-gp, but not MRP1 or LRP, might play a pivotal role in the pharmacologically-induced MDR phenotype of melanoma cells.     

Expression of MDR1/P-glycoprotein,the multidrug resistance protein MRP,and the lung-resistance protein LRP in multiple myeloma

The purpose of this study was to determine the incidence of three genes associated with multidrug resistance (MDR) in multiple myeloma in relation to treatment status. MDR1/Pgp (P-glycoprotein) expression was detected in 41% of 93 myeloma samples. Generally, the incidence of MDR1/Pgp expression was higher in pretreated samples, and treatments with doxorubicin and/or vincristine were more effective in MDR1/Pgp expression than with alkylating agents. A significant association was observed between MDR1/Pgp-positiveness and the ability of verapmil to increase doxorubicin sensitivity, suggesting functional relevance of MDR1/Pgp expression. MRP (multidrug resistance protein) expression was detected in 20.5% of 88 myeloma samples, in 26% at the mRNA level analyzed by quantitative reverse transriptase-polymerase chain reaction, and in only 3 of 79 samples by immunohistochemistry. LRP (lung-resistance protein) protein expression was observed in 12.5% of 72 myeloma samples. MRP and LRP expression was similar in samples with and without prior therapy. Approximately 80% of the myeloma samples with detectable mRNA expression of MDR1 and MRP exhibited low expression levels corresponding to <10% of the Pgp- and MRP-overexpressing multidrug-resistant human myeloma cell lines 8226/Dox6 and 8226/DOXint40c, respectively. Some normal bone marrow samples showed higher levels of MRP mRNA as compared to myeloma specimens, whereas MDR1 mRNA expression in normal bone marrow was much lower (≤ 5%) than that in 8226/Dox6. These findings indicate a requirement to develop single-cell assays for MRP detection in multiple myeloma that are more sensitive than immunohistochemistry and might be useful to evaluate the incidence of genes associated with MDR.     

Expression of mdr1, mrp and lrp genes in gastric carcinoma and their clinical significance

Objective: To explore the expression of mdr1, multidrug resistance-associated protein (MRP) and lung resistance protein (LRP) genes in human gastric cancer and their clinical significance. Methods: The mdr1 mRNA was assayed by RT-PCR, the MRP and LRP were detected by flow cytometry. Results: The positive rate of mdr1 mRNA was 44.4% (12/27), and the mean MRP and LRP expression were independent upon patient histologic type, nodal involvement, and TNM stage. The mdr1 mRNA expression in patients with serosa invasion was 30.0% (6/20), much lower than that without serosa invasion (85.7%). Conclusion: The multidrug resistance cells are present in primary gastric carcinomas prior to chemotherapy, and analysis of mdr1 gene, MRP, LRP may have guiding significance in the treatment of gastric carcinoma.     

Drug resistance features and S-phase fraction as possible determinants for drug response in a panel of human ovarian cancer xenografts

Multidrug resistance (MDR) and more specifically the expression of P-glycoprotein (Pgp) have been studied extensively in vitro. Unfortunately, it appears that the predictive value of MDR recognized in vitro is mostly an incorrect measure to determine the responsiveness of a particular tumour in the clinic. This misunderstood or overvalued role of MDR might explain the failure of strategies to reverse Pgp function by the use of modulators in solid tumours. To obtain more insight in in vivo drug resistance we investigated a panel of 15 human ovarian cancer xenografts consisting of the most common histological subtypes known in ovarian cancer patients. The response rate to cisplatin, cyclophosphamide and doxorubicin in the xenografts resembled the results of phase II trials with these agents in ovarian cancer patients. This resemblance justifies drug resistance studies in this experimental in vivo human tumour system. We determined the expression levels of MDR 1, MRP 1, LRP and topoisomerase IIalpha mRNA by the RNase protection assay and the presence of MRP1 and LRP proteins by immunohistochemistry. The S-phase fraction was investigated as a separate parameter by flow cytometry. In none of the 15 ovarian cancer xenografts was MDR 1 expression detectable. The expression levels of MRP 1 and LRP were low to moderate and resembled the presence of the MRP1 and LRP proteins. There was a weak, inverse relationship between the expression levels of LRP and sensitivity to cisplatin and cyclophosphamide (r = -0.44 and -0.45), but not to doxorubicin. The levels of topoisomerase IIalpha varied among the xenografts (0.73-2.66) and failed to correlate with doxorubicin resistance (r = 0.14). The S-phase fraction, however, showed a relation with the sensitivity to cisplatin (r = 0.66). Among the determinants studied in ovarian cancer in vivo, LRP mRNA and the S-phase fraction were the best predictive factors for drug response and most specifically for the activity of cisplatin.     

Reversal of drug resistance using hammerhead ribozymes against multidrug resistance-associated protein and multidrug resistance 1 gene

We examined the effects of suppressing multidrug resistance-associated protein (MRP) and multidrug resistance 1 (MDR1) gene expression in HCT-8DDP human colon cancer cell lines, which showed both cisplatin and multidrug resistance. Hammerhead ribozymes, designed to cleave MRP mRNA (anti-MRP Rz) and MDR1 mRNA (anti-MDR1 Rz), were transfected into the HCT-8DDP cells. Drug sensitivity was estimated by MTT assay in vitro. The HCT-8DDP/anti-MRP Rz cells were more sensitive to doxorubicin (DOX) and etoposide (VP-16) by 2.5- and 4.1-fold, respectively, compared with HCT-8DDP cells. The HCT-8DDP/anti-MDR Rz cells were more sensitive to DOX and VP-16 by 2.3- and 3.8-fold, respectively. The anti-MRP Rz and anti-MDR1 Rz significantly down-regulated resistance to DOX and VP-16, while anti-MRP Rz and anti-MDR1 Rz did not affect resistance to cisplatin, methotrexate and 5-fluorouracil. The hammerhead ribozyme-mediated specific suppression of MRP or MDR1 was sufficient to reverse multidrug resistance in the human colon cancer cell line.     

非小细胞肺癌多药耐药性病理学检测的临床意义

 目的 检测113例非小细胞肺癌（NSCLC）中谷胱甘肽巯基转移酶π（GST-π）、肺耐药相关蛋白（LRP）、DNA拓扑异构酶Ⅱa（TopoⅡa）、多药耐药相关蛋白（MRP）和多药耐药基因（MDRt）的表达，观察上述指标与肿瘤I临床病理因素的关系及各指标表达之间的相关性。方法 采用免疫组化SP法检测GST-π、LRP、TopoⅡa蛋白的表达；应用原位杂交技术检测MRP和MDR1 mRNA表达。结果 （1）GST-π、LRP、TopoⅡa蛋白及MRP、MDR1 mRNA在113例NSCLC中的阳性表达率分别为75.2％、80．5％、60．2％、79．6％、51．3％。其中LRP表达最高，MDR1表达最低。（2）LRP、TopoⅡa和MRP的表达分别与肿瘤组织学类型有关。（3）GST-π与MRP（P〈0．05）、LRP与MRP（P〈0．01）、MDR，与MRP（P〈0．01）的表达间具有相关性。结论 （1）多种耐药相关基因的过度表达及其相互作用可能是NSCLC产生原发MDR的重要原因。（2）LRP和MRP过度表达，TopoⅡa高表达和MRP低表达可能分别与肺腺癌、鳞癌的化疗敏感性有关。