Interaction of T4 and E2 on mammary carcinogenesis (ex vivo and in vitro)

Abstract

Hypothyroidism seems to be a protective factor against breast cancer (BCa) though long-term exposure or high doses of thyroxine (T4) may increase BCa risk. Our previous studies indicated that hypothyroidism prolonged the latency, reduced the incidence, and retarded the growth of tumors in rats, and that T4 may influence mammary carcinogenesis by interacting with other hormone pathways. In this study, our aim was to investigate the biological impact of T4 alone or combined with estradiol (E2) on mammary tumors of hypo- and euthyroid rats, as well as human BCa cell lines. Female Sprague-Dawley rats were treated with a single dose of dimethylbenzathracene and were divided into two groups: hypothyroidism (HypoT; 0.01% 6-N-propyl-2-thiouracil -PTU- in drinking water, n = 20) and EUT (untreated control, n = 20). At sacrifice, tumor explants were obtained and treated either with 10-9 M T4, E2, or a combination, or DMEM/ F12 only as control for 15 min to evaluate intracellular signaling pathways, and after 24 h to evaluate changes in the expression of proteins related to apoptosis and proliferation by immunohistochemistry and Western Blot. In vitro, MCF-7 cells were treated with the same treatments. We assessed cell proliferation via MTT assays, and cell viability using Trypan Blue and the expression of proteins related to proliferation and apoptosis by immunocytochemistry and Western Blot. In HypoT tumor, T4+E2 stimulated proliferation, as evidenced by increased expression of Ki67 and mitotic/apoptotic index. In addition, suppressed apoptotic processes, as indicated by reduced expression of caspases 3 and PARP. In vitro, T4 induced MCF7 cell proliferation (p=0.012), viability (p=0.025) after 24h treatment. T4+E2 also enhanced the proliferation (p= 0.013) and viability of MCF-7 after 48 h, and activated the pERK/ERK pathway similarly to T4 alone (p < 0.05). In conclusion, T4 tends to enhance the action of E2 in proliferation and the effect of their interaction depends on the thyroid status.