EF24 inhibits liver cancer growth and induces apoptosis both in subcutaneous HCC tumor model and orthotopic HCC models.

<p>(<b>a</b>). EF24 inhibits mice subcutaneous tumor growth in vivo. (<b>b</b>). Tumor size was measured after treated for three weeks. (<b>c</b>). EF24-treated animals resulted in a significantly lower tumor weight when compared with controls. (<b>d</b>). Tumor tissue removed from the animals were used for western blot analysis on the expressions of caspase-3, Cyclin B1, Cdc2, Bcl-2 and Bax. (<b>e</b>). Liver removed from the orthotopic HCC models, treated or untreated with EF24 for three weeks. (<b>f</b>). EF24 treatment groups significantly reduced the liver/body weight ratio compared to the control. (<b>g</b>). After treatmeant for three weeks, tumor tissue sections were stained with haematoxylin and eosion (HE). (<b>h</b>). Relative areas occupied by tumors in livers were calculated. (<b>i</b>). Tumor sections were stained with an anti-Ki-67 Ab to detect proliferating cells. (<b>j</b>). Cells expressing Ki-67 were counted to calculate the proliferation index, Assay was done in triplicate and p<0.01 is denoted by “*”.</p

EF24 inhibits angiogenesis and tumor cell survival signaling in liver cancer.

<p>(<b>a</b>). Lysates from EF24-treated (4 µM,) cells (Hepa1-6 and H22) used for western blot analysis on the expressions of ERK, p-ERK, Akt, p-Akt and COX-2. (<b>b</b>). HUVEC cells was treated with the indicated concentrations of EF24 for 24 h and 48 h. Cell growth was determined by cell counting kit-8 assay. (<b>c</b>). The proliferation of cells was also measured by crystal violet assay. (<b>d</b>). Tumor tissue lysates was used for western blotting to detect the levels of p-ERK, COX-2 and VEGF.</p

EF24 induces cell cycle arrest in mouse liver cancer.

<p>Cells were treated with either DMSO (control) or EF24 (4 µΜ) for 48 h were collected, flow cytometric analysis was performed for cell cycle distribution. (<b>a</b>). Representative flow cytometry graph for each treated or untreated groups. (<b>b</b>). Distribution of G2/M phase cells. Data represent mean ± SD of three independent experiments, *p<0.05 as compared with control group. (<b>c</b>). Immunoblot images of cell cycle regulatory molecules CycB1, Cdc2, p53, pp53, p21, MDM2 from treated (EF24, 4 µM, is denoted by “+”) and untreated (is denoted by “−”) cells.</p

EF24 inhibits cell proliferation and reduces cell viability.

<p>(<b>a</b>). Hepa1-6 and H22 cells were treated with the indicated concentrations of EF24 for 48 h and 72 h. Cell growth was determined by cell counting kit-8 assay. (<b>b</b>). Hepa1-6 cells and H22 cells incubated with 2 µM and 4 µM of EF24 for 48 h were analyzed for apoptosis. (<b>c</b>). Percentage of apoptotic cells as determined by flow cytometry of three independent experiments, * P<0.01 compared with the untreated (DMSO) cells. (<b>d</b>). and (<b>e</b>). Lysates from Hepa1-6 and H22 cells incubated with EF24 (4 µM) were analyzed by Western blotting for apoptosis-related proteins. (<b>f</b>). Hepa1-6 and H22 cells treated with EF24 and EF24 in combination with pan-caspase inhibitor z-VAD-fmk, Cell lysates were used for immunoblot assay for cleaved caspase-3.</p

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-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 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