P-glycoprotein down-regulates expression of breast cancer resistance protein in a drug-free state

This study investigated whether P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) are linked in terms of expression. RT-PCR and Western blot analyses showed that the lung cancer cell line SK-MES-1/WT expressed BCRP. In a drug-free state, BCRP expression was significantly down-regulated in doxorubicin-resistant SK-MES-1/DX1000 cells overexpressing Pgp. Pharmacological inhibitors (PSC833 or verapamil) or siRNA for Pgp inhibited the down-regulation of BCRP, which was confirmed by confocal microscopy. PSC833 induced the phosphorylation of c-Jun NH2-terminal kinase (JNK) and c-Jun, while the JNK inhibitor SP600125 inhibited this effect. Dominant negative c-Jun decreased the expression of BCRP, but increased that of Pgp. These results indicate that Pgp down-regulates BCRP expression in a drug-free state in which JNK/c-Jun is involved.     

Functionalized chalcones as selective inhibitors of P-glycoprotein and breast cancer resistance protein

A library of chalcones with basic functionalities were screened for inhibition of P-glycoprotein (Pgp, ABCB1) by the calcein-AM accumulation assay on MDCKII/MDR1 cells. Three members that had ring A substituted with 5-(1-ethylpiperidin-4-yl) and 2,4-dimethoxy groups were found to increase calcein-AM accumulation to a greater extent than verapamil, a Pgp inhibitor. These compounds were subsequently shown to enhance the uptake of doxorubicin by MCF-7 cells that over-expressed Pgp. However, when tested for inhibition of the breast cancer resistance protein (BCRP, ABCG2) by the mitoxantrone uptake assay, the same compounds fared poorly. In comparison, a non-basic chalcone (5-14, 3-(4-chlorophenyl)-1-(2,4-dimethoxyphenyl)prop-2-en-1-one) increased mitoxantrone uptake by BCRP over-expressing MCF-7 cells (MCF-7/MX) by more than 300% at 5 microM. Thus, introducing a basic group on the chalcone template enhanced Pgp inhibition at the expense of BCRP inhibition. The basic chalcones were also better Pgp inhibitors than their non-basic counterparts which may in turn be better BCRP inhibitors. Structure activity analysis showed that lipophilicity of the chalcones was not the overriding factor for Pgp inhibitory activity. Rather, good activity was associated with appropriately placed electron donor atoms, of which the meta-disubstituted dimethoxy motif on either ring A or B was of particular relevance. In spite of differing structural requirements for inhibition of Pgp and BCRP, chalcone 3-100 [3-(2,4-dimethoxyphenyl)-1-(4-(piperazin-1-yl)phenyl)prop-2-en-1-one] inhibited both Pgp and BCRP to a reasonable extent and may be a useful starting point for the design of dual inhibitors.     

Functional expression of the human breast cancer resistance protein in Pichia pastoris

We report functional expression of BCRP in Pichia pastoris in which BCRP was produced as a 62 kDa underglycosylated protein. BCRP expression level in P. pastoris was comparable to that in HEK cells. The basal BCRP ATPase activity in the yeast membranes was approximately 40-80 nmol Pi/min/mg protein, which can be modulated by known BCRP substrates and inhibitors. Photolabeling of BCRP with 8-azido[alpha-32P]ATP was dependent preferentially on the presence of Co2+ than Mg2+ and could be inhibited by a molar excess of ATP. Vanadate-induced trapping of 8-azido[alpha-32P]ADP by BCRP was much more significant in the presence of Co2+ than that with Mg2+. The Km and Vmax values of BCRP for [3H]E1S transport were 3.6+/-0.3 microM and 55.2+/-1.6 pmol/min/mg protein, respectively. This efficient and cost-effective expression system should facilitate large scale production and purification of BCRP for further structural and functional analyses.     

Flow cytometric analysis of breast cancer resistance protein expression and function

BACKGROUND: The breast cancer resistance protein (BCRP) is an ATP-binding cassette (ABC) half-transporter that mediates energy-dependent drug efflux. Assessing the clinical relevance of the BCRP will require sensitive and specific methods for detecting its expression and function that allow high-volume specimen throughput and employ widely available instrumentation. METHODS: The BXP-34 and BXP-21 monoclonal antibodies were evaluated for flow cytometric detection of BCRP expression. The modulation of efflux of rhodamine-123, 3,3'-diethyloxacarbocyanine iodide, doxorubicin, and mitoxantrone by fumitremorgin C was studied as an assay for BCRP function in BCRP-overexpressing cell lines and controls. RESULTS: BXP-34 and BXP-21 allowed detection of BCRP expression by flow cytometry in all BCRP-expressing cell lines. Mitoxantrone was the only substrate transported by BCRP in all lines, and with mitoxantrone at a 3-microM concentration, light emission (>670 nm) caused by excitation at 488 nm was sufficiently intense to allow detection of differences in retention associated with low levels of BCRP expression. CONCLUSIONS: Immunophenotyping with BXP-21 or BXP-34 and fumitremorgin C modulation of mitoxantrone retention allow detection of BCRP expression and function by flow cytometry with standard instrumentation. These assays will facilitate determination of the role of BCRP in clinical drug resistance.     

Reduced drug accumulation and multidrug resistance in human breast cancer cells without associated P-glycoprotein or MRP overexpression

MCF-7 human breast cancer cells selected in Adriamycin in the presence of verapamil developed a multidrug resistant phenotype, which was characterized by as much as 100,000-fold resistance to mitoxantrone, 667-fold resistance to daunorubicin, and 600-fold resistance to doxorubicin. Immunoblot and PCR analyses demonstrated no increase in MDR-1 or MRP expression in resistant cells, relative to parental cells. This phenotype is similar to one previously described in mitoxantrone-selected cells. The cells, designated MCF-7 AdVp, displayed a slower growth rate without alteration in topoisomerase IIα level or activity. Increased efflux and reduced accumulation of daunomycin and rhodamine were observed when compared to parental cells. Depletion of ATP resulted in complete abrogation of efflux of both daunomycin and rhodamine. No apparent alterations in subcellular daunorubicin distribution were observed by confocal microscopy. No differences were noted in intracellular pH. Molecular cloning studies using DNA differential display identified increased expression of the alpha subunit of the amiloride-sensitive sodium channel in resistant cells. Quantitative PCR studies demonstrated an eightfold overexpression of the alpha subunit of the Na+ channel in the resistant subline. This channel may be linked to the mechanism of drug resistance in the AdVp cells. The results presented here support the hypothesis that a novel energy-dependent protein is responsible for the efflux in the AdVp cells. Further identification awaits molecular cloning studies. J. Cell. Biochem. 65:513–526. © 1997 Wiley-Liss Inc.     

乳腺癌中AKT激活与耐药蛋白表达相关性

目的 明确乳腺癌中Akt的过度表达与P-gp、GST-π、TopoⅡ三种耐药蛋白的相关性.方法 采用免疫组织化学方法检测260例乳腺癌中Akt和P-gp、TopoⅡ、GST-π的表达情况.结果 乳腺癌中Akt、GST-π和TopoⅡ的表达与肿瘤大小相关,随着肿瘤体积增大,AKT、GST-π和TopoⅡ的阳性表达率增高;Akt和P-gp在伴有淋巴结转移的病例中高表达,且随着淋巴结的数目增多Akt表达越来越高;Akt、P-gp和GST-π的阳性表达患者预后差;Akt与P-gp、GST-π、Topo-Ⅱ阳性表达之间均呈显著相关性.结论 信号转导途径中Akt的激活与P-gp、GST-π、TopoⅡ三种耐药蛋白有相关性,AKT有可能在乳腺癌多药耐药的发生机制中起到一定的作用.     

Elevated expression of CUEDC2 protein confers endocrine resistance in breast cancer

Endocrine resistance is a major obstacle to hormonal therapy for breast cancers. Although reduced expression of estrogen receptor-α (ER-α) is a known contributing factor to endocrine resistance, the mechanism of ER-α downregulation in endocrine resistance is still not fully understood. Here we report that CUE domain-containing protein-2 (CUEDC2), a ubiquitin-binding motif-containing protein, is a key factor in endocrine resistance in breast cancer. We show that CUEDC2 modulates ER-α protein stability through the ubiquitin-proteasome pathway. Through the study of specimens from a large cohort of subjects with breast cancer, we found a strong inverse correlation between CUEDC2 and ER-α protein expression. Notably, subjects with tumors that highly expressed CUEDC2 had poor responsiveness to tamoxifen treatment and high potential for relapse. We further show that ectopic CUEDC2 expression impaired the responsiveness of breast cancer cells to tamoxifen. Therefore, our findings suggest that CUEDC2 is a crucial determinant of resistance to endocrine therapies in breast cancer.     

Glabratephrin通过抑制P-糖蛋白逆转三阴性乳腺癌的阿霉素耐药

三阴性乳腺癌是最具侵略性的乳腺癌之一。目前首要的治疗方案是化疗,通常以蒽环类药物如阿霉素为主。然而,由于P-糖蛋白(Pgp)的存在,化疗的疗效受到限制。Pgp是一种膜转运蛋白,可以排出阿霉素,减少其细胞积累和疗效。因此,如何开发安全有效的药物抑制Pgp的活性是一个巨大的挑战。来自意大利都灵大学的Chiara Riganti及其团队证明了一种来自灰毛豆的异戊二烯基黄酮Glabratephrin(Glab)能增加阿霉素在高Pgp含量的三阴性乳腺癌细胞中的积累和细胞毒性。     

Lactoferrin expression in human breast cancer

We analyzed lactoferrin expression in 78 samples from patients with sporadic breast cancer and found 31/78 negative for mRNA expression. Similar results were obtained by immuno-histochemical localization of the lactoferrin protein. We did not find relationship between lactoferrin expression and clinical parameters. We investigated for the absent lactoferrin expression in some cases of breast cancer. In 68 of the samples analyzed, we found an inverse correlation between estrogen receptor expression and lactoferrin expression (P < 0,0001), thus indicating that regulation by the estrogen receptor is not the main element responsible for the expression of lactoferrin in breast cancer. Analysis of methylation of the lactoferrin genomic DNA extracted from the same patients revealed that the degree of methylation does not explain the observed absence of lactoferrin. The 937 bp lactoferrin promoter was investigated for possible mutations. By single-strand conformation polymorphism analysis one polymorphic site was found and characterized.     

SYK expression in human breast cancer

BACKGROUND: Syk (Splenic Tyrosine Kinase) is an intracellular receptor protein kinase involved in cell proliferation, differentiation and phagocytosis. It has been studied in T and B lymphocytes, NK cells and platelets. The strong expression of Syk in mammary gland prompted research into its potential role in mammary carcinogenesis. There have been very few studies about its role in breast cancer with conflicting results. This study aims to investigate the hypothesis that Syk expression is down-regulated in breast cancer compared with ANCT and the association between its expression and clinicopathological parameters. MATERIALS AND METHODS: mRNA was extracted from 48 breast cancer specimens. Relative Syk to ribosomal RNA expression was determined by RT-PCR and Taqman methodology. Mann-Whitney U test was used to examine the association between Syk expression in cancer and ANCT. Spearman's rank correlation test was used to examine the association between Syk expression in tumours and patients' age, tumour size, tumour grade, estrogen and progesterone receptor status, lymph node metastasis, vascular invasion and clinical outcome. RESULTS: The median for the relative value of Syk expression was 0.17 and 0.18 (range: 0.12 - 0.56 and 0.0 - 1.77) for tumours and ANCT respectively. There was no significant association between Syk expression in cancers and ANCT (p= 0.598) nor between Syk expression in tumours and patients' age, tumour size, tumour grade, estrogen and progesterone receptor status, lymph node metastasis, vascular invasion or prognosis. CONCLUSION: This study shows that Syk mRNA expression does not seem to vary between breast tumours and ANCT. Furthermore, we observed no significant association between Syk expression and clinicopathological parameters.     

Estrogen-regulated protein in human breast cancer

An estrogen-regulated cytoplasmic protein has been purified from MCF-7 human breast cancer cells with anion exchange and monoclonal antibody affinity chromatographies. The purified protein has a monomeric mol. wt of 28,000 and isoelectric species with pI's between 5.9 and 6.0. Amino acid analysis indicates the protein is acidic, is probably hydrophilic, and contains unusually low amounts of methionine and half cystine. The monoclonal antibodies are of the IgG₁ subclass, exhibit a high affinity for the 28K protein, and recognize an antigenic site that is stable to sodium dodecyl sulfate (SDS) gel electrophoresis and electrophoretic transfer techniques. These monoclonal antibodies have been used to detect 28K in certain human breast tumors.     

乳腺癌耐药蛋白基因的转录调控机制

乳腺癌耐药蛋白(Breast cancer resistance protein, BCRP), 又名ABCG2, 是ATP结合盒(ATP-binding cas-sette, ABC)转运蛋白超家族成员之一, 在肿瘤多药耐药中具有十分重要的作用。BCRP基因启动子区无TATA盒, 含CAAT盒、AP1位点、AP2位点以及CpG岛下游的多个Sp-1位点。近年来的研究发现, 转录因子孕激素受体(PR)、雌激素受体(ER)、核因子-κB (NF-κB)、缺氧诱导因子(HIF)、Nrf2、芳香烃受体(AhR)、过氧化物酶体增殖活化受体(PPAR)和KLF5等可与BCRP启动子或增强子区的特定反应元件结合进而激活BCRP的转录。促炎细胞因子、生长因子、同源盒基因MSX2、Sonic hedgehog信号通路、Notch信号通路和RAR/RXR信号通路等均参与了BCRP的转录调控。此外, 启动子甲基化和组蛋白乙酰化在BCRP转录调控尤其是药物诱导BCRP表达中发挥重要作用。文章综述了这一研究领域的进展, 着重讨论了转录因子及表观遗传学在BCRP转录调控中的作用。     

Chloride channel-3 mediates multidrug resistance of cancer by upregulating P-glycoprotein expression

Chloride channel-3 (ClC-3), a member of the ClC family of voltage-gated Cl⁻ channels, is involved in the resistance of tumor cells to chemotherapeutic drugs. Here, we report a new mechanism for ClC-3 in mediating multidrug resistance (MDR). ClC-3 was highly expressed in the P-glycoprotein (P-gp)-dependent human lung adenocarcinoma cell line (A549)/paclitaxel (PTX) and the human breast carcinoma cell line (MCF-7)/doxorubicin (DOX) resistant cells. Changes in the ClC-3 expression resulted in the development of drug resistance in formerly drug-sensitive A549 or MCF-7 cells, and drug sensitivity in formerly drug-resistant A549/Taxol and MCF-7/DOX cells. Double transgenic MMTV-PyMT/CLCN3 mice with spontaneous mammary cancer and ClC-3 overexpression demonstrated drug resistance to PTX and DOX. ClC-3 expression upregulated the expression of MDR1 messenger RNA and P-gp by activating the nuclear factor-κB (NF-κB)-signaling pathway. These data suggest that ClC-3 expression in cancer cells induces MDR by upregulating NF-κB-signaling-dependent P-gp expression involving another new mechanism for ClC-3 in the development of drug resistance of cancers.     

Arachidonic acid metabolism by rabbit fetal membranes of various gestational ages

The ability of rabbit fetal membranes to convert arachidonic acid to both lipoxygenase and cyclooxygenase products was studied by separation and identification of products derived from incubations of 1-¹⁴ C-arachidonate with subcellular fractions obtained by differential centrifugation of tissue homogenates. Both amnion and splanchnopleure (the chorion-equivalent of the rabbit) produced a mixture of 11-, 12-, and 15-hydroxyeicosatetraenoic acids when stimulated by calcium ions; these products were produced in greater quantity in middle pregnancy (20–22d) than later (28–30d). Cyclooxygenase products included PGD₂, PGE₂, TxB₂ and PGF_2α, all of which were made more actively in late pregnancy than the middle in both amnion (which was more active) and chorion-equivalent. These data suggest that arachidonate metabolism by rabbit fetal membranes in middle pregnancy is directed primarily toward production of monohydroxy fatty acids, but that as pregnancy nears term, the PG-producing enzymes are induced, preparing the uterine smooth muscle for parturition.     

Arachidonic acid metabolism by rabbit fetal membranes of various gestational ages

The ability of rabbit fetal membranes to convert arachidonic acid to both lipoxygenase and cyclooxygenase products was studied by separation and identification of products derived from incubations of 1-14C-arachidonate with subcellular fractions obtained by differential centrifugation of tissue homogenates. Both amnion and splanchnopleure (the chorion-equivalent of the rabbit) produced a mixture of 11-, 12-, and 15-hydroxyeicosatetraenoic acids when stimulated by calcium ions; these products were produced in greater quantity in middle pregnancy (20-22d) than later (28-30d). Cyclooxygenase products included PGD2, PGE2, TxB2 and PGF2 alpha, all of which were made more actively in late pregnancy than the middle in both amnion (which was more active) and chorion-equivalent. These data suggest that arachidonate metabolism by rabbit fetal membranes in middle pregnancy is directed primarily toward production of monohydroxy fatty acids, but that as pregnancy nears term, the PG-producing enzymes are induced, preparing the uterine smooth muscle for parturition.     

Altered gene expression and methylation of the human chromosome 11 imprinted region in small for gestational age (SGA) placentae

Imprinted genes are known to be crucial for placental development and fetal growth in mammals, but no primary epigenetic abnormality in placenta has been documented to compromise human fetal growth. Imprinted genes demonstrate parent-of-origin-specific allelic expression that is epigenetically regulated i.e. extrinsic to the primary DNA sequence. To undertake an epigenetic analysis of poor fetal growth in placentae and cord blood tissues, we first established the tissue-specific patterns of methylation and imprinted gene expression for two imprinting clusters (KvDMR and H19 DMR) on chromosome 11p15 in placentae and neonatal blood for 20 control cases and 24 Small for Gestational Age (SGA) cases. We confirmed that, in normal human placenta, the H19 promoter is unmethylated. In contrast, most other human tissues show paternal methylation. In addition, we showed that the IGF2 DMR2, also paternally methylated in most human tissues, exhibits hypomethylation in placentae. However, in neonatal blood DNA, these two regions maintain the differential methylation status seen in most other tissues. Significantly, we have been able to demonstrate that placenta does maintain differential methylation at the imprinting control regions H19 DMR and KvDMR. Of note, in one SGA placenta, we found a methylation alteration at the H19 DMR and concomitant biallelic expression of the H19 gene, suggesting that loss of imprinting at H19 is one cause of poor fetal growth in humans. Of particular interest, we demonstrated also a decrease in IGF2 mRNA levels in all SGA placentae and showed that the decrease is, in most cases, independent of H19 regulation.     

Snail mRNA及其蛋白在乳腺癌中的表达研究

目的观察Snail蛋白及mRNA在人乳腺癌组织中的表达及其与临床病理特征的关系,并探讨它在乳腺癌发生、发展及转移中的作用。方法应用SP免疫组织化学和原位分子杂交方法检测Snail蛋白和Snail mRNA在70例乳腺浸润性导管癌、30例乳腺导管内癌、30例乳腺单纯性增生组织中的表达。结果①70例乳腺癌中,Snail蛋白和Snail mRNA阳性率分别为87.2%(61/70)、81.4%(57/70),分别高于乳腺导管内癌组织53.3%(16/30)、46.7%(14/30)和乳腺单纯性增生组织26.7%(8/30)、23.3%(7/30),三者相比有统计学意义(χ2=36.4,P<0.01;χ2=32.4,P<0.01)。②Snail蛋白和SnailmRNA在有淋巴结转移组中的阳性率为97.6%(40/41)、95.1%(39/41),无转移组阳性率为72.4%(21/29)、62.1%(18/29),两者相比有统计学意义(χ2=8.29,P<0.01);组织学分级Ⅲ级组明显高于Ⅰ、Ⅱ级组表达,但无统计学意义(χ2=0.72,P>0.05;χ2=0.98,P>0.05)。③Snail蛋白与Snail mRNA的表达与年龄、肿瘤大小均无关(P>0.05)。结论 Snail蛋白与Snail mRNA的表达呈正相关,与乳腺癌的发生发展、淋巴结转移密切相关,可作为判断乳腺癌预后、转移的生物学标志。     

Estradiol inhibits glucocorticoid receptor expression and induces glucocorticoid resistance in MCF-7 human breast cancer cells

Our study has shown that treatment of MCF-7 human breast cancer cells with 17-beta estradiol (E(2)) produced significant decreases in glucocorticoid receptor (GR) concentrations and GR mRNA levels. E(2) pre-treatment of MCF-7 cells stably transfected with the GR responsive pMTV-CAT reporter (MCF-7-MTV cells), caused significant attenuation of dexamethasone (DEX)-induced chloramphenicol acetyl transferase (CAT). In MCF-7 cells transiently transfected with [(GRE)(3)-Luc] reporter plasmid, E(2) pre-treatment significantly suppressed DEX-induced luciferase, which was abolished by the estrogen receptor antagonist ICI 182,780. We examined the effect of chronic E(2) treatment as well as E(2) withdrawal on GR function and abundance. MCF-7-MTV cells were treated with vehicle (control) or E(2) for up to 16 days. A third group received E(2) for 5 days followed by E(2) withdrawal from day 6 to 16. Chronic E(2) treatment almost totally abrogated DEX-induced CAT and reduced GR to very low levels. Interestingly, in the group subjected to E(2) withdrawal, neither the DEX response nor GR abundance recovered and reached control values suggesting that the estrogen mediated suppression is long lasting and could not be easily reversed. The E(2) induced resistance to glucocorticoid action may be of potential clinical significance in a number of settings including breast cancer, neuroendocrine response to stress and osteoporosis and could possibly contribute to the differences in glucocorticoid responsiveness among patients.