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。     

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.     

Drug resistance-associated markers P-glycoprotein, multidrug resistance-associated protein 1, multidrug resistance-associated protein 2, and lung resistance protein as prognostic factors in ovarian carcinoma.

Intrinsic and/or acquired resistance to chemotherapy is the major obstacle to overcome in the treatment of patients with ovarian carcinoma. The aim of the present study was to investigate the prognostic value of drug resistance-associated proteins P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), canalicular multispecific organic anion transporter (c-MOAT/MRP2), and lung resistance protein (LRP) in ovarian carcinoma. Expression of P-gp, MRP1, MRP2, and LRP was determined by immunohistochemistry of frozen tissue sections of 115 ovarian carcinoma patients and related to clinicopathological factors, response to chemotherapy, and progression-free survival. P-gp expression was observed in 20 of 115 (17%), MRP1 in 51 (44%), MRP2 in 19 (16%), and LRP in 85 (74%) tumors. Expression of MRP1 was related to MRP2 (P<0.0001) and P-gp (P<0.001) expression, whereas LRP expression was more frequently observed in patients with early stage (P<0.01), lower grade (P<0.05), and smaller residual tumor (P<0.05). Early stage (P<0.001), smaller residual tumor (P<0.001), and lower differentiation grade (P<0.05) were related to longer (progression-free) survival. P-gp, MRP1, MRP2, and LRP expression were neither related to response to first-line chemotherapy in 59 evaluable patients nor to progression-free survival in all patients. On multivariate analysis, only stage and residual tumor were independent prognostic factors for survival. In conclusion, in ovarian carcinoma, MRP1 expression is associated with MRP2 and P-gp expression, whereas LRP expression is associated with favorable clinicopathological characteristics. Assessment of P-gp, MRP1, MRP2, or LRP does not allow prediction of response to chemotherapy or survival in ovarian carcinoma.     

The clinical relevance of the expression of several multidrug-resistant-related genes in patients with primary acute myeloid leukemia

Multidrug resistance (MDR) is a complex phenomenon that includes the expression of many different genes regulating drug transport or metabolism, cellular repair or detoxification mechanisms. The co-expression of several genes could be at the basis of the resistant phenotype in vivo. In order to test a possible prognostic role of the expression and co-expression of several MDR-related genes (MDR1, topoisomerase IIalpha, topoisomerase IIbeta, MRP, GSTpi, LRP), 35 patients affected by acute myeloid leukemia (AML) were tested by RT-PCR assays. In our series, topoisomerase IIbeta was significantly co-expressed with MRP (p = 0.05), GSTpi (p = 0.017) and LRP (p = 0.005). GSTpi was co-expressed with LRP (p = 0.03) and MRP (p = 0.007); on the other hand, 53.8% of patients were LRP and MRP-positive (p = 0.02). The PCR-positivity did not differ according to biological/clinical characteristics of patients, including age; this latter was the only parameter conditioning the response and overall survival. Neither the expression nor the co-expression of the tested genes was significantly correlated with the response to the induction treatment and long-term outcome.     

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.     

Increased expression of the breast cancer resistance protein (BCRP) in relapsed or refractory acute myeloid leukemia (AML).

Expression of the multidrug resistance proteins P-glycoprotein, encoded by the MDR1 gene, multidrug resistance-associated protein (MRP1) and the lung resistance-related protein or major vault protein (LRP/MVP) is associated with clinical resistance to chemotherapy in acute myeloid leukemia (AML). Recently, the breast cancer-resistant protein (BCRP), the equivalent of mitoxantrone-resistant protein (MXR) or placental ABC transporter (ABCP), was described in AML. We investigated MDR1, MRP1, LRP/MVP and BCRP mRNA expression simultaneously in 20 paired clinical AML samples from diagnosis and relapse or refractory disease, using quantitative Taqman analysis. In addition, standard assays for P-glycoprotein expression and function were performed. BCRP was the only resistance protein that was expressed at a significantly higher RNA level (median 1.7-fold, P = 0.04) at relapsed/refractory state as compared to diagnosis. In contrast, LRP/MVP mRNA expression decreased as disease evolved (P = 0.02), whereas MDR1 and MRP1 mRNA levels were not different at relapse as compared to diagnosis. Also, at the protein level no difference of MDR1 between diagnosis and relapse was found. A significant co-expression of BCRP and MDR1 was found at diagnosis (r = 0.47, P = 0.04). The present results suggest that BCRP, but not MDR1, MRP1 or LRP/MVP is associated with clinical resistant disease in AML.     

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.     

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。     

Anticancer drug-mediated induction of multidrug resistance-associated genes and protein kinase C isozymes in the T-lymphoblastoid cell line CCRF-CEM and in blasts from patients with acute lymphoblastic leukemias.

The major determinants mediating drug resistance in acute lymphoblastic leukemias (ALL) unresponsive to chemotherapy, are still unclear. For example, it is still unknown whether selection or induction processes are responsible for drug resistance here or whether protein kinase C (PKC) isozymes contribute to the resistant phenotype. Therefore, inducibility of resistance factors or PKC isozymes genes was examined in CCRF-CEM cells treated with diverse anticancer drugs--adriamycin, camptothecin, etoposide or vincristine--at sublethal concentrations for 24 h. MDR1, MRP1, LRP and PKC isozyme alpha, beta(1), beta(2), epsilon, iota, eta, theta, zeta gene expression was determined by cDNA-PCR. We found significant dose-dependent, mostly combined, induction of the MDR1, MRP1 and LRP genes. Significantly enhanced gene expression of the majority of PKC isozyme genes was found after treatment with camptothecin. PKCzeta was upregulated throughout by each anticancer drug applied in this setting. A series of selected CCRF-CEM-derived multidrug resistance (MDR) sublines also showed enhanced expression of the PKC isozymes compared to the parental cell line. MDR1 and PKCeta gene expression levels were correlated highly significantly. Blasts from two patients with ALL during the first week of monotherapy with steroids revealed combined induction of the MDR1, multidrug resistance-associated protein 1 (MRP1), lung cancer resistance-related protein (LRP) and most PKC isozymes, predominantly PKCzeta. Another patient with T-ALL, who failed to respond to four months of intensive chemotherapy, showed an enhanced MRP1 gene expression combined with markedly overexpression of PKCeta and PKCtheta. Furthermore, the camptothecin and etoposide-mediated induction of resistance factors in the CCRF-CEM cell line could be suppressed by staurosporine, a rather unspecific inhibitor of protein kinases. However, selective inhibitors of PKC isozymes (bisindolylmaleimide GO 6850, indolocarbazole GO 6976) produced no significant effects here. Therefore, the PKC isozymes eta, theta and zeta are of interest as potential targets to overcome drug resistance in ALL.     

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     

MYCN-mediated regulation of the MRP1 promoter in human neuroblastoma

In the childhood cancer neuroblastoma (NB), the level of expression of the multidrug resistance-associated protein (MRP1) gene is strongly correlated with expression of the MYCN oncogene in primary NB tumors, suggesting that MRP1 may be a target for MYCN-mediated gene regulation. In this study, we show that MYCN induction in human NB cells results in increased MRP1 mRNA and protein levels, which in turn is accompanied by increased drug resistance and enhanced MRP1-mediated drug efflux. Furthermore, luciferase activity from MRP1 promoter/luciferase gene reporter constructs was significantly increased in NB cells with exogenous overexpression of MYCN, whereas activity was decreased in NB cells stably transfected with MYCN-antisense vectors. Decreased luciferase activity was observed with promoter constructs that lacked one or two E-box sequences or had E-box double point mutations, while a truncated MRP1 promoter lacking all three E-boxes exhibited only basal levels of activity. Specific electrophoretic mobility shifts of MRP1 E-box sequences were detected with nuclear extracts from NB cells with MYCN overexpression, and complex formation was inhibited with the addition of antibodies directed against MYCN or MYC. These findings indicate that by interacting with E-box elements within the promoter, MYCN can upregulate MRP1 expression and modulate drug resistance in NB.     

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.     

Copper-transporting P-type adenosine triphosphatase (ATP7B) as a cisplatin based chemoresistance marker in ovarian carcinoma: comparative analysis with expression of MDR1, MRP1, MRP2, LRP and BCRP

Intrinsic or acquired resistance to chemotherapy is the major obstacle to overcome in the treatment of patients with solid carcinoma. Cisplatin is one of the most effective chemotherapeutic agents for treating ovarian carcinoma. Recently, copper-transporting P-type adenosine triphosphatase (ATP7B) has been demonstrated as one of the genes responsible for cisplatin resistance in vitro. We hypothesized that the expression of ATP7B gene increases resistance to cisplatin in ovarian carcinoma and a priori knowledge of its expression is important for the choice of therapy. The aim of our study was to assess the role of ATP7B gene in ovarian carcinoma and compare its expression with those of multidrug resistance-related transporters such as MDR1, MRP1, MRP2, LRP and BCRP genes. The transporters' gene expression profiles from 82 patients treated with cisplatin-based chemotherapy after surgery were assessed by RT-PCR. We did not observe any significant correlation between ATP7B gene expression and those of MDR1, MRP1, MRP2, LRP or BCRP. The expression level of ATP7B gene was significantly increased (p < 0.05) in patients with moderately-/poorly-differentiated ovarian carcinomas treated with cisplatin-based chemotherapy, thus ATP7B may serve as an independent prognostic factor in these patients. In contrast, the expression level of MDR1, MRP1, MRP2, LRP and BCRP genes were not prognostic indicators of disease. These findings suggest that ATP7B gene may be considered as a novel chemoresistance marker and that inhibitor(s) of ATP7B might be useful, in patients with ovarian carcinoma treated with cisplatin-based chemotherapy.     

Topoisomerase IIalpha and other drug resistance markers in advanced non-small cell lung cancer

Resistance to chemotherapy is common in non-small cell lung cancer. The aim of this study was to investigate the prognostic impact of in vitro established drug resistance markers on the response to chemotherapy in patients with advanced non-small cell lung cancer. Samples of 38 patients were analyzed by immunohistochemical staining, for topoisomerase IIalpha and IIbeta, Ki-67, MRP and LRP. In addition, mutation analysis of the topoisomerase IIalpha gene, the B/DNBS and the Tyr804 region, was performed. Lung tumor biopsies were taken prior for treatment with one of the following regimens; cisplatin/paclitaxel, cisplatin/VM26 or VP16, or carboplatin/VP16/ifosfamide. Seventeen patients obtained a partial response, 12 had stable disease and nine patients had progressive disease. None of the investigated markers was related with overall response rate. In one sample a point mutation in the B/DNBS region of the topo IIalpha gene was detected which substitutes IIe(510) with Val. This tumor had a partial response to four courses of cisplatin/VP16 treatment. The survival analysis showed that the patients with high topo IIalpha expressing tumors had a significantly worse survival compared with the patients with low or intermediate topo IIalpha expressing tumors. In conclusion, no relation was observed between expression of topoisomerase IIalpha, IIbeta, Ki-67, MRP or LRP and response rate. Furthermore, worse survival was seen in patients with high topoisomerase IIalpha expressing tumors. In one tumor sample, a newly described mutation in the B/DNBS region of the topo IIalpha gene was detected, which does not appear to be related to drug resistance.     

Expression of LRP Gene in Breast Cancer Patients Correlated with MRP1 as Two Independent Predictive Biomarkers in Breast Cancer

Background: Breast cancer is the most common malignancy in women. Multidrug resistance (MDR) is still a greatobstacle of breast cancer chemotherapy. We have previously shown that multidrug resistance-associated protein 1 (MRP1)is associated with response to neoadjuvant chemotherapy. The lung resistance-related protein (LRP) is identified asa prognostic marker and response to treatment factor which has been studied mainly in hematological malignancy andleukemia. In this study, we aimed to analyze LRP expression and possible correlation between the expression level ofthis gene with MRP1 as a candidate marker for chemotherapy resistance. Materials and Methods: We collected 54breast tumors and adjacent normal tissues from Iranian breast cancer patients and Real time RT-PCR was employed tomeasure the gene expression level in our samples. Results: MRP1 and LRP expression level were significantly lowerin tumor tissues of the patients responding to chemotherapy compared to non-responding patients. No relation betweenthe expression level of either of these genes and clinicopathology markers was found. Conclusion: Our results suggestthat LRP gene expression is correlated to MRP1 in human breast cancer cells and may affect the clinical response totreatment.