17β-Estradiol inhibits testosterone-induced cell proliferation in HepG2 by modulating the relative ratios of 3 estrogen receptor isoforms to the androgen receptor

Sex hormones, especially 17 beta-estradiol (E2) and testosterone (TEST), play crucial roles in the oncogenesis and progression of liver cancer via hormone-related receptors. As women have a lower rate of hepatocellular carcinoma (HCC) than men, estrogens might attenuate the occurrence and development of HCC. This study aimed to investigate the inhibitory effects and mechanisms of E2 on TEST-induced HCC development; the HepG2 cell line was used as an in vitro model. Five endpoints, including cell viability, cell apoptosis, cell cycle, receptor protein expression, and messenger RNA transcription, were investigated. Different roles and the ratios of androgen receptor (AR) and 3 estrogen receptor (ER) subtypes were also estimated. Cell viability assay showed that co-treatment of E2 and TEST resulted in a significant inhibition of E2-induced or TEST-induced cell proliferation. Flow cytometry analysis revealed that combined treatment of E2 and TEST blocked the cell cycle in the G0/G1 phase as well as induced cell early apoptosis, characterized by decreased cyclin-dependent kinase transcription and the ratio of Bcl-2/Bax. Real-time quantitative polymerase chain reaction and Western blot analysis results further demonstrated that estrogen receptor estrogen receptor alpha 66 (ER alpha 66) and estrogen receptor beta (ER beta) were upregulated, whereas AR and estrogen receptor alpha 36 (ER alpha 36) were downregulated, irrespective of whether E2 and TEST were considered separately or together, whereas the combined treatment of E2 and TEST resulted in a decrease in the ER alpha 66/ER beta ratio, the ER alpha 66/ER alpha ratio, and the ER beta/ER alpha 36 ratio, but with an increase in the ER alpha 66/AR ratio, the ER alpha 36/AR ratio, and the ER beta/AR ratio. To sum up, E2 could inhibit TEST-induced cell proliferation by modulating the ratio of different hormone-related receptors.