Celecoxib was a gift from Pfizer Corp. Hong Kong Ltd. Aspirin was obtained from Sigma Chemical Co. (St. Louis, MO). Other chemicals were ordered from Sigma Chemical, if not stated.
Cell culture
MCF-7 cells stably transfected with human
CYP19 (MCF-7aro) were prepared as previously described [
25]. These cells were maintained in MEM medium (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (Invitrogen Life Technology, Rockville, MD) and the selection antibiotic G418 (500 μg/ml, USB, Cleveland, OH). They were incubated at 37°C in 5% carbon dioxide and routinely sub-cultured when reaching 80% confluency.
Part I. Animal experiment
This mouse model for postmenopausal breast carcinogenesis was described by Yue et al. [
26]. Six-week old female athymic mice were acquired from the Animal Facility of Chinese University of Hong Kong. These mice were ovariectomized and allowed 3 weeks to recover, and were fed purified phytoestrogen-free AIN-93G diet. They were transplanted with MCF-7aro cells and randomly assigned into 4 regimens: control mice (Control), mice injected with androstenedione (AD), mice injected with androstenedione and treated with celecoxib (AD + celecoxib) and mice injected with androstenedione and treated with aspirin (AD + aspirin). The AD, AD + celecoxib and AD + aspirin mice received daily
s.c. injections of androstenedione (0.1 mg dissolved in 0.1 ml 0.3% hydroxyl propyl cellulose). Control mice received the carrier solvent injection only. Celecoxib and aspirin were administered in the diet at 1500 ppm and 200 ppm, respectively. Before transplantation, MCF-7aro cells were maintained in a culture incubator as described above. The cells were trypsinized and suspended in matrigel matrix (BD Biosciences, San Jose CA) (10 mg/ml) at 3 × 10
7 cells/ml. One hundred μl of cells were injected into the two flanks of the animal. This experiment was approved by Department of Health, the Governemnt of the Hong Kong SAR (Ref (07–164) in DH/ORHI/8/2/1 pt.9), and Animal Experimentation Ethics Committee of the Chinese University of Hong Kong (Ref. 13/023/GRF).
The body weight, tumor size and food intake were monitored weekly throughout the study. Tumor volumes were measured by an electronic caliper and estimated according to the formula: π/6 × length × width × height, where length, width, and height were the three orthogonal diameters of the tumors. At the end of the study, the mice were euthanized by cervical dislocation. Livers and uteri were weighed. Tumors and serum were collected and stored at -80°C until assayed.
Quantitative real time PCR assay
The frozen tumor samples were pulverized in a Dounce homogenizer with liquid nitrogen. Total RNA was extracted from the sample using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The concentration and purity of RNA were determined by absorbance measured at 260/280 nm. First DNA strands were synthesized from 3 μg total RNA using 5× primers (Assay-on-Demand™, Applied Biosystems, Foster City, CA, USA) and MMLV reverse transcriptase (USB Corporation, Cleveland, OH, USA). Target fragments were quantified by DNA Engine Opticon II (MJ Research, Inc., Waltham, MA). Probes for amplification were obtained from Assay-on-Demand™, Applied Biosystems, i.e. the housekeeping U6 snRNA (Assay ID: 001973), has-miR-let-7c (Assay ID: 000379), has-miR-Let-7 g (Assay ID: 002282), has-miR-98 (Assay ID: 000577), has-miR-221 (Assay ID: 000524), has-miR-222 (Assay ID: 002276), has-miR-17-5P (Assay ID: 000393), has-miR-101 (Assay ID: 002253), has-miR-145 (Assay ID: 002278). We used the Real-time PCR Taqman Universal PCR Master Mix (Applied Biosystems) to set up the PCR reactions as described in the manual. Signals obtained from U6 were utilized for normalization, and relative gene expression were analyzed by using the 2
-ΔΔCT method [
27].
For the determination of MYC and E2F2 RNA expression, we used oligo-dT primers for the first strand synthesis, SYBR green PCR Master Mix Reagent kit (Applied Biosystems) for the reaction setup, and GAPDH for normalization. The gene-amplification primer sequences were shown as below. By analyzing the dissociation curves and gel images, these primers did not produce non-specific amplifications. The relative gene expression was also determined by the 2
-ΔΔCT method (see Table
1).
Table 1
List of primers designed for RT-PCR quantitation
MYC
| 5′-TCT TCC AGA TAT CCT CGC TG-3′ | 5′-TAT GAC CTC GAC TAC GAC TCG-3′ |
E2F2
| 5′-TTA CAG TCA GAG GCC TGG CT-3′ | 5′-TTC TAA TAC TCA TCC CTG TTT TTC C-3′ |
GAPDH
| 5′-GAG TCA ACG GAT TTG GTC GT-3′ | 5′-GAT CTC GCT CCT GGA AGA TG-3′ |
The frozen samples were pulverized in a Dounce homogenizer with added liquid nitrogen. The pulverized samples were then sonicated in lysis buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 40 mg/L PMSF, 0.5 mg/L aprotinin, 0.5 mg/L leupeptin, 1.1 mmol/L EDTA and 0.7 mg/L pepstatin) with a cell disruptor (Branson Ultrasonics Corp., Danbury, CT, U.S.A.) on ice for 30 s for protein extraction. Thirty μg of protein extract were separated on 10% SDS-PAGE and transferred onto an Immobilon PVDF membrane (Millipore, Bedford, MA). Anti-ERα, anti-COX-2, anti-CDK4, Cyclin A, E, anti-Bcl-xL, Bcl-2, Bax, Bak (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin primary (Sigma Chem) and secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology) were used for protein detection. The targeted proteins were visualized by autoradiography on a Biomax (Kodak®) film. The images were scanned and analyzed for optical density by using the computer software ImageJ (National Institute of Mental Health, Bethesda MD, USA).
Serum estradiol determination
Serum estradiol concentration was measured by using ELISA kits from Cayman Chemical Company (Ann Arbor, MI). The samples were added into a 96-well plate coated with antibody raised against estradiol. After incubating with the tracer and developing at room temperature, the absorbance was quantified using a microplate reader (FluroStar®, BMG Labtechnologies GmBH, Offenburg, Germany). The amount of estradiol could be read against a standard curve constructed with the hormone provided in the kit.
Part II. In vitroexperiments
CYP19 enzyme inhibition assay
Two pmol recombinant aromatase protein (human CYP19 Supersomes®, BD Gentest, Woburn, MA) was incubated with celecoxib or aspirin in the substrate-containing assay buffer (25 nM-[1β-3H(N)] androst-4-ene-3,17-dione (NET-926; Perkin-Elmer Life and Analytical Sciences, Boston, MA), 3 · 3 mM-MgCl2, 100 mM-KH2PO4 (pH 7 · 4)). The reaction was initiated by adding 1.3 mM-NADPH and incubated at 37°C for 15 min. An aliquot of the medium was mixed with chloroform and centrifuged at 10,000 g for 10 min at 4°C. The aqueous phase was transferred into a tube containing 500 μl of 5% activated charcoal. An aliquot of the supernatant was removed for scintillation counting after incubating for 30 min.
Verification of expression pattern in culture cells
MCF-7aro cells were seeded in culture dishes at 5 × 102 cells/mm2, and allowed to settle for 1 day before treatment began. They were co-treated with androstenedione and various concentrations of aspirin or celecoxib for 1–3 days with DMSO as the carrier solvent. The final concentration of solvent was 0.1% (vol/vol). Total protein or RNA was extracted and analyzed.
Relationship between the differentially expressed genes and miR-222/-98
MCF-7aro cells were cultured in OptiMEM (Invitrogen Life Technology) and transfected with miR-222 or miR-98 mimics (Invitogen Life Technology) in Lipofectamine 2000 (Invitrogen Life Technology). Six hr after the transfection, the culture medium was replaced with RPMI (phenol red free) supplemented with 10nM androstenedione and 5% charcoal-dextran treated fetal bovine serum (Biotechnics Research, CA USA). Total protein or RNA was extracted 72 hr after the medium change. MTT assays were also performed in separate experiments to investigate the effect on cell growth.
Statistical methods
The software package Prism® 5.0 (GraphPad Software, Inc., CA, USA) was employed for statistical analysis. For multiple group analysis, the data were analyzed by One-way ANOVA followed by Tukey’s Multiple Comparison if significant differences (P < 0.5) were observed.