Sodium oxamate

The cells were treated with 2-DG (0–10 mM), sodium oxamate (0–50 mM), or oligomycin (0–1.0 µg/mL; Santa Cruz, Dallas, TX, USA) for 24 h to test major metabolic pathways that are utilized by HepG2 cells. Each chemical was dissolved in DMSO and further diluted to final concentrations. Cells were treated with E2 (Sigma-Aldrich, St. Louis, MO, USA), ERα agonist PPT (Fisher Scientific), or ERβ agonist DPN (Fisher Scientific) for 48 h to examine effects of E2 and ERs. E2 and ERs were dissolved to 1 μM in DMSO and diluted to 10 nM in culture medium. Vehicle DMSO was the control treatment. The concentration ranges of these chemicals are commonly used in liver cancer research and our previous studies [13 (link),14 (link),28 (link),29 (link),30 (link)].

To obtain the inhibitory concentration 50 (IC₅₀) of the drugs, we followed the protocol described in (30 (link)). The cells were seeded in 96 well plates (7,000 cells per well), after 24 h, they were stimulated with the drugs at different concentrations: Doxorubicin (0.25, 0.5, 0.75, 1, 1.25, and 1.5 μM) (Doxolem ^®RU, 10 mg/5 ml), Metformin (0.001, 0.1, 5, 20, 35, 50, 65 mM) (Santa Cruz Biotechnology) and Sodium Oxamate (0.01, 0.5, 5, 15, 25, 35, 45 mM) (Santa Cruz Biotechnology). Individual treatments were performed with their respective IC₅₀ previously obtained. For the triple therapy IC₅₀, we choose the IC₅₀ of each drug, to recalculate a new IC₅₀ in combination, taking as a start point the individual IC₅₀. Cells were fixed with cold trichloroacetic acid at 10% and stained with Sulforhodamine B (MP BIOMEDICALS). The optical density was measured in a microplate reader (EPOCH, Biotek) at 510 nm. The IC₅₀ was determined from a linear regression (R2 = 0.92) to obtain the gradual dose–response graphs.