Bufalin suppresses sorafenib-induced Akt activation to reverse resistance to sorafenib in HCC cells.

<p>A-B, Huh7 cells were exposed to different concentrations of bufalin (A) or to 100 nM bufalin and/or 5 μM sorafenib (B) for 48 h. Untreated cells served as controls. Cell lysates were immunoblotted, and the density of each band was measured. Band densities were normalized to β-actin. The relative band density from untreated cells was defined as 1. (C) The Huh7 cells from (B) were immunostained with anti-p-Akt Ab (red) and DAPI (cellular nuclei, blue) and viewed with an inverted fluorescence microscope. The data represent three independent experiments. “*” (P<0.05) and “**” (P<0.001) vs. untreated control; “‡” (P<0.001) vs. sorafenib alone.</p

Bufalin synergizes with sorafenib to induce apoptosis in HCC cells.

<p>HepG2 and Huh7 cells were incubated with 100 nM bufalin and/or 5 μM sorafenib for 48 h. (A) The cells were stained with Annexin V/PI and subjected to flow cytometry to measure the apoptosis rate (%). (B) Representative images were taken of Huh7 cells stained with Annexin V/PI and viewed with a laser-scanning confocal microscope. (C) The activities of caspase-3 and caspase-9 were measured. The data represent three independent experiments. Untreated cells served as controls. “*” (P<0.05) and “**” (P<0.001) vs. untreated control; “‡” (P<0.001) vs. sorafenib alone; “#” (P<0.05) and “##” (P<0.001) vs. bufalin alone.</p

Bufalin-induced Akt inactivation is IRE1 dependent.

<p>(A) Huh7 cells were exposed to 100 nM bufalin for 12, 24, or 48 h. Untreated cells served as controls. Cell lysates were immunoblotted, and the density of each band was measured. Band densities were normalized to β-actin. (B) Huh7 cells were transfected with control, eIF2α, CHOP, or IRE1 siRNA for 24 h and then incubated with 100 nM bufalin for 24 h. Cell lysates were immunoblotted, and the density of each band was measured. Band densities were normalized to β-actin. The relative band density from control-siRNA transfected cells was defined as 1. (C) Huh7 cells were transfected with control or Akt siRNA for 24 h and then incubated with 100 nM bufalin for 24 h. Cell lysates were immunoblotted, and the density of each band was measured. Band densities were normalized to β-actin. The relative band density from control-siRNA transfected cells was defined as 1. (D) Huh7 cells were incubated with 100 nM bufalin and/or 5 μM sorafenib for 48 h. The cells were immunostained with Abs against IRE1 (red) and p-Akt (green) as well as with DAPI (cellular nuclei, blue). The data represent three independent experiments. N.S., not significant. “*” (P<0.05) and “**” (P<0.001) vs. untreated control; “##” represents P<0.001.</p

Inhibition of Akt enhances sorafenib-induced growth inhibition and apoptosis.

<p>A-B, HepG2 and Huh7 cells were exposed to 100 nM bufalin and/or 5 μM of sorafenib in the presence or absence of perifosine (10 μM) for 48 h. (A) Cell viability (%) was then compared with the corresponding untreated cells. (B) The percentages of apoptotic cells (%) were measured by flow cytometry. Untransfected cells served as controls. C-D, HepG2 and Huh7 cells were transfected with control or Akt siRNA for 24 h and then incubated with 100 nM bufalin, 5 μM sorafenib, or a combination of the two drugs for 24 h. (C) Cell viability (%) was compared with control siRNA-transfected cells. (D) The percentages of apoptotic cells (%) were measured by flow cytometry. Untransfected cells served as controls. The data represent three independent experiments. “**” represents P<0.001.</p

Bufalin reverses acquired resistance to sorafenib by downregulating p-Akt via IRE1 activation.

<p>(A) Huh7 and Huh7-Sora cells were cultured in complete medium, and the viability was examined after 24, 48, and 72 h in culture. (B) The above cells were exposed to increasing concentrations of sorafenib for 48 h. Untreated cells served as controls. Cell viability (%) was compared to the corresponding untreated cells. (C) The above cells were exposed to increasing concentrations of bufalin for 48 h. Untreated cells served as controls. Cell viability (%) was compared with the corresponding untreated cells. The black line indicates the IC50. (D) The lysates of cells from (A) were subjected to immunoblotting. Band densities were normalized to β-actin. The relative band density from Huh7 cells was defined as 1. (E) Huh7-Sora cells were transfected with control or IRE1 siRNA for 24 h and then incubated with 100 nM bufalin for 24 h. Cell lysates were immunoblotted, and the density of each band was measured. Band densities were normalized to β-actin. The relative band density from control cells was defined as 1. The data represent three independent experiments. N.S., not significant. “*” represents P<0.05, “**” represents P<0.001.</p

The siRNAs used in this study and their targeted genes.

<p>Abbreviations: IRE1, inositol-requiring enzyme 1; eIF2, eukaryotic initiation factor 2; CHOP, C/EBP-homologous protein.</p><p>The siRNAs used in this study and their targeted genes.</p

Bufalin synergizes with sorafenib to inhibit HCC cell growth.

<p>(A) HepG2 and Huh7 cells were exposed to different concentrations of bufalin and/or sorafenib for 48 h. (B) The above cells were incubated with 100 nM bufalin and/or 5 μM sorafenib for different periods. Cell viability (%) was then compared with the corresponding untreated cells. The data represent three independent experiments. “**” (P<0.001) vs. bufalin alone. “†” (P<0.05) and “‡” (P<0.001) vs. sorafenib alone.</p