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

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-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

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 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

Data_Sheet_1_Identification of Human B-1 Helper T Cells With a Th1-Like Memory Phenotype and High Integrin CD49d Expression.PDF

<p>Human B-1 cells have been proposed to be CD20⁺CD27⁺CD43⁺CD1c⁻ B cells found in the umbilical cord and adult peripheral blood, but their regulatory mechanisms have not been well elucidated. Previously, we reported that mouse CD49d^high CD4⁺ T cells could enhance the secretion of natural antibodies by B-1 cells. In this study, we aimed to investigate the presence and helper functions of the human equivalents of murine CD49d^high CD4⁺ T cells. Here, we showed that human CD49d^high CD4⁺ T cells found in the peritoneal cavity (PEC), spleen, and peripheral blood can enhance the production of IgM antibodies by B-1 cells. As revealed in mouse, CD49d^high CD4⁺ T cells were more abundant in the PEC and showed a higher tendency to form conjugates with B cells than CD49d^low CD4⁺ T cells. Moreover, CD49d^high CD4⁺ T cells showed a Th1-like memory phenotype, characterized by high expression of CD44 and CXCR3; low expression of CD62L and CCR7; rapid production of IFN-γ, tumor necrosis factor-α, and IL-2 upon stimulation with phorbol myristate acetate and ionomycin; and rapid proliferation upon stimulation with anti-CD3 and anti-CD28 antibodies. These cells also expressed high levels of PD-1, ICOS, and CD5, suggesting that they are undergoing chronic stimulation. Remarkably, CD49d^high CD4⁺ T cells specifically helped B-1 cells, but not follicular memory B cells (CD27⁺ CD43⁻CD1c⁻) or marginal zone B cells (CD27⁺CD43⁻CD1c⁺), produce IgM and IgG antibodies. In parallel, the titer of human anti-blood group A IgM was positively correlated with the frequency of CD49d^high CD4⁺ T cells. In conclusion, we identified human CD49d^high CD4⁺ T cells with a Th1-like memory phenotype that secrete Th1 proinflammatory cytokines and help B-1 cells secrete antibodies, thereby aiding in primary defense. We suggest that these CD49d^high CD4⁺ T cells are a unique type of B-cell helper T cells distinct from follicular helper T cells.</p