Retrospective biological samples
For AGE analysis, serum samples were obtained from the Hollings Cancer Center Tissue Biorepository at the Medical University of South Carolina (MUSC). In total, 20 well and 20 poorly differentiated serum samples (1 ml of each) were selected from female breast cancer patients which had been stored between 1 and 5 years since diagnosis.
Tissue microarray’s were obtained from US BioMax (Rockville, MD) and contained 96 samples of non-cancer, hyperplastic, and cancerous tissue from 48 patients. Pathological data available from each patient included tumor grade and receptor status. Tissue for mass spectrometry analysis was obtained from breast cancer patients recruited in Baltimore hospitals between 1993 and 2003, as previously described [15, 16].
Immunohistochemical (IHC) staining
IHC staining was performed to examine AGE levels in tissue as previously published . IHC scores were calculated using the formula: intensity X % positive tumor cells. Intensity staining was scored as follows: 1—weak, 2—intermediate, 3—strong, and 4—very strong. Percent positive tumor cells were scored as follows: 1—less than 10%, 2—10–30%, 3—30–60%, and 4—60–100% positive cells by a pathologist.
To examine the levels of AGEs in serum, 96-well format Oxi-select ELISA’s (Cell Biolabs, San Diego, CA) were used as directed by the manufacturer. All samples were normalized to total protein concentration .
Mass spectrometry analysis
Carboxymethyllysine (CML) and carboxyethyllysine (CEL) and the internal standards (IS) CML-13C4/CEL-13C4 were obtained from Sigma-Aldrich/Santa Cruz. The CML/CEL stock solutions were prepared in water as 1 mg/mL solutions. The CML/CEL calibration standards, 0.8–100 ng/mL, were prepared by serial dilution of the stocks with PBS. The working IS solution (10 ng/mL each of the 13C4) was prepared by diluting the IS stocks with 0.24N trichloroacetic acid (TCA) solution. The tissue samples were wet weighted and homogenized in PBS using the Bead Ruptor (Omni), followed by centrifugation (10 min/16 kg). 15 µL of the tissue supernatant or calibration standards were vortex mixed with 75 µL IS solution. After centrifugation (10 min/16 kg), the solutions were transferred to injection vials for LC/MS/MS. LC was performed with a Shimadzu 20AC-XR system. The injection volume was 40 µL. Separation was achieved with a 2 × 150 mm, 3 µm SS-C18 column (Imtakt). Mobile phase A was 0.1% formic acid in water and mobile phase B was 50 mM ammonium acetate/80% methanol. The flow rate was 300 µL/min and peaks were eluted with a 8-min gradient. MS/MS was performed with a TSQ Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific) operating in selected reaction monitoring (SRM) mode with positive electrospray ionization. The target peaks were detected using the following m/z precursor > product ions: CML (205 > 84); CML-13C4 (209 > 88); CEL (219 > 84); CEL-13C4 (223 > 88). CML and CEL in the supernatant (Y ng/mL) were determined using the Thermo Xcalibur software. Calibration curves, constructed by plotting the peak area ratios vs. standards, were fitted by linear regressions (R2 = 0.99). The peak area ratios were calculated by dividing the peak areas of CML or CEL by the peak area of CML-13C4 or CEL-13C4, respectively.
MCF7 and T47D cell lines were purchased from ATCC (Manassas, VA). Cells were incubated at 37 °C, 5% CO2 in their respective media. Cells were cultured up to 30 passages before being replaced from low passage stocks. Mycoplasma-negative cultures were ensured by PCR testing. Cells were monitored throughout with consistent morphology and doubling-time. T47D cells were incubated in RPMI (Fisher Scientific, Fair Lawn, NJ) with 10% fetal bovine serum (Fisher Scientific) and 1% Penicillin/Streptomycin (Fisher Scientific). MCF7 cells were incubated in DMEM/High Glucose media (Fisher Scientific) containing 10% FBS and 1% Penicillin/Streptomycin with the following additives: 1% MEM Non-essential Amino Acids, 1% Sodium Pyruvate, 1% Sodium Bicarbonate, and 1% insulin (all obtained from Life Technologies, Grand Island, NY).
Exogenous AGE treatment
Exogenous BSA-AGE and control BSA were produced as previously published . For examination of AGE treatment, cells (400,000) were seeded into each well of a 6-well plate in serum-free media overnight before treatment with AGE metabolite (50ug/ml). Effects of AGE treatment on the phosphorylation of ERα, AKT, and ERK was assessed by Western blot as indicated below. Pharmacological targeting of the MAPK/ERK and PI3K/AKT pathways in the presence of AGE was achieved in serum-free media using the molecular inhibitors U0126 (ERK) and LY294002 (AKT) (Cell signaling Technology, Danvers, MA) at a concentration of 10uM for 12 h before exposure to AGE metabolites. To examine the effects of AGE metabolite on tamoxifen resistance, cells (3,000) were seeded into each well of a 96-well plate and were treated with the active metabolite of tamoxifen, 4-hydroxytamoxifen (0, 10 µM) (Sigma-Aldrich, St. Louis, MO) in combination with varying doses of AGEs (0, 5 µg/mL, 10 µg/mL, 50 µg/mL). After 24, 48, and 72 h, SRB staining was used to quantify cell growth as described previously .
Western Blot analysis
For isolation of total protein, cells at 70–80% confluence were washed twice with ice cold 1xPBS, and were lysed in radio-immunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (Sigma). Equal amounts of total protein (50 µg) were resolved by 10% SDS-PAGE and subjected to Western blot analyses using ECL system (Pierce-Fisher Scientific, Rockford, IL). Total protein lysates were examined using primary antibodies against total ERα, phosphorylated ERα-ser118 (p-ERα (ser118)), phosphorylated ERα-ser167 (p-ERα (ser167)), total ERK, phosphorylated-ERK (pERK), total AKT, phosphorylated-AKT (pAKT), and GAPDH (all obtained from Cell Signaling Technology).
Intervention design and dietary assessment
See supplementary methods.
See supplementary methods.