乳腺癌上皮-间质转化后引发BCRP介导的多药耐药的研究

背景与目的:研究发现肿瘤的侵袭浸润和转移增强是上皮-间质转化(epithelial mesenchymal transition, EMT)所致,而EMT的发生也与肿瘤细胞多药耐药现象的发生密切相关.本研究通过分析乳腺癌组织和细胞中EMT与乳腺癌耐药蛋白表达的相关性,探讨EMT对乳腺癌中乳腺癌耐药蛋白(breast cancer resistant protein, BCRP)介导多药耐药的影响.方法:免疫组织化学方法检测乳腺癌组织中Snail、BCRP的表达:构建Snail真核表达载体pCDNA3.1-Snail,转染至MCF-7细胞,采用免疫荧光、Western blot、Real-time PCR检测转录抑制因子snail、上皮标志物E-钙粘素、间质标志物vimentin以及多药耐药蛋白BCRP的表达;MTT法检测细胞对米托蒽醌的耐药指数.结果:免疫组化结果显示乳腺癌组织中Snail、BCRP的表达呈显著相关:免疫荧光、Western blot、Real-time PCR显示与亲本MCF-7细胞相比,转染Snail后的MCF-7细胞中E-cadherin表达水平明显降低,而snail、vimentin和BCRP的表达水平明显增加;MTT结果显示细胞对米托蒽醌的耐药指数增加至9.93.结论:转染snail真核表达载体pCDNA3.1-snail可使乳腺癌MCF-7细胞发生EMT, 并导致细胞中BCRP表达增加,引发BCRP介导的多药耐药.     

In vitro and in vivo evaluation of WK-X-34, a novel inhibitor of P-glycoprotein and BCRP, using radio imaging techniques

Overexpression of the multidrug resistance proteins P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) results in treatment failure of many malignancies including ovarian cancer. Dual inhibition of Pgp and BCRP may restore the sensitivity of resistant cells to anticancer drugs. We report the synthesis and characterization of a novel anthranilic-acid based Pgp and BCRP modulator, WK-X-34. In vitro inhibition of Pgp activity was evaluated using 99mTc-Sestamibi and daunorubicin accumulation in Pgp overexpressing human ovarian cancer cells (A2780/Adr) and its sensitive counterpart (A2780/wt). Interaction with BCRP was examined with a mitoxantrone-efflux assay in BCRP-overexpressing MCF7/mx cells, with flow cytometry. Interactions with the multidrug resistance associated proteins (MRP) were evaluated in transfected MRP1, MRP2 and MRP3 cell lines, using a 5-CFDA efflux assay. In vivo 99mTc-Sestamibi imaging of human ovarian cancer xenografts was used to evaluate the in vivo efficacy of WK-X-34 in mice. Daunorubicin accumulation in A2780/Adr cells was inhibited by WK-X-34 at nanomolar concentrations (IC50: 82.1 +/- 6 nM). WK-X-34 inhibited mitoxantrone accumulation in BCRP-overexpressing cells at micromolar concentrations (IC50 = 26.5 +/- 4.6 microM), whereas WK-X-34 did not significantly alter 5-CFDA accumulation in MRP transfected cells. In vivo, uptake of 99mTc-Sestamibi was significantly increased in A2780/Adr xenograft tumors, brain and intestine (AUCs(0-4h) 136%, 147% and 138%; p < 0.05) in mice dosed with WK-X-34 (20 mg/kg i.p.). WK-X-34 selectively modulates Pgp and BCRP in vitro and in vivo in multidrug resistant ovarian cancer cells, and thus may have potential utility in the treatment of multidrug resistant tumors.     

Fumitremorgin C reverses multidrug resistance in cells transfected with the breast cancer resistance protein

Fumitremorgin C (FTC) is a potent and specific chemosensitizing agent in cell lines selected for resistance to mitoxantrone that do not overexpress P-glycoprotein or multidrug resistance protein. The gene encoding a novel transporter, the breast cancer resistance protein (BCRP), was recently found to be overexpressed in a mitoxantrone-selected human colon cell line, S1-M1-3.2, which was used to identify FTC. Because the drug-selected cell line may contain multiple alterations contributing to the multidrug resistance phenotype, we examined the effect of FTC on MCF-7 cells transfected with the BCRP gene. We report that FTC almost completely reverses resistance mediated by BCRP in vitro and is a pharmacological probe for the expression and molecular action of this transporter.     

乳腺癌耐药蛋白--肿瘤多药耐药研究新进展

乳腺癌耐药蛋白（breast cancer resistance protein,BCRP)是近年发现的与肿瘤多药耐药有关的新的药物排出泵。BCRP是含655个氨基酸残基的跨膜蛋白,属于ABC转运蛋白超家族的成员,BCRP仅有6个跨膜区和1个ATP的结合位点,故被称为不完整转运分子,推测BCRP通过组成同二聚体或异二聚体构成跨膜通道而发挥功能,过表达BCRP的肿瘤细胞株对米托蒽醌,阿霉素,柔红霉素,鬼臼乙叉甙,拓扑替康,CPT－11等产生交叉耐药,而对长春新碱,紫杉醇无交叉耐药,GF120918和Fumitremorgin C能有效逆转过表达BCRP的肿瘤细胞株的耐药性,并与细胞内药物蓄积量呈正相关,人体正常组织中胎盘合体滋养层细胞,小肠和结肠粘膜上皮细胞,胆小管膜,乳腺小叶及血管内皮细胞和干细胞均能检测到BCRP的表达,推测BCRP具有抑制消化道吸收某些外源性物质（包括抗癌药和有毒物质）,参与形成胎盘屏障等生理功能,BCRP与急性髓性白血病,非小细胞肺癌,乳腺癌等多种肿瘤的临床化疗敏感性有关。     

Atypical multidrug resistance: breast cancer resistance protein messenger RNA expression in mitoxantrone-selected cell lines

BACKGROUND: Human cancer cell lines grown in the presence of the cytotoxic agent mitoxantrone frequently develop resistance associated with a reduction in intracellular drug accumulation without increased expression of the known drug resistance transporters P-glycoprotein and multidrug resistance protein (also known as multidrug resistance-associated protein). Breast cancer resistance protein (BCRP) is a recently described adenosine triphosphate-binding cassette transporter associated with resistance to mitoxantrone and anthracyclines. This study was undertaken to test the prevalence of BCRP overexpression in cell lines selected for growth in the presence of mitoxantrone. METHODS: Total cellular RNA or poly A+ RNA and genomic DNA were isolated from parental and drug-selected cell lines. Expression of BCRP messenger RNA (mRNA) and amplification of the BCRP gene were analyzed by northern and Southern blot hybridization, respectively. RESULTS: A variety of drug-resistant human cancer cell lines derived by selection with mitoxantrone markedly overexpressed BCRP mRNA; these cell lines included sublines of human breast carcinoma (MCF-7), colon carcinoma (S1 and HT29), gastric carcinoma (EPG85-257), fibrosarcoma (EPF86-079), and myeloma (8226) origins. Analysis of genomic DNA from BCRP-overexpressing MCF-7/MX cells demonstrated that the BCRP gene was also amplified in these cells. CONCLUSIONS: Overexpression of BCRP mRNA is frequently observed in multidrug-resistant cell lines selected with mitoxantrone, suggesting that BCRP is likely to be a major cellular defense mechanism elicited in response to exposure to this drug. It is likely that BCRP is the putative "mitoxantrone transporter" hypothesized to be present in these cell lines.     

MiR-487a resensitizes mitoxantrone (MX)-resistant breast cancer cells (MCF-7/MX) to MX by targeting breast cancer resistance protein (BCRP/ABCG2)

Breast cancer resistance protein (BCRP/ABCG2) specifically transports various chemotherapeutic agents and is involved in the development of multidrug resistance (MDR) in cancer cells. MicroRNAs (miRNAs) can play an important role in modulating the sensitivity of cancer cells to chemotherapeutic agents. Therefore, after confirming that BCRP was increased in the mitoxantrone (MX)-resistant MCF-7 breast cancer cell line MCF-7/MX compared with its parental sensitive MCF-7 cell line, we aimed to explore the miRNAs that regulate BCRP expression and sensitize breast cancer cells to chemotherapeutic agents. In the present study, bioinformatic analysis indicated that miR-487a was one of the miRNAs that could bind to the 3′ untranslated region (3′UTR) of BCRP. Quantitative RT-PCR (qRT-PCR) analysis demonstrated that the expression of miR-487a was reduced in MCF-7/MX cells, and a luciferase reporter assay demonstrated that miR-487a directly bound to the 3′UTR of BCRP. Moreover, ectopic miR-487a down-regulated BCRP expression at the mRNA and protein levels, increasing the intracellular accumulation and cytotoxicity of MX in resistant MCF-7/MX breast cancer cells. Meanwhile, inhibition of miR-487a increased BCRP expression at the mRNA and protein levels and induced MX resistance in sensitive MCF-7 breast cancer cells. Furthermore, the reduced expression of BCRP and increased antitumor effects of MX were also detected in MCF-7/MX xenograft tumors treated with the miR-487a agmir. Thus, our results suggested that miR-487a can directly regulate BCRP expression and reverse chemotherapeutic drug resistance in a subset of breast cancers.     

Anticancer Effects of the Nitric Oxide-Modified Saquinavir Derivative Saquinavir-NO against Multidrug-Resistant Cancer Cells

The human immunodeficiency virus (HIV) protease inhibitor saquinavir shows anticancer activity. Although its nitric oxide-modified derivative saquinavir-NO (saq-NO) was less toxic to normal cells, it exerted stronger inhibition of B16 melanoma growth in syngeneic C57BL/6 mice than saquinavir did. Saq-NO has been shown to block proliferation, upregulate p53 expression, and promote differentiation of C6 glioma and B16 cells. The anticancer activity of substances is frequently hampered by cancer cell chemoresistance mechanisms. Therefore, we here investigated the roles of p53 and the ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein 1 (BCRP1) in cancer cell sensitivity to saq-NO to get more information about the potential of saq-NO as anticancer drug. Saq-NO exerted anticancer effects in lower concentrations than saquinavir in a panel of human cancer cell lines. Neither p53 mutation or depletion nor expression of P-gp, MRP1, or BCRP1 affected anticancer activity of saq-NO or saquinavir. Moreover, saq-NO sensitized P-gp-, MRP1-, or BCRP1-expressing cancer cells to chemotherapy. Saq-NO induced enhanced sensitization of P-gp- or MRP1-expressing cancer cells to chemotherapy compared with saquinavir, whereas both substances similarly sensitized BCRP1-expressing cells. Washout kinetics and ABC transporter ATPase activities demonstrated that saq-NO is a substrate of P-gp as well as of MRP1. These data support the further investigation of saq-NO as an anticancer drug, especially in multidrug-resistant tumors.     

MiR-181a enhances drug sensitivity in mitoxantone-resistant breast cancer cells by targeting breast cancer resistance protein (BCRP/ABCG2)

Breast cancer resistance protein (BCRP)/ATP-binding cassette subfamily G member 2 (ABCG2) mediates multidrug resistance (MDR) in breast cancers. In this study, we aimed to investigate the role of microRNAs in regulation of BCRP expression and BCRP-mediated drug resistance in breast cancer cells. Microarray analysis was performed to determine the differential expression patterns of miRNAs that target BCRP between the MX-resistant breast cancer cell line MCF-7/MX and its parental MX-sensitive cell line MCF-7. MiR-181a was found to be the most significantly down-regulated miRNA in MCF-7/MX cells. Luciferase activity assay showed that miR-181a mimics inhibited BCRP expression by targeting the 3′ untranslated region (UTR) of the BCRP mRNA. Overexpression of miR-181a down-regulated BCRP expression, and sensitized MX-resistant MCF-7/MX cells to MX. In a nude mouse xenograft model, intratumoral injection of miR-181a mimics inhibited BCRP expression, and enhanced the antitumor activity of MX. In addition, miR-181a inhibitors up-regulated BCRP expression, and rendered MX-sensitive MCF-7 cells resistant to MX. These findings suggest that miR-181a regulates BCRP expression via binding to the 3′-UTR of BCRP mRNA. MiR-181a is critical for regulation of BCRP-mediated resistance to MX. MiR-181a may be a potential target for preventing and reversing drug resistance in breast cancer.     

Tetrandrine enhances cytotoxicity of cisplatin in human drug-resistant esophageal squamous carcinoma cells by inhibition of multidrug resistance-associated protein 1

Multidrug resistance is one of the major causes limiting the efficacy of chemotherapeutic agents to control esophageal cancer. Herein, we investigated that the effect and mechanism of tetrandrine (TET) in the human esophageal squamous carcinoma cisplatin-resistant cell line YES-2/DDP. The human esophageal squamous carcinoma cisplatin-resistant cell line YES-2/DDP was isolated by stepwise selection in increasing concentrations of cisplatin. The CCK-8 method was carried out to measure the cell viability when cells were exposed to TET with or without cisplatin, and the IC50 and resistance index (RI) of cisplatin was then calculated. Real-time RT-PCR and western blotting were used to detect the mRNA and protein expression of multidrug resistance?1 (MDR1), multidrug resistance-associated protein?1 (MRP1) and breast cancer resistance protein (BCRP), respectively. Flow cytometry was adopted to determine CMFDA efflux and cell apoptosis, respectively. The resulting cell line YES-2/DDP was 16.4-fold resistant to cisplatin, the cytotoxicity of cisplatin to YES-2/DDP cells was enhanced by TET in a dose-dependent manner. Further, it was found that the expression of MDR1 and BCRP was similar in different treated cells. In contrast, the expression of MRP1 was markedly increased in YES-2/DDP cells, which was dose-dependently decreased by TET. In agreement with the results, MRP1 activity was also reversed by TET. In conclusion, TET possesses a reversal effect on drug resistance in YES-2/DDP cells through downregulation of MRP1, and has the potential to be an adjunct to chemotherapy for esophageal cancer.     

Guggulsterone and bexarotene induce secretion of exosome‐associated breast cancer resistance protein and reduce doxorubicin resistance in MDA‐MB‐231 cells

Many breast cancer cells acquire multidrug resistance (MDR) mediated by ABC transporters such as breast cancer resistance protein (BCRP/ABCG2). Here we show that incubation of human breast cancer MDA‐MB‐231 cells with farnesoid X receptor antagonist guggulsterone (gug) and retinoid X receptor agonist bexarotene (bex) elevated ceramide, a sphingolipid known to induce exosome secretion. The gug+bex combination reduced cellular levels of BCRP to 20% of control cells by inducing its association and secretion with exosomes. Exogenous C6 ceramide also induced secretion of BCRP‐associated exosomes, while siRNA‐mediated knockdown or GW4869‐mediated inhibition of neutral sphingomyelinase 2 (nSMase2), an enzyme generating ceramide, restored cellular BCRP. Immunocytochemistry showed that ceramide elevation and concurrent loss of cellular BCRP was prominent in Aldefluor‐labeled breast cancer stem‐like cells. These cells no longer excluded the BCRP substrate Hoechst 33342 and showed caspase activation and apoptosis induction. Consistent with reduced BCRP, ABC transporter assays showed that gug+bex increased doxorubicin retention and that the combination of gug+bex with doxorubicin enhanced cell death by more than fivefold. Taken together, our results suggest a novel mechanism by which ceramide induces BCRP secretion and reduces MDR, which may be useful as adjuvant drug treatment for sensitizing breast cancer cells and cancer stem cells to chemotherapy.     

Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2)

Observations of functional adenosine triphosphate (ATP)-dependent drug efflux in certain multidrug-resistant cancer cell lines without overexpression of P-glycoprotein or multidrug resistance protein (MRP) family members suggested the existence of another ATP-binding cassette (ABC) transporter capable of causing cancer drug resistance. In one such cell line (MCF-7/AdrVp), the overexpression of a novel member of the G subfamily of ABC transporters was found. The new transporter was termed the breast cancer resistance protein (BCRP), because of its identification in MCF-7 human breast carcinoma cells. BCRP is a 655 amino-acid polypeptide, formally designated as ABCG2. Like all members of the ABC G (white) subfamily, BCRP is a half transporter. Transfection and enforced overexpression of BCRP in drug-sensitive MCF-7 or MDA-MB-231 cells recapitulates the drug-resistance phenotype of MCF-7/AdrVp cells, consistent with current evidence suggesting that functional BCRP is a homodimer. BCRP maps to chromosome 4q22, downstream from a TATA-less promoter. The spectrum of anticancer drugs effluxed by BCRP includes mitoxantrone, camptothecin-derived and indolocarbazole topoisomerase I inhibitors, methotrexate, flavopiridol, and quinazoline ErbB1 inhibitors. Transport of anthracyclines is variable and appears to depend on the presence of a BCRP mutation at codon 482. Potent and specific inhibitors of BCRP are now being developed, opening the door to clinical applications of BCRP inhibition. Owing to tissue localization in the placenta, bile canaliculi, colon, small bowel, and brain microvessel endothelium, BCRP may play a role in protecting the organism from potentially harmful xenobiotics. BCRP expression has also been demonstrated in pluripotential "side population" stem cells, responsible for the characteristic ability of these cells to exclude Hoechst 33342 dye, and possibly for the maintenance of the stem cell phenotype. Studies are emerging on the role of BCRP expression in drug resistance in clinical cancers. More prospective studies are needed, preferably combining BCRP protein or mRNA quantification with functional assays, in order to determine the contribution of BCRP to drug resistance in human cancers.     

Expressions of Topoisomerase IIα and BCRP in Metastatic Cells are Associated with Overall Survival in Small Cell Lung Cancer Patients

The aim of this study was to investigate the mRNA expression levels of multidrug resistance-associated proteins in chemo-na?ve metastatic lung cancer cells and to determine the correlation with response to chemotherapy and overall survival. Metastatic cells were obtained by transbronchial fine needle aspiration biopsy of enlarged mediastinal lymph nodes in 14 patients with small cell lung cancer (SCLC) and 7 patients with non-small cell lung cancer (NSCLC). After cytological confirmation of lung cancer type, total RNA was extracted from biopsy samples and reverse transcribed to cDNA, and real-time PCR for the genes of interest [P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), breast cancer resistance protein (BCRP), lung resistance protein (LRP) and topoisomerase IIα (TOPIIα)], was performed. We observed significantly decreased expression of BCRP and significantly increased expression of TOPIIα in metastatic SCLC cells compared to NSCLC. Furthermore, in SCLC high topoisomerase IIα and low BCRP expression levels positively correlated with longer overall survival. Our results showed higher expression levels of BCRP as well as lower levels of topoisomerase IIα in chemo-na?ve metastatic cells in NSCLC than in SCLC. These results correlate with previous observations that metastatic SCLC cells at the beginning of chemotherapy are potentially more sensitive to chemotherapeutic agents while in metastatic NSCLC cells resistance is usually inherent. We also showed that altered levels of topoisomerase IIα and BCRP in SCLC are important factors that contribute to resistance to chemotherapeutics that interfere with the enzyme and/or DNA and are highly associated with overall survival.     

Breast cancer resistance protein (BCRP) in acute leukemia

Multidrug resistance, cross-resistance to structurally and functionally unrelated drugs, is an important cause of treatment failure in acute leukemia. Multidrug resistance can result from the overexpression of ATP-dependent efflux pumps, such as P-glycoprotein and members of the multidrug resistance associated protein (MRP) family. Recently a novel transporter has been identified, which is called breast cancer resistance protein (BCRP), ABCG2 or mitoxantrone resistance protein. BCRP confers resistance to chemotherapeutic agents, such as mitoxantrone, doxorubicin and daunorubicin. This review describes BCRP detection techniques and the normal physiology of BCRP. The role of BCRP in the physiology of hematopoietic stem cells is addressed as well as the involvement of BCRP in multidrug resistance in acute leukemia. In AML and ALL, several studies showed that BCRP is expressed and functionally active at low, but variable levels. However, further studies are warranted to investigate its effect on clinical outcome, and explore whether patients could benefit from the combination of BCRP inhibitors and chemotherapy.     

乳腺癌组织中四种耐药相关蛋白的表达及其临床意义

目的:研究包括P-糖蛋白(P-glycoprotein,PGP)、多药耐药相关蛋白-1(Multidrug resistance-associated protein1,MRP1)、肺耐药相关蛋白(Lung resistance-related protein,LRP)和乳腺癌耐药蛋白(Breast cancer resistance protein,BCRP)在乳腺癌组织中的表达,并评估其在乳腺癌预后中的作用。方法:采用免疫组织化学方法检测50例经手术切除的乳腺癌组织中PGP、MRP1、LRP和BCRP的表达,并分析其与临床、病理特征的关系及其对预后的影响。结果:(1)各种耐药蛋白在乳腺癌组织中均有不同程度的表达,PGP、MRP1、LRP和BCRP的表达率分别为82·0%(41/50)、86·0%(43/50)、80·0%(40/50)和58·0%(29/50);(2)除MRP1表达与淋巴结转移状况有关以及BCRP表达与激素受体有关外(P<0·05),PGP、MRP1、LRP和BCRP表达与月经状况、肿瘤大小、淋巴结转移、组织分级和激素受体状况均无关(P>0·05);(3)Kaplan-Meier生存分析结果表明PGP、MRP1、LRP和BCRP表达均与无病生存期明显相关(P<0·05),但只有PGP表达与总生存期显著相关(P<0·01),而MRP1、LRP和BCRP表达与总生存期无关(P>0·05);(4)Cox多因素分析中,肿瘤大小和腋淋巴结转移与无病生存期和总生存期明显相关(P<0·05),但PGP表达仅与总生存期明显相关(P<0·05)。结论:乳腺癌组织中具有多种耐药蛋白的过度表达,其中PGP有可能成为判断乳腺癌患者预后的独立指标。     

Modulation of P-glycoprotein but not MRP1- or BCRP-mediated drug resistance by LY335979

Our study examines the ability of LY335979 (Zosuquidar trihydrochloride) to modulate 3 distinct ABC transporters that are mechanisms of drug resistance: P-glycoprotein (Pgp, ABCB1), multidrug resistance associated protein (MRP1, ABCC2) and breast cancer resistance protein (BCRP, ABCG2). Pgp-mediated resistance can be modulated by coadministration with the highly potent, selective inhibitor, LY335979. Modulation of resistance by mitoxantrone and vinorelbine, 2 drugs used to treat certain solid tumors, was examined in a 3-day cytotoxicity assay using a panel of HL60 leukemia cell lines or MCF-7 breast cancer transfectants. LY335979, at 0.5 microM, substantially reversed mitoxantrone resistance and fully reversed vinorelbine resistance of Pgp-expressing HL60/Vinc cells. However, LY335979 did not modulate drug resistance in the MRP1-expressing HL60/ADR or drug-sensitive parental HL60 cells. To ascertain if LY335979 modulates BCRP-mediated drug resistance, the sensitivity of 26-fold mitoxantrone resistant, BCRP-transfected MCF-7 cells was evaluated. Addition of 5 microM LY335979, a concentration approximately 100-fold higher than the affinity of Pgp, had little to no effect on the BCRP transfectant. [(125)I]Iodomycin photolabeled Pgp in CEM/VLB(100) membranes and was inhibited by 5 microM LY335979 and GF120918. No photolabeling of MRP or BCRP occurred in H69AR or MCF-7/BCRP membranes, respectively. These results further demonstrate that LY335979 is highly specific for Pgp and does not modulate MRP1- or BCRP-mediated resistance and can be used in combination with mitoxantrone and vinorelbine in tumor cells.     

表达BCRP的胚肾细胞系HEK293/BCRP的建立及生物学特征

目的:建立HEK293/BCRP多药耐药细胞系并研究其生物学功能。方法:构建有BCRP基因的表达载体,利用脂质体转染方法将载体转入HEK293细胞,并用RT-PCR、间接免疫荧光染色和流式细胞术分析检测转染细胞系BCRP的表达,体外细胞毒试验检测其对米托蒽醌等的耐药指数,流式细胞术和激光共聚焦分别检测其对罗丹明123和阿霉素的外排作用。结果:HEK293/BCRP细胞系在BCRP mRNA和蛋白表达均明显升高(P<0.05),对米托蒽醌的耐药指数达112.07倍,经1.5 h和3 h外排,细胞内罗丹明123浓度分别降低了42.25%和69.01%,激光共聚焦显示细胞内阿霉素浓度降低。结论:成功构建了表达BCRP的胚肾细胞系HEK293/BCRP,并对多种抗肿瘤药物具有耐受性。