Plasma pharmacokinetics and tissue distribution of the breast cancer resistance protein (BCRP/ABCG2) inhibitor fumitremorgin C in SCID mice bearing T8 tumors

Multidrug resistance (MDR) remains a major obstacle in the treatment of human cancers. The recently discovered breast cancer resistance protein (BCRP/ABCG2) has been found to be an important mediator of chemotherapeutic MDR. Fumitremorgin C (FTC) is a selective and potent inhibitor of BCRP that completely inhibits and reverses BCRP-mediated resistance at micromolar concentrations. We report a study of the pharmacokinetics and tissue distribution of FTC when administered intravenously (IV) at a dose of 25 mg/kg to female SCID mice bearing the BCRP-overexpressing human ovarian xenograft Igrov1/T8 tumors. Plasma pharmacokinetics and tissue distribution of FTC in various organs and tissues were studied. In addition, the effect of FTC administration on the expression of BCRP in T8 tumors was also assessed by RT-PCR. Administration of a single FTC IV dose did not appear to cause any major toxicities. The resulting pharmacokinetic data were fit to a two-compartment model using NONMEM and the FTC clearance was determined to be 0.55 ml/min (25.0 ml/min/kg) with a 56% inter-animal variability. Area under the plasma concentration time curve was determined by Bailers method and was calculated to be 1128±111 g min/ml. FTC was widely distributed in all tissues assayed with highest concentrations found in lungs, liver and kidney in decreasing order, respectively. FTC did not appear to have any effect on the expression of BCRP in T8 tumors. Less than 2% of the administered dose was recovered in the urine and feces after 24 h, suggesting hepatic metabolism as a primary mechanism of elimination. The current study can be used as a basis for future animal or in vivo studies with FTC designed to further understand the impact of BCRP on drug resistance.     

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.     

Folate deprivation induces BCRP (ABCG2) expression and mitoxantrone resistance in Caco-2 cells

Folates can induce the expression and activity of the breast-cancer-resistance-protein (BCRP) and the multidrug-resistance-protein-1 (MRP1). Our aim was to study the time-dependent effect of folate deprivation/supplementation on (i) BCRP and MRP expression and (ii) on drug resistance mediated by these transporters. Therefore Caco-2 colon cancer cells usually grown in standard RPMI-medium containing supraphysiological folic acid (FA) concentrations (2.3 muM; high-folate, HF) were gradually adapted to more physiological folate concentrations (1 nM leucovorin (LV) or 1 nM FA; low-folate, LF), resulting in the sublines Caco-2-LF/LV and Caco-2-LF/FA. Caco-2-LF/LV and LF/FA cells exhibited a maximal increase of 5.2- and 9.6-fold for BCRP-mRNA and 3.9- and 5.7-fold for BCRP protein expression, respectively, but no major changes on MRP expression. Overexpression of BCRP in the LF-cells resulted in 3.6- to 6.3-fold resistance to mitoxantrone (MR), which was completely reverted by the BCRP inhibitor Ko143. On the other hand, LF-adapted cells were markedly more sensitive to methotrexate than the HF-counterpart, both after 4-hr (9,870- and 23,923-fold for Caco-2-LF/LV and LF/FA, respectively) and 72-hr (11- and 22-fold for Caco-2-LF/LV and LF/FA, respectively) exposure. Immunofluorescent staining observed with a confocal-laser-scan-microscope revealed that in Caco-2 cells (both HF and LF), BCRP is mainly located in the cytoplasm. In conclusion, folate deprivation induces BCRP expression associated with MR resistance in Caco-2 cells. The intracellular localization of BCRP in these cells suggests that this transporter is not primarily extruding its substrates out of the cell, but rather to an intracellular compartment where folates can be kept as storage.     

Reversal of breast cancer resistance protein (BCRP/ABCG2)-mediated drug resistance by novobiocin, a coumermycin antibiotic

Breast cancer resistance protein (BCRP/ABCG2) of an ATP-binding cassette half-transporter confers resistance against mitoxantrone and camptothecin derivatives of topotecan and irinotecan. Novobiocin, a coumermycin antibiotic, is known to enhance anticancer drug sensitivity of cancer cells in vitro and in vivo, the mechanism of which remains undetermined. Here we focused on drug efflux pump and examined whether novobiocin reversed drug resistance in multidrug-resistant cells highly expressing BCRP. To explore the reversal mechanisms, intracellular drug accumulation was measured by flow cytometry, and a topotecan transport study using plasma membrane vesicles was performed. We used PC-6/SN2-5H2 small cell lung cancer and MCF-7/MX breast cancer cells selected with SN-38 of the active irinotecan metabolite and mitoxantrone, respectively, and the BCRP cDNA transfectant MCF-7/clone 8 cells. These cells expressed high levels of BCRP mRNA but not other known transporters. Compared to the parental PC-6 cells, PC-6/SN2-5H2 cells were 141-, 173- and 57.2-fold resistant to topotecan, SN-38 and mitoxantrone, respectively. Novobiocin at 60 microM decreased the degree of the above resistance by approximately 26-fold in PC-6/SN2-5H2 cells, and similarly reversed resistance in MCF-7/MX, MCF-7/clone 8 and un-selected NCI-H460 cells highly expressing BCRP. Furthermore, novobiocin increased the intracellular topotecan accumulation in these cells and inhibited the topotecan transport into the membrane vesicles of PC-6/SN2-5H2 cells. No effects of novobiocin in these assay were observed in the parental PC-6 and MCF-7 cells. The kinetic parameters in the transport study indicated that novobiocin was a inhibitor for BCRP, resulting in competitive inhibition of BCRP-mediated topotecan transport. These findings suggest that novobiocin effectively overcomes BCRP-mediated drug resistance at acceptable concentrations.     

Role of BCRP as a biomarker for predicting resistance to 5-fluorouracil in breast cancer

Purpose  Chemotherapy is not only important but also necessary for the patient of breast cancer. Breast cancer resistance protein (BCRP), an atypical drug efflux pump, mediates multidrug resistance in breast cancer. The aim of this study is to search new substrate of BCRP. The result will guide the drug selection of chemotherapy in BCRP-positive breast cancer. Methods  PA317/Tet-on/TRE-BCRP cell induced with doxycycline was used to screen the possible substrates of BCRP by MTT assay. The suspicious substrate [5-fluorouracil (5-Fu)] was further confirmed in PA317 and breast cancer cell MCF-7 by HLCP, apoptosis assay (staining and FACS) and RNAi technique. Results  Mitoxantrone, 5-Fu, adriamycin, Methotrexate, Pirarubicin, and Etoposide were identified as substrates of BCRP. However, Paclitaxel, Vincristine, Vindesine, Mitomycin C, and cisplatin were not mediated by BCRP. 5-Fu was identified as substrate of BCRP for the first time. The further study showed that the intracellular retention dose of 5-Fu and the 5-Fu induced cellular apoptosis all decreased when BCRP highly expressed. Furthermore, 5-Fu accumulation and 5-Fu induced DNA damage increased when BCRP was silenced by RNAi in breast cancer cells. Conclusions  5-Fluorouracil may be a specific substrate which can be bound by BCRP. BCRP can predict the sensitivity of breast cancer to 5-Fu. And BCRP-targeted therapy will reverse the resistance of breast cancer to 5-Fu. Jianhui Yuan and Hui Lv were contributed equally to this work.     

BCRP/ABCG2 levels account for the resistance to topoisomerase I inhibitors and reversal effects by gefitinib in non-small cell lung cancer

Purpose: Breast cancer resistance protein (BCRP) confers resistance against topoisomerase I inhibitors in cancer cells. Very recently, we reported that gefitinib reverses BCRP-mediated drug resistance by direct inhibition. However, it remains undetermined how much BCRP contributes to the resistance to topoisomerase I inhibitors in non-small cell lung cancer (NSCLC). The present study was designed to examine whether BCRP levels in NSCLC cells are correlated with the resistance to topoisomerase I inhibitors and the reversal effect by gefitinib. Methods: BCRP levels and its function were evaluated by Western blotting and flowcytometry, respectively. Gefitinib-insensitive NSCLC cells expressed various levels of BCRP, which were closely correlated not only with the IC₅₀ values of SN-38 (r=0.874, P<0.05) and those of topotecan (r=0.968, P<0.001), but also with the reversal effects of 1 μM gefitinib on SN-38 resistance (r=0.956, P<0.001) and topotecan resistance (r=0.977, P=0.0001). Results: BCRP levels accounted for between 80 and 90% of the variation in the resistance to topoisomerase I inhibitors and the reversal effects by gefitinib. Also, gefitinib increased intracellular topotecan accumulation in proportion to the BCRP levels. Conclusions: These findings suggest that BCRP is the most important molecule responsible for topoisomerase I inhibitor resistance, and that the development of BCRP inhibitors is an effective approach for overcoming this resistance. In addition, the examination of BCRP levels in NSCLC tissues may identify an optimal patient population for treatment with topoisomerase I inhibitors alone or in combination with BCRP inhibitors.S. Nagashima and H. Soda contributed equally to this study.     

乳腺癌耐药蛋白在癌组织中的表达及临床意义

背景与目的:乳腺癌耐药蛋白(BCRP)是新发现的多药耐药相关的膜转运蛋白,了解耐药蛋白在乳腺癌组织中的表达,对乳腺癌患者的治疗是有重要意义。我们研究了BCRP在乳腺癌组织中的表达以及它在乳腺癌化疗指导中的潜在意义,评估其在判断乳腺癌化疗敏感性中的作用。方法:应用流式细胞术检测31例原发乳腺癌组织中BCRP的表达,并分析其与临床病理特征的关系及对预后的影响。结果:BCRP在乳腺癌组织中的表达(0.282581±0.183686)与正常对照组(0.03125±1.000905)比较差别有非常显著性(P<0.01)。乳腺癌组织BCRP与ER、PR、c-erb-B-2、EGFR阴阳性值均数的比较,以及腋淋巴结无转移、腋淋巴结转移组之间BCRP表达值差均无显著性。结论:BCRP在乳腺癌组织中具有一定的表达水平,是独立于ER、PR、c-erb-B-2、EGFR的细胞膜蛋白。BCRP高表达者采用表阿霉素为辅的联合化疗可取得满意结果。     

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.     

乳腺癌耐药蛋白在乳腺癌原发灶和转移灶中表达的比较

目的研究乳腺癌耐药蛋白(breast cancer resistance protein,BCRP)在乳腺癌原发灶和转移灶中的表达并进行比较,分析两者间有无差异及其与预后的关系.方法采用免疫组织化学方法(IHC)检测44例手术切除的乳腺癌原发灶组织以及相应的淋巴结转移灶中,BCRP的表达.结果(1)BCRP在乳腺癌原发灶组织中的高表达率为43.2%(19/44例),在淋巴结转移灶中的高表达率为56.8%(25/44例);(2)经统计分析两者之间的表达并无差异(P＞0.05);(3)Kaplan-Meier生存分析结果表明,乳腺癌原发灶和转移灶中BCRP表达与患者的无病和总生存期皆无关(P＞0.05).结论BCRP在乳腺癌组织中具有一定的表达水平,原发灶与转移灶之间表达无明显差异且与预后无关.     

乳腺癌耐药蛋白的研究进展

乳腺癌耐药蛋白(BVRP)是新近发现的一种肿瘤耐药相关蛋白．与P-gp和多药耐药相关蛋白(MRP)同属ABC转运蛋白超家族。本文对BCRP的发现、基因表达特征、与造血干细胞分化的关系、转运底物及其耐药逆转和临床意义等方面的研究进展进行综述。     

Wild-type breast cancer resistance protein (BCRP/ABCG2) is a methotrexate polyglutamate transporter

The existence of an ATP-dependent methotrexate (MTX) efflux mechanism has long been postulated; however, until recently, the molecular components were largely unknown. We have previously demonstrated a role for the ATP-binding cassette transporter breast cancer resistance protein (BCRP) in MTX resistance (Volk et al., Cancer Res., 62: 5035-5040, 2002). Resistance to this antifolate directly correlated with BCRP expression, and was reversible by the BCRP inhibitors fumitremorgin C and GF120918. Here, we provide evidence for BCRP as a MTX-transporter using an in vitro membrane vesicle system. Inside-out membrane vesicles were generated from both drug-selected and stably transfected cell lines expressing either wild-type (Arg482) or mutant (Gly482) variants of BCRP. In the presence of the wild-type variant of BCRP, transport of MTX into vesicles was ATP-dependent, osmotically sensitive, and inhibited by fumitremorgin C. In contrast, no transport was observed in vesicles containing the mutant form of BCRP. Wild-type BCRP appeared to have low affinity, but high capacity, for the transport of MTX, with an estimated K(m) of 680 micro M and a V(max) of 2400 pmol/mg/min. MTX accumulation was greatly decreased by mitoxantrone, a known BCRP substrate, suggesting competition for transport. Furthermore, and in contrast to the multidrug resistance-associated proteins, BCRP also transported significant amounts of polyglutamylated MTX. Although transport gradually decreased as the polyglutamate chain length increased, both MTX-Glu(2) and MTX-Glu(3) were substrates for BCRP. Together, these data demonstrate that BCRP is a MTX and MTX-polyglutamate transporter and reveal a possible mechanism by which it confers resistance.     

MiR-100 resensitizes docetaxel-resistant human lung adenocarcinoma cells (SPC-A1) to docetaxel by targeting Plk1

MicroRNAs (miRNAs) expression correlates with biological characteristics of both normal cells and cancer cells, but their roles in cancer chemoresistance remain unclear. By microarray analysis, miR-100 was found significantly down-regulated in docetaxel-resistant SPC-A1/DTX cells compared with parental SPC-A1 cells. Ectopic miR-100 expression resensitized SPC-A1/DTX cells to docetaxel by suppression of cell proliferation and induction of cell arrest in G(2)/M phase and apoptosis. Knock-down of Plk1, which was a direct target of miR-100, yielded similar effects as that of ectopic miR-100 expression. The inverse correlation between miR-100 and Plk1 expression was also detected in nude mice SPC-A1/DTX tumor xenografts and clinical lung adenocarcinoma tissues and was proved to be related with the in vivo response to docetaxel. Thus, our results suggested that down-regulation of miR-100 could lead to Plk1 over-expression and eventually to docetaxel chemoresistance of human lung adenocarcinoma.     

Cyclosporin A, tacrolimus and sirolimus are potent inhibitors of the human breast cancer resistance protein (ABCG2) and reverse resistance to mitoxantrone and topotecan

Purpose: Several studies have demonstrated significant interactions between immunosuppressants (e.g., cyclosporin A) and chemotherapeutic drugs that are BCRP substrates (e.g., irinotecan), resulting in increased bioavailability and reduced clearance of these agents. One possible mechanism underlying this observation is that the immunosuppressants modulate the pharmacokinetics of these drugs by inhibiting BCRP. Therefore, the aim of this study was to determine whether the immunosuppressants cyclosporin A, tacrolimus and sirolimus are inhibitors and/or substrates of BCRP. Methods: First, the effect of the immunosuppressants on BCRP efflux activity in BCRP-expressing HEK cells was measured by flow cytometry. Results: Cyclosporin A, tacrolimus and sirolimus significantly inhibited BCRP-mediated efflux of pheophorbide A, mitoxantrone and BODIPY-prazosin. The EC₅₀ values of cyclosporin A, tacrolimus and sirolimus for inhibition of BCRP-mediated pheophorbide A efflux were 4.3±1.9 μM, 3.6±1.8 μM and 1.9±0.4 μM, respectively. Cyclosporin A, tacrolimus and sirolimus also effectively reversed resistance of HEK cells to topotecan and mitoxantrone conferred by BCRP. When direct efflux of cyclosporin A, tacrolimus and sirolimus was measured, these compounds were found not to be transported by BCRP. Consistent with this finding, BCRP did not confer resistance to the immunosuppressants in HEK cells. Conclusion: These results indicate that cyclosporin A, tacrolimus and sirolimus are effective inhibitors but not substrates of BCRP. These findings could explain the altered pharmacokinetics of BCRP substrate drugs when co-administered with the immunosuppressants and suggest that pharmacokinetic modulation by the immunosuppressants may improve the therapeutic outcome of these drugs. Grant support: We gratefully acknowledge financial support from NIH grant HD044404 (to QM and JDU) and VA Merit Review grant (to DDR)     

MiR-422a weakened breast cancer stem cells properties by targeting PLP2

Objective: This study investigated miR-422a and PLP2 expressions in breast cancer cells and breast cancer stem cells (BCSCs). Besides, their influences on polymorphism changes were observed.

Methods: Flow cytometry and fluorescence-activated cell sorting was performed and CD24^?/CD44⁺ cells were sorted from breast cancer cells and recognized as BCSCs. Microarray was applied to search for the differentially expressed miRNAs and mRNAs between MCF7 and BCSCs. The aberrant expression of miR-422a and PLP2 was further confirmed by RT-qPCR and the direct targeted relationship was verified by dual-luciferase reporter assay. After in vitro transfection, the expression of miR-422a and PLP2 were manipulated and biological functions of BMSCs were compared with CCK-8, colony formation and sphere formation assay. The tumorigenesis ability of transfected BMSCs was also investigated in NOD/SCID tumor mice models.

Results: BMSCs were successfully established from MCF7 cells and miR-422a expression was downregulated while PLP2 level decreased in BMSCs. MiR-422a directly targets the 3′UTR of PLP2 and suppressed its expression. Besides, the up-regulation of miR-422a contributed to weakened ability of proliferation and microsphere formation of BMSCs, while PLP2 overexpression facilitated those biological abilities. Tumorigenesis of BMSCs in mice models was impaired by either overexpression of miR-442a or silencing of PLP2.

Conclusion: Up-regulation of miR-422a attenuated microsphere formation, proliferation and tumor formation of breast cancer stem cells via suppressing the PLP2 expression.     

腺苷酸活化蛋白激酶增强乳腺癌对多柔比星化疗敏感性的机制

背景与目的：腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)在调控细胞代谢和能量平衡方面起着重要作用,并与细胞增殖、生存和多种信号通路密切相关。近年来发现AMPK参与肿瘤的抑制和耐药。该研究旨在探讨AMPK对多柔比星抑制乳腺癌作用的影响及其机制。方法：采用四甲基偶氮唑蓝(meth-ylthiazolyl tetrazolium,MTT)法检测多柔比星作用后对MCF-7/adr、MCF-7/adr-vector及MCF-7/adr-AMPKα细胞增殖的影响;Hoechst染色法观察各组细胞凋亡形态;流式细胞术(flow cytometry,FCM)检测各组细胞凋亡率;荧光酶标仪检测3组细胞多柔比星累积量;蛋白[质]印迹法(Western blot)检测各组细胞中耐药蛋白及凋亡相关蛋白的表达。结果：多柔比星对MCF-7/adr细胞增殖抑制作用呈剂量和时间依赖性,其作用24、48 h的IC50值分别为(36.8±2.1)和(28.8±1.3)μg/mL。过表达AMPKα可增加多柔比星对MCF-7/adr细胞的生长抑制作用,呈剂量和时间依赖性,其作用24、48 h的IC50值分别为(16.0±0.7)和(4.2±0.2)μg/mL。荧光形态分析发现多柔比星联合AMPKα能诱导MCF-7/adr细胞凋亡。1.0μg/mL多柔比星作用48 h后,MCF-7/adr、MCF-7/adr-vector及MCF-7/adr-AMPKα细胞的凋亡率分别为(12.0±1.4)%、(12.7±1.6)%和(32.0±4.2)%,MCF-7/adr细胞中AMPKα过表达明显提高MCF-7/adr细胞对多柔比星的敏感性。荧光酶标仪检测显示,过表达AMPKα能明显提高细胞内多柔比星的累积量,具有浓度依赖性。Western blot实验结果显示,与MCF-7/adr和MCF-7/adr-vector细胞比较,MCF-7/adr-AMPKα细胞中Bax、细胞色素c(Cyto c)的释放、caspase-3和多聚腺苷二磷酸-核糖聚合酶降解产物(cleaved PARP)蛋白表达明显增加,而细胞外排泵P-糖蛋白(P-gp)和Bcl-2蛋白表达降低。结论：AMPKα可通过抑制耐药细胞外排泵以及调控凋亡相关蛋白的表达,从而增强乳腺癌耐药细胞对多柔比星的化疗敏感性。     

Sensitization of ABCG2-overexpressing cells to conventional chemotherapeutic agent by sunitinib was associated with inhibiting the function of ABCG2

Sunitinib is an ATP-competitive multi-targeted tyrosine kinase inhibitor. In this study, we evaluated the possible interaction of sunitinib with P-glycoprotein (P-gp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), breast cancer resistance protein (BCRP, ABCG2) and lung-resistance protein (LRP) in vitro. Our results showed that sunitinib completely reverse drug resistance mediated by ABCG2 at a non-toxic concentration of 2.5 μM and has no significant reversal effect on ABCB1-, ABCC1- and LRP-mediated drug resistance, although a small synergetic effect was observed in combining sunitinib and conventional chemotherapeutic agents in ABCB1 overexpressing MCF-7/adr and parental sensitive MCF-7 cells, ABCC1 overexpressing C-A120 and parental sensitive KB-3-1 cells. Sunitinib significantly increased intracellular accumulation of rhodamine 123 and doxorubicin and remarkably inhibited the efflux of rhodamine 123 and methotrexate by ABCG2 in ABCG2-overexpressing cells, and also profoundly inhibited the transport of [³H]-methotrexate by ABCG2. However, sunitinib did not affect the expression of ABCG2 at mRNA or protein levels. In addition, sunitinib did not block the phosphorylation of Akt and Erk1/2 in ABCG2-overexpressing or parental sensitive cells. Overall, we conclude that sunitinib reverses ABCG2-mediated MDR through inhibiting the drug efflux function of ABCG2. These findings may be useful for cancer combinational therapy with sunitinib in the clinic.     

Synergistic apoptosis of MCF-7 breast cancer cells by 2-methoxyestradiol and bis(ethyl)norspermine

2-Methoxyestradiol (2ME) is an estradiol metabolite with anti-tumor and anti-angiogenic properties. We studied the effect of 2ME on apoptosis of MCF-7 breast cancer cells and explored a combination therapy using 2ME and a polyamine analogue, bis(ethyl)norspermine (BE-3-3-3). Determination of viable cells on day 4 of treatment with 2ME/BE-3-3-3 combinations showed synergistic effects by Chou–Talalay analysis. APO-BRDU analysis showed that there was only 1.5 ± 0.5% apoptosis at 200 nM 2ME and 3.7 ± 1.7% in the presence of 2.5 μM BE-3-3-3. Combination of 200 nM 2ME and 2.5 μM BE-3-3-3 resulted in 52.2 ± 2.6% apoptosis. Up to 90% of the cells underwent apoptosis in the presence of 1000 nM 2ME and 2.5 μM BE-3-3-3. Combination treatments resulted in total disruption of microtubules and depletion of putrescine, spermidine and spermine. In addition, phosphorylation of Akt and nuclear localization of cyclin D1 were altered by 2ME/BE-3-3-3 combination. Our results suggest an important strategy to induce apoptosis of breast cancer cells, with potential applications in therapy.     

Breast cancer resistance protein (BCRP) and excision repair cross complement-1 (ERCC1) expression in esophageal cancers and response to cisplatin and irinotecan based chemotherapy

Background

Esophageal cancer patients face a dismal outcome despite tri-modality management and median survival remains 15-18 months. Breast cancer resistance protein (BCRP) is an ATP-dependent efflux protein associated with chemotherapy resistance. The role of BCRP expression in esophageal cancer and normal esophageal cells is not known. Excision repair cross complement-1 (ERCC1) overexpression has been correlated with poorer response to cisplatin based chemotherapy. We examined the expression of BCRP and ERCC1 in patients with esophageal cancer and correlated it with survival in patients receiving irinotecan and cisplatin based chemotherapy.

Methods

With IRB approval, 40 cases of esophageal cancer diagnosed from 2004-2008, were stained for BCRP and ERCC1 expression by immunohistochemistry and scored by a pathologist blinded to clinical data. Baseline demographics, therapy given and survival data were collected and correlated with BCRP and ERCC1 expression. Fisher’s exact test was used to determine association between BCRP and ERCC1 expression and demographics. Cox proportional hazards model was used for association of BCRP and ERCC1 with survival.

Results

On immunohistochemistry, 30/40 cancers (75%) expressed BCRP. Interestingly, down-regulation of BCRP expression in tumor compared with normal cells was seen in 40% of patients. ERCC1 positivity was seen in 15/30 cases (50%). Median overall survival (OS) was 19 months with no difference in survival between BCRP positive and negative patients (P=0.13) or ERCC1 positive and negative patients (P=0.85). Estimated hazard ratio (HR) of death for BRCP positive patients was 2.29 (95% CI: 0.79-6.64) and for ERCC1 positive patients was 1.09 (95% CI: 0.46-2.56). There was no association of BCRP and ERCC1 expression with disease stage, age, gender or histology. For patients who received cisplatin and irinotecan as first line chemotherapy, there was no difference in survival based on BCRP or ERCC1 status.

Conclusions

BCRP expression is seen in a majority of esophageal cancers and normal esophageal mucosa. ERCC1 expression is seen in about half of the patients with esophageal cancer. Irinotecan based studies with esophageal and gastric cancer suggest response rates of 14-65%. Whether the 40% of tumors in our study found with down regulation of BCRP expression, constitute a majority of these responders needs to be prospectively validated in a larger data set. It should include markers such as ERCC1 predicting response to 5-fluorouracil and platinum based chemotherapy, to enable individualizing therapy for this cancer.     

托瑞米芬逆转乳腺癌耐药蛋白介导的多药耐药机制

目的 探讨托瑞米芬逆转乳腺癌耐药蛋白(BCRP)介导的多药耐药机制。方法 通过基因扩增,构建分别由BCRP启动子和巨细胞病毒(CMV)启动子启动表达BCRP的重组质粒pcDNA3-Promoter-BCRP和作为对照的质粒pcDNA3-CMV-BCRP,将其分别转染雌激素受体α（ERα）阳性的MCF-7和ERα阴性的MDA-MB-231乳腺癌细胞系,建立由BCRP启动子和CMV启动子启动表达BCRP的4种耐药细胞系MCF-7/Promoter-BCRP、MCF-7/CMV-BCRP、MDA-MB-231/PromoterBCRP和MDA-MB-231/CMV-BCRP。在耐药细胞培养基中加入托瑞米芬,通过逆转录聚合酶链反应(RT-PCR)、Western blot、外排实验以及细胞毒性实验观察托瑞米芬对不同细胞系的耐药逆转效果。结果与空白对照组（未加药物）相比,托瑞米芬以剂量依赖方式抑制BCRP mRNA的表达,0.1、1和10 μmol/L托瑞米芬处理组MCF-7/Promoter-BCRP细胞中BCRP mRNA的表达水平分别下调29.5％(P＜0.05)、68.1％ (P＜0.01)和97.4％(P＜0.01);MCF-7/Promoter-BCRP细胞经托瑞米芬和17β-雌二醇联合处理后,细胞中BCRP mRNA的相对表达水平为64.2％±1.3％,明显高于托瑞米芬单独处理组(3.8％±0.2％,P＜0.01)。托瑞米芬对各组细胞系中BCRP蛋白表达的调控作用与mRNA相似。经托瑞米芬处理后,MCF-7/Promoter-BCRP细胞内米托蒽醌的荧光强度显著增强,外排米托蒽醌的能力降低了 47.3％ (P ＜0.05);经托瑞米芬和17β-雌二醇联合处理后,MCF-7/Promoter-BCRP细胞内米托蒽醌的荧光强度明显低于托瑞米芬单独处理组,外排米托蒽醌的能力升高了61.5％。托瑞米芬可有效逆转MCF-7/Promoter-BCRP细胞对米托蒽醌的耐药性。上述作用在MCF-7/CMV-BCRP、MDA-MB-231/Promoter-BCRP和MDA-MB-231/CMV-BCRP细胞中未能体现。结论 托瑞米芬可能通过ERot的介导与BCRP启动子上游调控序列中的ERE结合,负性调节BCRP的表达,抑制BCRP蛋白的功能,在体外有效逆转BCRP介导的多药耐药。