Effect of emodin combined with various amount of rhodamine 123 or verapamil on inhibition of P-gp.

<p>K562/ADM cells were incubated with emodin (0.25–20 μM), together with 1, 3, 10 μM of rhodamine 123, respectively (A); The <i>K</i>_<i>i</i> of emodin was calculated and plotted against the concentrations of rhodamine 123 (B). K562/ADM cells were incubated with emodin (0.25–20 μM) with 0.5, 1, 2 μM of verapamil and 1 μM of rhodamine 123 (C). The calculated values of <i>K</i>_<i>i</i> for emodin were plotted against the concentrations of verapamil (D). Data were represented as the mean ± S.D of three independent experiments.</p

Free binding energies and residues identified to interact with emodin dependent on the binding sites.

<p>Free binding energies and residues identified to interact with emodin dependent on the binding sites.</p

Effects of emodin on rhodamine 123 uptake in Caco-2 and K562/ADM cells.

<p>The concentration-dependent effects of emodin on rhodamine 123 accumulation in Caco-2 (A) and K562/ADM (B) cells were investigated, respectively at final concentrations of 10, 50, 100, 200 μM; 0.25, 0.5, 1, 2.5, 5, 20 μM. Data were represented as the mean ± S.D. of three independent experiments. *<i>p</i> < 0.05; ** <i>p</i>< 0.01.</p

Intracellular amount of emodin in K562/ADM and K562 cells.

<p>Both cells were incubated with various concentrations of emodin (0.1–10 μM) at 37°C or 4°C for 2 h (A); K562/ADM cells were co-treated with 0, 1, 3, 10 μM of verapamil or 0, 2, 4, 10 μM of cyclosporine A and emodin (5 μM) for 2 h (B). RNAi of P-gp (C). A-: K562/ADM cells transfected with negative control siRNA; AL: K562/ADM cells transfected with Lipofectamine RNAiMAX Reagent; AP: K562/ADM cells transfected with P-gp siRNA; A: K562/ADM cells; S: K562 cells. After transfection with P-gp siRNA, intracellular amount of emodin (5 μM) was enhanced compared with other control groups (D). Data were represented as the mean ± S.D. of three independent experiments. *<i>P</i> < 0.05; ** <i>P</i> < 0.01.</p

Effect of emodin on expression of P-gp protein in K562/ADM cells.

<p>The cells were incubated in absence or presence of adriamycin or emodin for 48 h. The protein expression was determined by western blot analysis (A) and the relative expression of P-gp was calculated (B). *<i>p</i>< 0.05; **<i>p</i>< 0.01.</p

Effect of emodin on adriamycin-induced apoptosis in K562/ADM cells measured by annexin V-APC/DAPI double-staining assay.

<p>The K562/ADM cells were treated with adriamycin (10 μM), emodin (10 and 20 μM) or verapamil (10 μM) alone;, or adriamycin (10 μM) combined with emodin (10 and 20 μM) or verapamil (10 μM) for 48 h. Then cells were stained with annexin V-APC and DAPI before being subjected to flow cytometry for analysis. Histogram represented the means ± S.D. values for apoptotic cells obtained from three independent experiments. *<i>P</i> < 0.05; ** <i>P</i> < 0.01.</p

Effect of emodin on adriamycin cytotoxicity in K562/ADM cells.

<p>Effect of emodin on adriamycin cytotoxicity in K562/ADM cells.</p

Effect of emodin on the viability of K562, K562/ADM cells and Caco-2 cells.

<p>The K562 (A), K562 /ADM (B) cells were treated with emodin at different doses for 24, 48 or 72 h, Caco-2 (C) cells for 48 h respectively. Data were expressed as means ± standard deviations (S.D.) of three independent experiments.</p

Docking views of emodin with P-gp.

<p>The three-dimensional diagrams show the interactions and preferred conformation of emodin on the R site (A) and the M site (B) with labeled amino residues which significantly contributed for binding free energy. Emodin is colored in yellow. The hydrogen-bond is represented in purple dotted line. The π-π interaction is represented in green dotted lines. The two-dimensional diagrams display the interactions of emodin to the amino acid residues at the emodin on the R site (A') and the M site (B') of P-gp.</p