Figure 2.

The effect of C-1311 on the CYP3A4 level and enzymatic activity in CHO-HR-3A4 cells. (A) Representative immunoblots of the level of cytochrome P450 3A4 in CHO-HR-3A4 cells exposed to C-1311 for the indicated time. CHO-HR-3A4 cells were exposed to C-1311 at the concentration corresponding to the IC₈₀ (0.06 μmol/L). After the indicated time, cells were lysed, and total protein was extracted, separated by SDS-PAGE, electrotransferred to a nitrocellulose membrane, and subjected to immunoblotting with anti-CYP3A4 antibody. n=3. (B) The time course of C-1311 influence on CYP3A4 activity in CHO-HR-3A4 cells. Cells were treated with two different concentrations of C-1311, 0.06 μmol/L (corresponding to the IC₈₀) and 1.0 μmol/L (corresponding to 50×IC₅₀), for the times indicated. (C) The concentration-dependent influence of C-1311 on CYP3A4 enzymatic activity in CHO-HR-3A4 cells. Cells were treated with 0.01, 0.1, 1.0, 5, 10, 25, and 50 μmol/L for 24 h. After the treatment, the media was changed, and 50 μmol/L testosterone solution was added for 24 h. The conversion of testosterone to 6-β-hydroxytestosterone was measured using RT-HPLC and calculated as the activity of CYP3A4 versus control (untreated cells). Values are the mean±SD. ^cP<0.01 vs control non-treated cells, n=3, one-way ANOVA test with Dunnett's post hoc test.