Sex Hormone‐Dependent Modulation of Nrf2 and Its Association With Apoptosis, Metastasis, and Drug Resistance in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) exhibits a gender disparity, with a 2- to fourfold higher incidence in men, as attributed by previous studies to estrogen-mediated hepatoprotection versus the tumor-promoting role of androgens. This study investigated the effects of these sex hormones-testosterone and β-estradiol-on Nrf2 expression and its relation to key factors in metastasis, apoptosis, and drug resistance in HepG2 HCC cell line. HepG2 cells were exposed to testosterone or β-estradiol at viability-equivalent doses. Oxidative stress was quantified by total oxidant status (TOS), malondialdehyde (MDA), and total antioxidant capacity (TAC) assays. Apoptosis was evaluated by Annexin V/PI flow cytometry. mRNA levels of Nrf2, MRP-1 (multidrug resistance protein 1), MMP-9 (matrix metalloproteinase-9), BCL2, and SIRT1 were assessed via qRT-PCR. At viability-equivalent doses, β-estradiol elicited dose-dependent apoptosis in HepG2 cells (total: 47.4% at V₅₀ vs. 32.7% for testosterone; p < 0.001), predominantly late-stage (34.5% vs. 21.8%; p < 0.0001). Concomitantly, β-estradiol decreased TOS (maximal at V₄₀, p < 0.05) and MDA (all doses, p < 0.05) while increasing TAC (medium/high doses, p < 0.05)-effects not observed with testosterone (p > 0.05). β-estradiol induced downregulation of Nrf2, BCL2, MMP-9, and MRP-1 mRNA (moderate/high doses; p < 0.05 vs. control). Conversely, testosterone upregulated SIRT1 across doses (p < 0.05), unaffected by β-estradiol (p > 0.05). β-Estradiol mitigates oxidative stress, induces apoptosis, and suppresses pro-survival, metastatic, and chemoresistant pathways-contrasting testosterone's minimal effects. These findings align with HCC's male predominance and highlight the hormone-modulated strategies' potential for future therapeutic exploration.