The estradiol metabolite, 2-methoxyestradiol inhibits proliferation of human cultured airway smooth muscle

The extent of the anti-mitogenic actions of, ßz-adrenoceptor agonists and glucocorticoids on proliferation of human cultured airway smooth muscle cells (HASM) depends on the mitogen and its concentration. There is a need for new agents with greater capacity to control this aspect of asthma pathology. We examined inhibition of HASM proliferation by the antiangiogenic estradiol (E2) metabolite, 2-methoxyestradiol (2-MEO) and its relationship to estrogen receptor (ER) binding, cell cycle signal transduction systems including ERK activity and cyclin D1 protein levels. Methods: DMA synthesis was measured by [3H]-thymidine incorporation. Cyclin D1 protein and message levels were measured by Western and Northern blotting, respectively. Radioligand binding assays were carried out on rat uterine cytosol (ER preparation). Results: 2-MEO (1-10>M) reduced DMA synthesis in response to thrombin (pICso 5.21 ±0.04, n=8) with greater potency than its precursor, 2-hydroxyestradiol (20HE2, 5.03±0.08, n=5). Conversely, 20HE2 was more potent in displacing 0.2 nM [3H]-E2 from ER (pICso 9.49±0.02) than 2-MEO (7.50±0.15), which showed a lower affinity than E2 (9.89±0.24). 2-Methoxyestriol (2-MEL) and 2-methoxyestrone (2-MEN), which have substitutions on 2-MEO resulting in an electronegative charge cloud below the steroid D ring plane, enhanced DMA synthesis and had negligible affinity for ER (pIC.: 2-MEL, 5.56±0.08; 2-MEN, 4.97±0.34). 2-MEO (10 p.M) reduced cyclin D1 protein, but not mRNA levels, had no effect on ERK activity and lost inhibitory activity when added 6 h after thrombin. Salbutamol or dexamethasone inhibited S-phase entry when added up to 18-20 h after thrombin. Conclusions: 2-MEO inhibits HASM proliferation by mechanisms that differ from those of salbutamol or dexamethasone and has potential as a novel anti-asthma agent.