• Open Access

Progesterone receptor downregulates breast cancer resistance protein expression via binding to the progesterone response element in breast cancer

Authors


To whom correspondence should be addressed.
E-mail: zhougy@sdu.edu.cn

Abstract

Breast cancer resistance protein (BCRP) plays a major role in multidrug resistance (MDR). Sequence analysis reveals there is a novel progesterone response element (PRE) in the BCRP promoter, suggesting progesterone receptor (PR) may have a function in the regulation of BCRP expression. We examined the expressions of BCRP, PR, estrogen receptor α (ERα), androgen receptor (AR) and Her-2 in 95 breast cancer samples by immunohistochemistry. Then, to identify the role of PR in the regulation of BCRP expression, two constructs encoding full length BCRP cDNA driven by putative PRE promoter or constitutive CMV promoter were transfected into MCF-7 and MDA-MB-231, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to detect the expression of BCRP. Further electrophoretic mobility shift assay (EMSA) was used to verify the nuclear protein-DNA specific binding. We innovatively found that the expression of BCRP negatively related with that of PR and ERα in breast cancer by immunohistochemistry. While at a cellular level, after being treated by progesterone and 17β-estradiol, BCRPmRNA and protein levels were significantly reduced in a concentration-dependent manner in MCF-7/P-BCRP cells with PR bound to the identified PRE in BCRP promoter. Our results demonstrated that active PR inactived BCRP expression via progesterone-PR complexes binding to PRE in BCRP promoter in breast cancer cells. (Cancer Sci 2012; 103: 959–967)

To date, breast cancer is the most prevalent type of malignant tumor in women, and the multidrug resistance (MDR) is a major obstacle in successful chemotherapy. Breast cancer resistance protein (BCRP) is identified first from drug selected human breast cancer cells (MCF-7/AdrVp).[1] It overexpresses in many cancer cell lines and belongs to the subfamily of G of the ATP-binding cassette (ABC) transporter superfamily.[2-4] It can efflux a variety of chemotheraputic agents including mitoxantrone, topotecan and anthracyclines out of the cell.[5] Therefore, the investigation of mechanism of BCRP expression may bring out better treatment measures for cancer therapy.

Recent reports suggest that the BCRP expression may be adjusted at the promoter level by steroid hormones, such as estrogen, progesterone and testosterone via classical pathways, but the data have some conflicts.[6-8]

Hormone-receptor (HR) status has a major role in the prognosis and treatment of breast cancer patients. Estrogen plays an important role in the regulation of progesterone receptor (PR) in both mammary gland and breast cancer. However, there are still 2.00–8.12% of breast cancer patients immunohistochemically with ER–/PR+ and it often occurs in young or middle-aged women. The ER–/PR+ tumors are larger in size and appear more aggressive compared with the group of ER+/PR+.[9] Therefore, in those ER negative breast cancer cells, the PR expression level should be greatly compromised, which implies that the role of PR remains an enigma.

Our group has firstly revealed that toremifen can pre-transcriptionally mediate BCRP expression.[10] Recent research revealed that progesterone response element (PRE) in BCRP promoter was just the estrogen response element (ERE),[11] but little was known about the role that the novel element played in the BCRP-mediated MDR pathway in breast tumor cells.

In this study we transfected BCRP cDNA in MCF-7 and MDA-MB-231 breast cancer cells, and characterized the role of PRE in BCRP-mediated MDR. The results provided new insights into the regulatory mechanism of BCRP in human breast cancers by progesterone.

Materials and Methods

Patients, tissue samples and immunohistochemistry

This study was conducted on a total of 95 breast samples at the Department of Pathology of Qilu Hospital of Shandong University from 2007 to 2010, which were histopathologically diagnosed as invasive ductal carcinoma. Paraffin-embedded, 4-μm thickness, breast cancer sections were dewaxed and subjected to antigen retrieval by microwave irradiation for 20 min in 0.01 m citrate buffer (pH 6.0). Endogenous peroxidase was blocked with 0.3% H2O2 in methanol for 20 min. Then slides were incubated in primary antibodies to BCRP (clone BXP21, dilution 1:40, Abcam (Cambridge, MA, USA), estrogen receptor α (ERα) (dilution 1:250, ZSGB-Bio, Beijing, China), PR (dilution 1:200, ProMab, Richmond, Canada), androgen receptor (AR) (dilution 1:250, ZSGB-Bio) and Her-2 (dilution 1:250, ZSGB-Bio) overnight at 4°C. After being washed with PBS, the primary antibody was detected with appropriate secondary antibody for 25 min at 37°C. Following washes, slides were incubated in two-step plus Poly-HRP Anti- Mouse/Rabbit IgG Detection System (PV-9000, ZSGB-Bio) according to the manufacturer's recommendations and visualized using DAB (PV-9000, ZSGB-Bio), then rinsed in distilled water and counterstained with hematoxylin. A dual semi-quantitative scale combining staining intensity and percentage of positive cells was used to evaluate the protein staining. Briefly, staining of BCRP was scored semi-quantitatively for intensity (0, no expression; 1, weak; 2, moderate; 3, strong) and for the percentage of positive cells (0, <10%; 1, 10–40%; 2, 40–70%; and 3, ≥70%). The staining of PR and ERα were scored semi-quantitatively for intensity (0, no expression; 1, weak; 2, moderate; 3, strong) and for the percentage of positive cells (1, 0–1/100; 2, 1/100–1/10; 3, 1/10–1/3; 4, 1/3–2/3; and 5, 1/3-1 ).[12] The score of immunohistochemistry (IHC) ≥2 was defined as positive and <2 as negative. The staining of AR for the percentage of stained tumor cells was divided into four classes: 0, 0%; 1, 1–10%; 2, 11–50%; 3, 51–80%; positive tumor cells and 4, ≥80%. The intensity of staining was the same as above.[13] The score of IHC >3 was defined as positive and ≤3 as negative. Scoring of Her-2 was done as reported.[14] Normal breast epithelium and capillary were used as positive controls. All of the slides were examined and scored independently by two pathologists without any knowledge of the patient's clinical data. The procedures were in accordance with the ethical standards of the responsible committee on human experimentation.

Chemicals, reagents and antibodies

Progesterone, 17β-estradiol (E2), 5α-Androstan-17β-ol-3-one (DHT), mitoxantrone, dimethyl sulfoxide (DMSO) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI medium1640, with [+] L-glutamine and [–] Phenol Red medium and FBS were purchased from GIBCO (Invitrogen, Carlsbad, CA, USA). The BXP-21 (anti-BCRP antibody) was purchased from Abcam. The anti-PR antibody was purchased from ProMab. The anti-β-actin antibody was from Beyotime (Haimen, Jiangsu, China).

Cell lines and culture conditions

Human breast carcinoma cell lines, MCF-7 and MDA-MB-231, were obtained from the American National Cancer Institute. Cells were maintained in RPMI medium1640, with [+] L-glutamine and [–] Phenol Red, supplemented with 10% (v/v) FBS, 100 μL/mL penicillin and 100 μL/mL streptomycin in humidity atmosphere with 5% CO2 at 37°C.

RNA extraction, PCR amplification and plasmid construction

Total RNA was extracted from cell lines using Trizol reagent (TaKaRa, Kyoto, Japan). cDNA synthesis and semi-quantitative reverse transcriptase-PCR (RT-PCR) reactions were performed as previously described.[15] The primers used for amplifying the human BCRP cDNA are listed in Table 1. The amplified full-length BCRP cDNA with CMV promoter was inserted into pEGFP-C1 vector (Invitrogen) with Apa I/BamH I digestion to construct pEGFP/C-BCRP. To generate pEGFP/P-BCRP construct, BCRP promoter was firstly cloned into pMD20-T vector (TaKaRa), and then sub-cloned into pEGFP/C-BCRP construct with SnaB I/NheI digestion to destroy the CMV promoter. All constructs were sequenced to confirm the right promoter and cDNA sequences.

Table 1. Primer sequences for reverse transcription-polymerase chain reaction (RT-PCR)
GeneName and sequence
  1. BCRP, breast cancer resistance protein.

BCRP promoterSense primer 5′-GCCAGTGACGGCGACCAA -3′
Antisense primer 5′-AGCGCTGACACGAACTTCCTAA-3′
Wild-type human BCRP cDNASense primer 5′-GCTGCGGCCGCGAACTGGGTAGGATTTAGG-3′
Antisense primer 5′-CCCGCGGCCGCTGAAAGAACCCAAGACAT-3′
BCRP cDNASense primer 5′-TGGCTGTCATGGCTTCAGTA-3′
Antisense primer 5′-GCCACGTGATTCTTCCACAA-3′
β-actin cDNASense primer 5′-ACCCCCACTGAAAAAGATGA-3′
Antisense primer 5′-ATCTTCAAACCTCCATGATG-3′

Transfection and cell lysate preparation

Human breast carcinoma cell lines (MCF-7 and MDA-MB-231) were transient-transfected with those recombinant pEGFP/BCRP vectors using lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. To examine BCRP expression in the transfected cells treated with progesterone, E2 and DHT, the medium was replaced with fresh medium including different reagent at various concentration. Then, the cells were harvested after 24 h for mRNA isolation or 48 h for immunoblotting.

Semi-quantitative RT-PCR

Total RNA was isolated from cell lines using Trizol reagent (TaKaRa) according to the manufacturer's instructions. First-strand cDNA was synthesized with 1 μg of total RNA using One-step RT-PCR Kit (TOYOBO, ShangHai, China). The BCRP mRNAs were amplified with 2×EasyTaq PCR SuperMix (Transgene, Beijing, China) according to the following protocol: pre-denaturation at 94°C for 5 min, 29 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, elongation at 72°C for 30 s, and final extension at 72°C for 10 min. The primers used for amplifying the human BCRP cDNA and β-Actin are listed in Table 1.

Western blotting

Total proteins were extracted with radio-immunoprecipitation assay (RIPA). lysis buffer for 20 min on ice.(16) Centrifugation was at 12 000 g for 15 min at 4°C and the extracted proteins were boiled at 100°C for 5 min and then subjected to SDS-PAGE. The fractionized proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5% fat-free milk in TBS at room temperature for 2 h, the membrane was incubated with antibodies against human BCRP, human PR or human β-Actin overnight at 4°C. Afterwards, the membranes were washed three times with TBST at room temperature and then incubated with a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody at 37°C for 40 min. Finally, the membranes were visualized using an enhanced chemiluminescence kit (Millipore) according to the manufacturer's instruction.

Nuclear protein extraction and electrophoretic mobility shift assay (EMSA)

Nuclear proteins were prepared from MCF-7 and MDA-MB-231 cells using nuclear and cytoplasmic protein extraction kit and quantified with kit (Beyotime Institute of Biotechnology, Beijing, China) according to the recommendation.

Nuclear protein/DNA-binding reactions were performed in a volume of 25 μL containing 5 μg of nuclear extract protein. 3′-biotin-labeled oligonucleotides and unlabeled probe were synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). The sequence of oligonucleotides containing PRE of BCRP gene and mutated PRE (mutPRE) were shown in Table 2. Nuclear extract (5 μg) was incubated in an EMSA/Gel-Shift binding buffer (Beyotime) according to the recommendation and 0.2 nmole of labeled DNA probe. The mixture was incubated at room temperature for another 25 min. Specific binding was inhibited by the addition of 20- and 50-fold excess of unlabeled probe or 50-fold excess of unlabeled mutERE probe. For antibody supershift assay, 2 μL of anti-PR antibody was added in the binding reaction. Nondenaturing 5% polyacrylamide gels were pre-electrophoresed for 40 min in 0.5×Tris borate-EDTA (TBE) buffer before loading the binding reaction samples. Then, the samples were electrophoresed in 0.5×TBE buffer and transferred onto a positively charged nylon membrane (Hybond-N+). Transferred DNAs were cross-linked to the membrane for 10 min and detected using horseradish peroxidase-conjugated streptavidin and visualized using an enhanced chemiluminescence kit (Millipore).

Table 2. Oligonucleotides used for electrophoretic mobility-shift assay (EMSA)
ProbeSequencePosition
  1. PRE, progesterone response element.

PRE5′-ACGGCAGGTGACCC-3′–187/–173
5′-TGCCGTCCACTGGG-3′ 
mutPRE5′-ACGGCATCTGACCC-3′ 
5′-TGCCGTAGCTGGG-3′ 

Statistical analysis

Data analysis was performed with SPSS software version 18.0. Results are presented as mean ± SD. The correlations between immunohistochemical expression and the clinical variables were evaluated by the Chi-squared test. Comparison between groups were made using one-way anova followed by Student's paired t-test to determine the difference in significance. Values of P < 0.05 were considered significant.

Results

Expression of BCRP, PR, ERα, AR and Her-2 protein in breast cancer tissue

Only membrane staining of cell was considered BCRP positive and nuclear staining of the cell was judged as PR, ERα or AR positive in breast cancer tissue. The ductal epithelium of breast tissue and the endothelial cells were used as internal controls. As shown in Table 3, 29 of 95 patients were BCRP positive (30.53%), 45 of 95 were PR positive (47.37%), 65 of 95 were ER positive (68.42%), 58 of 95 were AR positive (61.05%) and 15 of 95 were Her-2 positive (15.79%) (Fig. 1). Moreover, expression of BCRP was negatively associated with that of PR (= 0.045) and ERα (= 0.017) and positively corrected with lymph node metastasis in breast invasive ductal carcinoma (= 0.025). While the expression of BCRP was not associated with that of AR and Her-2, also not corrected with patient age and tumor size.

Table 3. Results of immunohistochemistry
  BCRPP-value
nPositiveNegative
  1. AR, androgen receptor; BRCP, breast cancer resistance protein; ER, estrogen receptor; LN, lymph node; PR, progesterone receptor.

PRPositive459360.045
Negative502030
ERPositive6515500.017
Negative301416
ARPositive5817410.809
Negative34826
Her-2Positive155100.769
Negative802456
LNN02917220.025
N1661244
Age≤503718190.128
>50581840
Size≤24012281.000
>2551738
Figure 1.

Immunochemistry staining by anti-breast cancer resistance protein (BCRP) monoclonal antibody, anti-progesterone receptor (PR) and anti-ERα monoclonal antibody to identify the expression of BCRP, PR and estrogen receptor α in breast cancer tissue, respectively. (a,b) Representative image of membranous staining pattern of BCRP (200× and 400×, respectively). (c,d) Nuclear staining was negative for both PR and ERα (400×). (e,f) Nuclear staining was positive for both PR and ERα (400×). (g,h) Expression of BCRP was negative (400×).

Expression of PR, ERα, AR and BCRP protein in MCF-7 and MDA-MB-231 cells

We examined PR, ERα and AR expression in MCF-7 and MDA-MB-231 cell with western blotting. The results showed that MCF-7 cells expressed a high level of PR and ERα and a low level of AR, while in MDA-MB-231 cells, the level was very low (Fig. 2a). We observed the BCRP protein expression after transient-transfected cells with pEGFP/BCRP to test the function of the constructive plasmids. Only reagent that exhibited at least 60% transfection efficiency of plasmid delivery can pass to the next steps.

Figure 2.

Expression of progesterone receptor (PR), ERα, AR and breast cancer resistance protein (BCRP) in MCF-7 and MDA-MB-231 cells. (a) Expression of PR, ERα and AR in MCF-7 and MDA-MB-231 cells. MCF-7 and MDA-MB-231 were harvested and total proteins were extracted. To evaluate the expression of PR, ERα and AR protein, western blotting and densitometric analysis was performed. (b) Constructive plasmids pEGFP/BCRP transient-transfected MCF-7 and MDA-MB-231 cells. After being treated for 48 h, cells were harvested and total proteins were extracted. To evaluate BCRP expression level, western blotting and densitometric analysis were performed. β-Actin was analyzed in parallel as an internal control.

In Figure 2b,c, we can conclude that in both cell lines, the BCRP protein level dramatically elevated in the pEGFP/P-BCRP groups controlling to that in the pEGFP/C-BCRP or pEGFP empty group (P < 0.01), respectively. Furthermore, the level had no significant difference among pEGFP/C-BCRP, pEGFP empty and untransfected MCF-7 group (P > 0.05). These results suggested that the constructive BCRP promoter included PRE (pEGFP/P-BCRP) was indispensible for promoter induced BCRP activation.

Effects of progesterone, E2 and DHT treatment on exogenous BCRP mRNA expression

First, we investigated the BCRP mRNA level in pEGFP/BCRP transfected MCF-7 and MDA-MB-231 cells with RT-PCR. In MCF-7/P-BCRP cells, progesterone could inhibit the BCRP mRNA levels by 12.5%, 19.8%, 32.43%, 42.6% and 50.1%, respectively, compared with the control cells (P < 0.01) (Fig. 3a,e); E2 could inhibit the BCRP mRNA levels by 20.89%, 51.08%, 58.21%, 56.05% and 35.16% (P 0.05 at 10−13m group and others P < 0.01) (Fig. 4a,i); While after being treated by DHT, BCRP mRNA levels could be upregulated by 26.82% only at 5 × 10−7 m (P < 0.05) group (Fig. 4e,k). In MCF-7/C-BCRP cells, both progesterone and DHT did not have significant effects on BCRP mRNA level (P > 0.05) (Figs 3b,e and 4f,k), while E2 could promote the expression of BCRP mRNA (P < 0.01, respectively), but there was no significance between different concentration groups (Fig. 4b,i). In MDA-MB-231/P-BCRP and MDA-MB-231/C-BCRP cells, there were no significant changes with the addition of progesterone (Fig. 3c,d,f), E2 (Fig. 4c,d,j) and DHT (P > 0.05), except in 10−8 and 5×10−8 m group of DHT, which could upregulate the BCRP mRNA levels in MDA-MB-231/C-BCRP cells (P < 0.05, respectively) (Fig. 4g,h,l).

Figure 3.

Progesterone inhibited the exogenous breast cancer resistance protein (BCRP) mRNA level. Cells were cultured in a range of concentration of progesterone for 24 h (a–d). Densitometric analysis was then performed (e,f). Total RNA was extracted and the relative BCRP mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR) in comparison to β-Actin. Data shown are from three independent experiments.

Figure 4.

E2 inhibited the exogenous breast cancer resistance protein (BCRP) mRNA level and 5α-Androstan-17β-ol-3-one (DHT) stimulated the exogenous BCRP mRNA level at high concentration. Cells were cultured in a range of concentration of E2 or DHT for 24 h (a–h). Densitometric analysis was then performed (i–l). Total RNA was extracted and the relative BCRP mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR) in comparison to β-Actin. Data shown were from three independent experiments.

Effects of progesterone, E2 and DHT treatment on exogenous BCRP protein expression

To determine the effect of progesterone, E2 and DHT on the expression of BCRP protein, the pEGFP/BCRP transfected cells were incubated with reagents at different concentrations for 48 h. Then the cells were harvested and total proteins were extracted for western blotting analysis. In MCF-7/P-BCRP cells, the results showed that the addition of progesterone could lead to a suppression of BCRP protein expression by 33.3%, 50.0%, 59.5%, 66.7% and 80.9%, respectively, (P < 0.01) (Fig. 5a,e). The addition of E2 also lead to suppression by 46.94%, 59.51%, 67.34% and 33.04% at the range of 10−13 to 10−7 m, but the protein level increased by 52.66% at 10−5 m (P < 0.01, respectively) (Fig. 6a,i), while the addition of DHT stimulated the levels by 60.68%, 72.38%, 75.33%, 84.33% and 77.33% (P < 0.01, respectively) (Fig. 6e,k). In MCF-7/C-BCRP cells, E2 provoked the protein expression by 73.41%, 77.87%, 82.97%, 81.43% and 79.00% (10−13, 10−11 and 10−5 m P < 0.05; 10−9 and 10−7 m P < 0.01) (Fig. 6b,i), while progesterone (Fig. 5b,e) and DHT (Fig. 6f,k) showed little impact on BCRP protein levels (P > 0.05). Moreover, progesterone had little influence on BCRP protein in both MDA-MB-231/P-BCRP and MDA-MB-231/C-BCRP cells (P > 0.05) (Fig. 5c,d,f). But E2 and DHT could increase the protein levels in some groups (Fig. 6c,d,j; Fig 6g,h,l).

Figure 5.

Progesterone inhibited exogenous breast cancer resistance protein (BCRP) expression. After treatment with progesterone at the indicated final concentrations for 48 h, cells were harvested and total proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blotting with anti-BCRP antibody and anti-β-Actin antibody (a–d). Densitometric analysis was then performed (e,f).

Figure 6.

E2 inhibited exogenous breast cancer resistance protein (BCRP) expression at lower concentrations, but increased BCRP protein level at higher concentrations. While DHT could upregulate BCRP expression at the translation level. After treatment with E2 or DHT at the indicated final concentrations for 48 h, cells were harvested and total proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blotting with anti-BCRP antibody and anti-β-Actin antibody (a–h). Densitometric analysis was then performed (i–l).

Specific binding of PR to indentified PRE in the promoter of BCRP gene

EMSA were performed to examine whether the PRE in BCRP gene could be distinguished by PR. As shown, it was found that the specific binding complex was verified in PR-positive MCF-7, but not in PR-negative MDA-MB-231 with the consensus oligonucleotide containing PRE sequence and anti-PR antibody. The complex was supershifted on the addition of the anti-PR antibody. The DNA-protein complexes were completed partly or completely by adding 20- and 50-fold molar excess of unlabeled PRE probe. However, the complexes were not affected by adding 50-fold unlabeled mutPRE probe (Fig. 7). The results suggested that PR could have specifically recognized the putative PRE in the human BCRP gene.

Figure 7.

Specific binding of progesterone receptor (PR) protein to the identified progesterone response element (PRE) with electrophoretic mobility shift assay (EMSA). (a) Nuclear protein extracts isolated from MCF-7 and MDA-MB-231 cells. Lane A, nuclear extract incubated with mouse anti-PR antibody prior to biotinylated oligonucleotide containing PRE probe was added to detect supershift of the DNA protein complexes. Lane B, oligonucleotide with nuclear extract, the reaction containing 50-fold excess of unlabeled double-stranded mutated PRE (mutPRE). Lanes C and D, competition of biotinylated PRE probe with 50- and 20-fold molar excess of unlabeled oligonucleotide PRE probe, respectively. Lane E, specific DNA-protein complexes incubated with the biotinylated oligonucleotide containing PRE. Lane F, probe control alone. (b) Nuclear extracts from MDA-MB-231 cells were incubated with the biotinylated or unlabeled oligonucleotide.

Discussion

Drug resistance remains a major impediment to the effective treatment of breast cancer. Multidrug resistance (MDR) is usually related to the increasing expression of ABC transporters that consume energy to transport substrate drugs out of the cell, resulting in decreasing the drug concentration in the cell. P-glycoprotein (P-gp), BCRP and multidrug resistance associated protein (MRP) are three primary types of ABC transporters in human relating to the MDR phenomenon.[17-19] Though the expression level of BCRP is not as high as P-gp in malignant tumors, except pumped out chemotherapeutic drugs, BCRP is correlated with response in cases treated with some kinds of anticancer drug, which are not the substrates of BCRP.[16, 20] So it is inciting exploration of the mechanism of BCRP-mediated MDR.

BCRP is localized to 4q22 in chromosome and made up by 16 exons and 15 introns encoding a 72 kDa, 655-amino acid protein.[19] BCRP is designated as a half transporter and has the function of transporting various structurally and functionally unrelated anticancer agents across cell membranes against concentration gradient with the hydrolysis of ATP.[21] But studies of BCRP expression are controversial in cancer. Faneyte et al.[22] reported that BCRP was expressed in normal ductal cells, while it was negative in carcinoma cells. On the other hand, the cytoplasmic staining was considered to be positive.[23] Though the BCRP protein is synthesized in cytoplasm, it must be transported to the cell membrane to play its function. Currently, the knowledge about the structure-function relationship is limited. Wang first verified six transmembrane segments for BCRP, which had an important implication for comprehension of the interactions of substrate with BCRP and efflux mechanism.[24] So the status of BCRP may impact its function. In this experiment, only the brown staining on cell membrane can be judged as BCRP positive. The result showed that BCRP expression was low and had negative relation with that of PR.

Many studies found that the steroid hormone nuclear receptors were involved in the regulation of BCRP in tumors.[2, 25] It was reported that the expression of BCRP was decreased by E2 on transcriptional and translation levels via activating the ERβ at the blood–brain barrier (BBB).[26] In this study, we found the expression of BCRP had negative correlation with that of PR and ERα, while it had positive correlation with lymph node metastasis. Furthermore, it had no correlation with the expression of AR and Her-2, as well as patient age and tumor size. The data revealed that PR and ERα were involved in the modulation of expression of BCRP and its overexpression may induce the metastasis of the tumor.

Wang has verified a novel response element, named PRE, the same element as ERE,[11, 27] which was localized in the BCRP promoter and could interact with both PR and ERα. However, its role in breast cancer cells remains unknown. In the current study, to elaborate the molecular mechanisms by which steroid hormone nuclear receptors regulated BCRP expression, we constructed plasmids containing BCRP cDNA driven by endogenous PRE promoter, taking constitutive CMV promoter as a control. We then transfected MCF-7 cells and MDA-MB-231 cells with the two types of plasmids.

Progesterone, E2 and testosterone are important steroid hormones in women. The physiological levels of endogenous hormones of oestrogen, progesterone and testosterone were 1.8 × 10−9–4.8 × 10−11 m, 0–1 × 10−8 m and 7 × 10−103.1 × 10−9 m, respectively. It was reported, in postmenopausal cases diagnosed with invasive breast adenocarcinoma, that the serum level of total ostradiol ranged from 4.6 × 10−11 to 7.4 × 10−11 m, and that of progesterone was from 5 × 10−7 to 3 × 10−6 m.[28] We designed a range of steroid hormones concentrations among subjects in our study that was wide enough to detect a trend compared with the typical level of circulating hormones.

By treating the transfected cells with a various range of progesterone, E2 and DHT to investigate the mechanism of BCRP-mediated drug resistance, we observed that both progesterone and E2 downregulated BCRP gene expression at the transcriptional level in MCF-7/P-BCRP cells. But in the E2-treated group, although the mRNA level was downregulated at 10−5 m compared with the vehicle control, it slightly increased compared with 10−7 m. Furthermore, at the protein level, both progesterone and E2 can inhibit the expression of BCRP except E2 at 10−5 m increased BCRP protein by 52.66% compared with vehicle control. Moreover, DHT lower than 5 × 10−7 m had no effect on BCRP mRNA level, but significantly increased BCRP expression at the translation level. While in MCF-7/C-BCRP cells, progesterone and DHT had little effect on BCRP expression and E2 could increase BCRP expression. Moreover, in MDA-MB-231/P-BCRP and MDA-MB-231/C-BCRP cells, treatment with progesterone also induced little change in BCRP expression; E2 had no influence on BCRP mRNA level, but upregulated BCRP protein expression at certain concentrations. DHT could increase BCRP expression at both the mRNA level and protein level. This suggested that both progesterone and E2, at the physiological level, markedly downregulated exogenous BCRP expression in ERα+/PR+ MCF-7 cells, but not in ERα–/PR– MDA-MB-231 cells and the different pattern of inhibition of BCRP level in the two cell lines might result from the endogenous PRE promoter or constitutive CMV promoter of BCRP. Though immunochemical studies showed that AR was highly expressed in breast cancer tissue, it was low in MCF-7 cells. DHT treatment-increased BCRP expression might be caused by it stimulating the AR level in MCF-7/BCRP cells.[29]

Our group have reported that ERα mediated BCRP expression via ERE in breast cancer cells. Further EMSA confirmed that the putative PRE in the promoter region of the BCRP gene and PR were essential for inhibiting BCRP expression at a transcriptional level. These observations indicated that progesterone might bind to the PRE in the BCRP promoter through progesterone-PR complexes and decrease BCRP mRNA expression and protein level.[11] Collectively, we conjectured that the mechanism of progesterone that inhibited BCRP expression might be pre-transcriptional regulation, that was to say, progesterone-PR complex was directly bound to the identified PRE and interacted with some kind of coinhibitor protein and components of the transcription regulator, resulting in decreased transcription.[30]

In this study, we first found that the expression of BCRP protein was negatively related with that of PR and ERα in breast caner. Further research in cell lines revealed that transcription inhibition was involved in the regulation of BCRP expression by both progesterone and E2. This process was modulated by progesterone-PR or E2-ERα complex binding to the PRE or ERE in BCRP promoter via the typical pathway to inactivate transcription of the human BCRP gene, leading to reversing BCRP-mediated drug resistance in breast cancer cells. It was well known that estrogen stimulates the growth of breast cancer cells, but the function of progesterone in breast cancer remained controversial. Our studies provide the potential clinical guidance to treat BCRP-mediated MDR with progesterone in breast cancer. The result is consistent with the phenomenon that whatever the therapeutic drug, the sensitive cells in the carcinoma are eliminated; at the same time the drug resistance cells survive. Cancers with a higher percent of drug resistance cells will lead to poor treatment outcomes.[31]

Acknowledgements

This work was supported by the Natural Science Foundation of Shandong Province of China (No. ZR2009CQ009), Graduate's Independent Innovation Foundation of Shandong University (GIIFSDU) to X. W. (No. 21300070613175) and partly supported by the National Natural Science Youth Foundation of China (No. 30901456).

Disclosure Statement

The authors have no conflict of interest.

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