PMC

Schematic of TAM metabolism.

Analysis of glucuronidation activities against trans-4-OH-TAM and trans-endoxifen in HLM stratified by UGT2B7 genotypes. Glucuronidation assays were performed and 4-OH-TAM and endoxifen glucuronides separated by UPLC as described previously. (A) trans-4-OH-TAM and UGT2B7 codon 268 genotypes; (B) trans-endoxifen and UGT2B7 codon 268 genotypes. Comparative analysis was performed using the wild-type UGT2B7^268His as the referent; ^*P < 0.001; ^**P < 0.002, and error bars represent standard error.

Competitive binding assay of TAM and metabolites of TAM. The cytosolic fraction of MCF-7 cells was incubated (500 μL total reaction) for 18 h at 4°C with the indicated concentration of competitor (10⁻⁹ to 10⁻⁶ M) or [³H]-labeled E₂ (10⁻¹¹ to 10⁻⁶ M). After incubation, 5% dextran-coated charcoal was added to adsorb unbound ligand for 15 min at 4°C, and radioactivity was measured in the supernatant. Data are expressed as the percentage of specific binding of [³H]-E₂ for the ER when competitor was not present. Numbers in parentheses, RBA to ER for each test compound, with all data normalized to the RBA of E₂ (set at 100). (A) T4, trans-4-OH-TAM; T4O, trans-TAM-4-O-glucuronide, T4N, trans-4-OH-TAM-N-glucuronide; (B) C4, cis-4-OH-TAM; C4O, cis-TAM-4-O-glucuronide; C4N, cis-4-OH-TAM-N-glucuronide; (C) TE, trans-endoxifen; TEO, trans-endoxifen-O- glucuronide; CE, cis-endoxifen; CEO, cis-endoxifen-O-glucuronide; (D) TN, TAM-N-glucuronide.

Effect of TAM and TAM metabolites on PGR gene expression in MCF-7 cells. Cells were incubated with 1 × 10⁻¹⁰ M E₂ for 12 h at 37°C in 5% CO₂. Total RNA was extracted and PGR mRNA levels were determined by RT-PCR as described in the Methods section. PGR mRNA levels were expressed as the fold-induction of PGR mRNA levels observed for untreated cells (media). (A) PGR gene expression in E₂-induced MCF-7 cells treated with TAM and 4-OH-TAM isomers or glucuronides; (B) PGR gene expression in E₂-induced MCF-7 cells treated with endoxifen isomers or glucuronides. The E₂ positive control, the media alone, and vehicle negative control are shown on both panels. E₂, cells incubated with 1 × 10⁻¹⁰ M E₂; vehicle, cells incubated with 0.1% DMSO; trans-4-OH, cells incubated with trans-4-OH- TAM; cis-4-OH, cells incubated with cis-4-OH-TAM, trans-4-OH-O, cells incubated with trans-TAM-4-O-glucuronide; cis-4-OH-O, cells incubated with cis-TAM-4-O-glucuronide; trans-4-OH-N, cells incubated with trans-4-OH-TAM-N⁺-glucuronide; cis-4-OH-N, cells incubated with cis-4-OH-TAM-N⁺-glucuronide; trans-E, cells incubated with trans-endoxifen; cis-E, cells incubated with cis-endoxifen; trans-E-O, cells incubated with trans-endoxifen-O-glucuronide; cis-E-O, cells incubated with cis-endoxifen-O-glucuronide; TAM, cells incubated with TAM; TAM-N, cells incubated with TAM-N-glucuronide. The figure legend describes the concentration of TAM or TAM metabolite used in this experiment. The mean ± standard error is shown for three experiments.