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BK21 Project Team, College of Pharmacy, Chosun University, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, South Korea
(Requests for offprints should be addressed to K W Kang; Email: kwkang{at}chosun.ac.kr)
H K Choi and J W Yang contributed equally to this work
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Introduction |
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Breast cancer is the most common malignancy in western women. The ability to reduce breast tumor growth through the administration of anti-estrogens has played a key role in the endocrine therapy of breast cancer. A non-steroidal anti-estrogen, tamoxifen (TAM), is a representative drug that has been approved for the chemoprevention of breast cancer and is the most widely used anti-estrogen in estrogen receptor-positive breast cancer patients (Rose et al. 1985). Although most patients are initially responsive, the acquisition of resistance to TAM is the main problem of anti-estrogen therapy (Clemons et al. 2002).
In order to establish a TAM-resistant breast cancer cell line, Gottardis & Jordan (1988) administered TAM long term to nude mice bearing MCF-7 cells, and the isolated MCF-7 cells became TAM resistant. In addition, a continuous culture of human breast cancer cell lines with TAM can cause the development of cell lines that are resistant to TAM. It was reported that a long-term culture of MCF-7 cells with TAM or 4-hydroxytamoxifen results in the formation of TAM-resistant cells (Badia et al. 2000, Knowlden et al. 2003). We have also established MCF-7-derived TAM-resistant cell line (TAMR-MCF-7 cells) by the long-term ( > 9 months) culture of MCF-7 cells with 4-hydroxytamoxifen.
Previous studies have shown that the transition of chemotherapy-responsive cancer cells to chemotherapy-resistant cancer cells is accompanied mainly by the increased expression of ABC transporters (Lage 2003). Considering that the next line of chemotherapy against TAM-resistant breast cancer involves a methotrexate- or cisplatin-containing regimen (Blumenschein et al. 1997, Assikis et al. 2003), a study on whether the expression levels of the ABC transporter can be changed in TAM-resistant breast cancer cells might lead to a better understanding of the pathological implications of TAM resistance. This study shows for the first time that the expression of MRP2 is higher in TAMR-MCF-7 cells when compared with those in MCF-7 cells and demonstrates that the phosphatidylinositol 3-kinase (PI3-kinase)-dependent activation of PXR is essential for the induction of MRP2 in TAM-resistant breast cancer cells.
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Materials and methods |
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The anti-MRP2, anti-MDR1, and anti-PXR antibodies were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-aromatase antibody was purchased from Abcam (Cambridge, MA, USA). The horseradish peroxidase-conjugated donkey anti-rabbit IgG, anti-goat IgG, and alkaline phosphatase-conjugated donkey anti-mouse IgG were obtained from Jackson Immunoresearch Laboratories (West Grove, PA, USA). The 5-bromo-4-chloro-3-indoylphosphate/nitroblue tetrazolium was acquired from Life Technologies. The antibodies for phosphorylated Akt and Akt antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Most of the reagents used for molecular studies were obtained from Sigma. The p2635-MRP2-Luc reporter plasmids containing human MRP2 promoter region ( – 2635 bp), p491-MRP2-Luc, and p491-MRP2-Luc-Mut were kindly provided by Dr Uchiumi T (Kyushu University, Fukuoka, Japan; Tanaka et al. 1999). The PXR reporter plasmids containing three copies of the pregnane X receptor (PXR)-responsive elements from the CYP3A23 gene and pGL3-MRP2-1 (rat MRP2 gene promoter) were kind gifts from Dr Edward PA (University of California Los Angeles, Los Angeles, CA, USA).
Cell culture and establishment of TAM-resistant MCF-7 cells
The MCF-7 cells were cultured at 37 ° C in 5% CO2/95% air in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 µ g/ml streptomycin. The TAM-resistant MCF-7 cells (TAM-R) were established using the methodology reported elsewhere (Knowlden et al. 2003). Briefly, the MCF-7 cells were washed with PBS, and the culture medium was changed with phenol red-free DMEM containing 10% charcoal-stripped steroid-depleted FBS (Hyclone, Logan, UT, USA) and 4-hydroxytamoxifen (0.1 µ M). The cells were continuously exposed to this treatment regimen for 2 weeks and the concentration of 4-hydroxytamoxifen was gradually increased up to 3 µ M over a 9-month period. Initially, the cell growth rates were reduced. However, after exposure to the medium for 9 months, the rate of cell growth gradually increased, showing the establishment of a TAM-resistant cell line.
MTT cell viability assay
To determine the cell viabilities, the cells were plated at 104 cells/well in 96-well plates. For the cytotoxicity determination by 4-hydroxytamoxifen, MCF-7, and TAMR-MCF-7 cells were incubated in the FBS-free medium with or without 4-hydroxytamoxifen (0.1–30 µ M) for 24 h. To determine the effect of aromatase inhibitor on testosterone-mediated cell proliferation, MCF-7, and TAMR-MCF-7 cells were cultured in phenol red-free DMEM containing 5% charcoal-stripped steroid-depleted FBS in the presence or absence of testosterone (0.1 µ M)/4-hydroxyander-stenedione (0.1–1 µ M) for 48 h. The viable adherent cells were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; 2 mg/ml) for 4 h. The media were then removed and the formazan crystals produced were dissolved by adding 200 µ l dimethylsulfoxide/well. The absorbance was assayed at 540 nm. The cell viability was expressed as the relative ratios to the untreated control cells.
Preparation of nuclear extracts
The nuclear extracts were prepared essentially according to a previously published method (Schreiber et al. 1990). Briefly, the cells in the dishes were washed with ice-cold PBS. The cells were then scraped, transferred to microtubes, and allowed to swell after adding 100 µ l of a hypotonic buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.5% Nonidet P-40, 1 mM dithiothreitol (DTT), and 0.5 mM phenyl-methylsulfonyl fluoride (PMSF). The lysates were incubated for 10 min on ice and centrifuged at 7200 g for 5 min at 4 ° C. Pellets containing the crude nuclei were resuspended in 50 µ l of an extraction buffer containing 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 10 mM DTT, and 1 mM PMSF and incubated for 30 min on ice. The samples were centrifuged at 15 800 g for 10 min to obtain the supernatants containing the nuclear fractions. The nuclear fractions were stored at – 80 ° C until needed.
Immunoblot analysis
After washing with sterile PBS, the MCF-7 or TAMR-MCF-7 cells were lysed in EBC lysis buffer containing 20 mM Tris–Cl (pH 7.5), 1% Triton X-100, 137 mM sodium chloride, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 25 mM ß-glycerophosphate, 2 mM sodium pyrophosphate, 1 mM PMSF, and 1 µ g/ml leupeptin. The cell lysates were centrifuged at 10 000 g for 10 min to remove the debris, and the proteins were fractionated using a 10% separating gel. The fractionated proteins were then transferred electrophoretically to nitrocellulose paper and the proteins were immunoblotted with the specific antibodies. Horseradish peroxidase- or alkaline phosphatase-conjugated anti-IgG antibody were used as the secondary antibodies. The nitrocellulose papers were developed using 5-bromo-4-chloro-3-indolylpho-sphate/4-nitroblue tetrazolium or an ECL chemiluminescence system.
Calcein efflux assay
Calcein-AM is a non-fluorescent and lipophilic acetoxymethylester of calcein. In the cytoplasm, calcein-AM is metabolized by esterases yielding hydrophilic and fluorescent calcein that cannot diffuse out of the cells (Vellonen et al. 2004). Since calcein is a substrate of MRP1 and MRP2, but not of MDR1 (Bauer et al. 2003), the only route for calcein to escape from the cell is by MRP1- or MRP2-mediated efflux. MCF-7 and TAMR-MCF-7 cells were seeded in 24-well plates. After 80% confluency reached, the cells were incubated in FBS-free DMEM for 18 h. The culture medium was changed with Hank’s balanced salt solution and the cells were preincubated at 37 ° C for 15 min. After incubation of the cells with 2 µ M calcein-AM for 20 min, the medium was completely removed and the cells were lysed in EBC lysis buffer. The calcein fluorescene retention in cell lysates was measured using the excitation and emission wavelengths of 496 and 516 nm respectively. The fluorescence values were divided by total protein contents of each sample.
RT-PCR
The total RNA was isolated from the cells using a total RNA isolation kit (RNAgents, Promega). The total RNA (1.0 µ g) was reverse transcribed using an oligo(dT) 18-mer as a primer and M-MLV reverse transcriptase (Bioneer, Eumsung, South Korea) to produce the cDNA. PCR was performed using the selective primers for the human PXR (sense primer: 5'-TCCGGAAAGATCTGTGCTCTT-3' , anti-sense primer: 5'-AGGGAGATCTGGTCCTCGAT-3' ; Masuyama et al. 2003), human MDR1 (sense primer: 5'-TGCTCAGACAGGATGTGAGTTG-3' , antisense primer: 5'-TAGCCCCTTTAACTTGAGCAGC-3' ) and S16 ribosomal protein (S16r) genes (sense: 5'-TCCAAGGGTCCGCTGCAGTC-3' , antisense: 5'-CGTTCACCTTGATGAGCCCATT-3' ). The PCRs were carried out for 35 (PXR) or 40 cycles (MDR1) under the following conditions: denaturation at 98 ° C for 10 s, annealing at 60 ° C (PXR) or 51 ° C (MDR1) for 0.5 min, and elongation at 72 ° C for 1 min. The band intensities of the amplified DNAs were compared after visualization using a u.v. transilluminator.
Reporter gene analysis
The promoter activity was determined using a dual-luciferase reporter assay system (Promega). Briefly, the cells (3 x 105 cells/well) were replated in 12-well plates overnight and transiently transfected with the p-MRP2-Luc plasmids/pRL-SV plasmid (Renilla luciferase expression for normalization; Promega) or the PXR reporter plasmid/pRL-SV plasmid using Genejuice reagent (Novagen, Madison, WI, USA). The cells were then incubated in the culture medium without serum for 18 h, and the firefly and R. luciferase activities in the cell lysates were measured using a luminometer (Turner Designs; TD-20, CA). The relative luciferase activities were calculated by normalizing the promoter-driven firefly luciferase activity versus R. luciferase.
Statistical analysis
Scanning densitometry was performed using an Image Scan & Analysis System (FLA-7000, Fujifilm, Japan). One-way ANOVA was used to assess the significant differences between the treatment groups. For each significant effect of treatment, the Newman–Keuls test was used to compare the multiple group means. The criterion for statistical significance was set at either P < 0.05 or P < 0.01.
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Results |
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The acquisition of TAM resistance in TAMR-MCF-7 cells was verified using a MTT assay. 4-hydroxytamoxifen caused a concentration-dependent decrease in the cell viability of MCF-7 cells (Fig. 1A
). In contrast, the cell viability of TAMR-MCF-7 cells was not changed by up to 30 µ M 4-hydroxytamoxifen (Fig. 1A).
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). The basal MRP2 levels were also low in the control MCF-7 cells. In contrast, the protein expression was higher in the TAMR-MCF-7 cells (Fig. 1C). The cellular retention of calcein, a substrate of MRP1 and MRP2 (Bauer et al. 2003), was determined to confirm the functional transport activity of MRP2. As shown in Fig. 1D, the cellular uptake ratio of calcein was 11.7-fold lower in the TAMR-MCF-7 cells, when compared with the control MCF-7 cells. There was a reasonable correlation between the cellular uptake of calcein and the MRP2 expression level in both the cell types. These suggest that the regulation of MRP2 expression is coupled with the process of acquiring TAM resistance, indicating that long-term exposure of breast cancer cells to TAM may cause additional changes in the uptake of chemotherapeutic agents. Involvement of PXR in the enhanced MRP2 expression in TAM-resistant breast cancer cell
Reporter gene analyses were performed using p2635-MRP2-Luc reporter plasmid, which contained the luciferase structural gene and a – 2.6 kb human MRP2 promoter, in order to determine whether transcriptional activation is involved in the over-expression of MRP2 gene in TAMR-MCF-7 cells. In addition, an attempt was made to identify the transcription factors involved in MRP2 gene transactivation. In comparison with the control MCF-7 cells, a 2.2-fold increase in the luciferase activity was observed in TAMR-MCF-7 cells transfected with p2635-MRP2-Luc plasmid (Fig. 2A), which suggests that the induction of MRP2 in the TAM-resistant breast cancer cells is mediated through the transcriptional activation of the MRP2 gene. Since PXR is believed to be an essential transcription factor for the induction of MRP2 (Teng & Piquette-Miller 2005), and the roles of PXR in the inducible expression of MRP2 have been examined extensively (Kast et al. 2002, Anapolsky et al. 2006), this study compared the reporter activities in both MCF-7 and TAMR-MCF-7 cells using the PXR reporter plasmid (three copies of the PXR-responsive elements from CYP3A23 gene; Kast et al. 2002). The reporter activity was significantly higher in the TAMR-MCF-7 cells than in the control MCF-7 cells (Fig. 2B). In addition, the luciferase reporter gene of rat MRP2 gene promoter, which contains farnesoid X-activated receptor (FXR)/PXR-binding region, was 7.4-fold induced in the TAMR-MCF-7 cells (Fig. 2C).
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). Consistent with the western blot analysis result, RT-PCR analysis showed that the PXR mRNA level was also increased in the TAMR-MCF-7 cells (Fig. 2D). The mRNA level of the S16 ribosomal protein was comparable in all samples (Fig. 2D). Therefore, PXR activation might be one possible mechanism for the higher MRP2 expression levels in TAMR-MCF-7 cells. No role of CCAAT/enhancer-binding protein ß (C/EBPß ? activation in MRP2 induction
It has been also suggested that the C/EBPß-binding region ( – 356 to – 343 bp) in human MRP2 promoter is involved in the basal expression of the MRP2 gene (Tanaka et al. 1999). C/EBPß is mainly located in the cytoplasmic fraction and is translocated to the nucleus upon activation. The nuclear C/EBPß levels were measured by subcellular fractionation and western blot analysis in order to determine whether C/EBPß activation is essential for the enhanced MRP2 expression in TAMR-MCF-7 cells. The nuclear levels of C/EBPß were higher in the TAM-resistant breast cancer cells than in the control cells (Fig. 3A). In contrast, C/EBP
was not altered by the long-term exposure of MCF-7 cells to TAM (Fig. 3A). The total amount of C/EBPß in the total cell lysates from TAMR-MCF-7 cells was also higher than those from the control MCF-7 cells (Fig. 3A). Therefore, the nuclear accumulation of C/EBPß in TAMR-MCF-7 cells may be due to the increased expression. A promoter mutation study was performed to clarify the role of C/EBPß activation in the transactivation of MRP2 in TAMR-MCF-7 cells. The reporter activities of both p491-MRP2-Luc ( – 491 bp human MRP2 promoter) and p491-MRP2-Luc-Mut constructs were compared. The p491-MRP2-Luc-Mut construct contains a promoter sequence with a specific mutation in the C/EBP-binding site (AACTTTGAAAGCC ( – 365 to – 343 bp) was converted to AACTT-TAACCGCC; Tanaka et al. 1999). The intensity of the luciferase activity by p491-MRP2-Luc-Mut in TAMR-MCF-7 cells was similar to that by p491-MRP2-Luci. This suggests that C/EBPß activation does not play an important role in the induction of MRP2 in TAM-resistant breast cancer cells (Fig. 3B).
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The physiological function of MRP2 is coupled with phase II conjugation enzymes because the conjugated metabolites can be pumped out through the ABC transporters. A series of our experiments demonstrated that PI3-kinase plays a key role in the induction of the phase II conjugation genes (Kang et al. 2001a,b). A recent study also suggested that PI3-kinase is involved in the transcriptional regulation of the MRP2 gene in Caco-2 cells (Jakubicova et al. 2005). The levels of phosphorylated Akt (a downstream kinase of PI3-kinase) were measured in order to determine whether PI3-kinase is activated in TAM-resistant breast cancer cells. PI3-kinase was persistently activated in the TAMR-MCF-7 cells, while minimal levels of phosphorylated Akt were observed in the MCF-7 cells (Fig. 4A). The role of PI3-kinase in the induction of MRP2 in TAMR-MCF-7 cells was then assessed using specific chemical inhibitor. Pretreatment of these cells with LY294002 (20 µ M) significantly blocked the induction of MRP2 in TAMR-MCF-7 cells (Fig. 4B).
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, pretreating the cells with LY294002 (20 µ M) almost completely inhibited the PXR reporter activity in TAMR-MCF-7 cells (Fig. 4C). To confirm the precise role of PI3-kinase, the PXR reporter activity was monitored in cells overexpressing p85 regulatory subunit of PI3-kinase (Fig. 4C). Overexpression of the p85 subunit suppressed PXR-driven reporter activity in TAMR-MCF-7 cells, whereas pCMV5 overexpression (Mock transfection) failed to alter the reporter activity. These results suggest that the PI3-kinase pathway plays a key role in controlling the PXR activity and PXR-dependent MRP2 expression in TAM-resistant breast cancer cells. Aromatase expression is not altered in TAM-resistant breast cancer cell
Clinical application of aromatase inhibitors in adjuvant therapy for hormone-sensitive breast cancer has rapidly grown over the past few years. Unlike TAM, which antagonizes estrogen receptor and subsequently inhibits the transcription of estrogen-responsive genes, aromatase inhibitors block synthesis of estrogens through the inhibition of aromatase catalyzing the conversion of C19 androgens to estrogens (Smith & Dowsett 2003). We further determined the effect of an aromatase inhibitor, 4-hydroxyandrostenedione (Formestane), on the cell proliferation for 48 h in testosterone-containing culture medium. The inhibition percentage of cell proliferation by 4-hydroxyandrostenedione appeared to be comparable in both the MCF-7 and TAMR-MCF-7 cells (Fig. 5A). Cell proliferation in the presence of testosterone was ~70% inhibited by 1 µ M 4-hydroxyandrostenedione in both the cell types (Fig. 5A). We also found that the protein expression level of aromatase in TAMR-MCF-7 cells was similar to that in control MCF-7 cells (Fig. 5B). These results show that transcriptional or translational regulation of aromatase is not altered during the acquisition of TAM resistance and imply that aromatase inhibitor may be active to inhibit the growth of TAM-resistant breast cancer cells.
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Discussion |
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A number of studies have been carried out on the changes in MRP2 expression under pathological conditions. MRP2 expression was significantly lower in obstructive cholestasis and chronic inflammation (Denson et al. 2002, Teng & Piquette-Miller 2005). Higher levels of MRP2 expression has been frequently observed in several types of cancer tissues including human colorectal carcinoma (Hinoshita et al. 2000), hepatoma (Nies et al. 2001) and lung cancers (Young et al. 1999), which might be associated with their resistance to chemotherapeutic agents. Since MRP2 is involved in the efflux of various chemotherapeutic agents, these findings indicate that breast cancer cells exposed to TAM long term may acquire additional resistance to other anti-cancer agents through the increased MRP2 activity.
The transcriptional regulation of MRP2 gene is not completely understood. The C/EBPß-binding region in the human MRP2 promoter is essential for the transactivation of the MRP2 gene (Tanaka et al. 1999). This study found that the long-term exposure of MCF-7 cells to TAM results in the upregulation of C/EBPß . However, the promoter reporter activity was not reduced in the TAMR-MCF-7 cells transfected with p-491-MRP2-Luc-Mut containing a specific mutation in the C/EBPß-binding site, which supported the hypothesis that C/EBPß activation in TAM-resistant breast cancer cells is not related to the induction of MRP2. In the rat MRP2 promoter, FXR binds to an unusual 26 bp sequence, which contains an inverted repeat of the AGTTCA motif (ER-8; Kast et al. 2002). It has been found that this sequence also interacts with PXR and the constitutive androstane receptor. Indeed, most of the PXR ligands such as rifampicin and hyperforin have been shown to upregulate MRP2 expression in humans, mice, and rats (Fromm et al. 2000, Kast et al. 2002, Anapolsky et al. 2006). Excess glucocorticoid concentrations increase the MRP2 mRNA levels, which implicates glucocorticoid-inducible PXR activation (Kliewer et al. 1998). It was found in this study that PXR was persistently activated in TAMR-MCF-7 cells. The reporter activity of ER-8 containing the rat MRP2 promoter in TAMR-MCF-7 cells was also higher than that in the control MCF-7 cells (Fig. 2C). Therefore, MRP2 overexpression in TAM-resistant breast cancer cells might result from PXR activation. Although MDR1 expression could not be detected in the control MCF-7 and TAMR-MCF-7 cells, the reporter activity of MDR1 promoter was considerably higher in the TAMR-MCF-7 cells after transfection with the reporter containing the human MDR1 promoter region (data not shown). The most important transcriptional event for the expression of MDR1 gene is PXR binding to the DR4 region (Geick et al. 2001). This shows that PXR activation during the acquisition of TAM resistance is the main regulator of the genes encoding MRP2 and MDR1, which contributes significantly to the additional chemoresistance. It has been reported that endocrine-disrupting chemicals activate PXR-mediated transcription, presumably through an interaction with co-activators (Masuyama et al. 2000). The long-term exposure of breast cancer cells to TAM might cause persistent PXR activation in a similar manner.
In this study, the increase ratio of MRP2 reporter activity by – 2635 promoter region in TAMR-MCF-7 cells was obviously decreased in comparison with that by the – 491 promoter. This would imply that there is some very potent repressor element between – 2635 and – 491 promoter region of human MRP2 gene. Tanaka et al.(1999) previously showed that the reporter activity of – 1659 MRP2 promoter decreased in HepG2 cells when compared with that of – 491 MRP2 promoter and they suggested that a putative silencer element was located in the – 1659 ~ – 491 promoter region. Possible transcription factors bind to the region are C/EBP , HNF-3ß , Evi-1 and AP-1; one of these proteins may function as a repressor for the transcription of human MRP2 gene.
During the development of the TAM resistance phenotype, the cell survival signaling pathways such as PI3-kinase and ERK can be activated, which might be one of the key issues in the transcriptional regulation of various MRPs. Jakubikoba et al.(2005) recently showed that PI3-kinase, but not ERK, was essential for the expression of isothiocyanate-inducible MRP2. In this study, the PI3-kinase was potently activated in TAMR-MCF-7 cells and either chemical inhibition or transfection with the p85 subunit significantly suppressed the expression of MRP2 in TAM-resistant breast cancer cells. Considering the findings that the percentage inhibition of the PXR reporter activity by LY294002 or p85 overexpression was > 80% and PI3-kinase inhibition did not cause complete loss of MRP2 expression in TAMR-MCF-7 cells, it would be possible that distinct kinase(s) and transcription factor(s) might also be involved in the regulation of MRP2 expression in TAM-resistant breast cancer cells.
Overall, this study showed that PXR is activated in TAMR-MCF-7 cells, and that the persistently activated PI3-kinase plays a role in activating PXR and PXR-dependent MRP2 expression. These observations might have clinical implications in highlighting the possibility of patients undergoing long-term TAM therapy acquiring additional chemoresistance.
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Acknowledgements |
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