FIGURE 3

FIGURE 3

Coinhibition of NF-κB and c-JUN synergistically suppressed HCC growth and in vitro and in vivo. (A) The dose-response curves for each inhibitor alone or in combination. For the combination, the x-axis represents the concentration of JNK-IN-8. (B) Isobologram for the combination of JNK-IN-8 and JSH-23 in BEL-7402 cells. The red line represents the intercept line of the IC50 values for JNK-IN-8 and JSH-23 alone, and the “×” indicates the IC50 value for the two inhibitors at a dose ratio of 1:8. CI < 1, = 1, and > 1 indicate synergism, an additive effect, and antagonism, respectively. (C–E,F–H,I–K,L–N) Treatments administered to a zebrafish tumor xenograft model were vehicle control, JNK-IN-8 (10 μM) alone, JSH-23 (5 μM) alone, and JNK-IN-8 (10 μM) combined with JSH-23 (5 μM). BEL-7402 cells were labeled with CM-DiI red fluorescence dye, and white arrowheads indicate disseminated tumor foci in 5-dpi zebrafish embryos. (O–Q) Quantification of the tumor mass, the inhibition rate, and the number of disseminated tumor foci. (R–Ac) Representative bright field and fluorescent images of control embryos or embryos treated with JSH-23 at 4 days post-fertilization (dpf). In control embryos, SIVs developed as a smooth basket-like structure over the yolk at 4 dpf (V,Z, yellow dashed lines). In contrast, the treatment of embryos with JSH-23 resulted in specific defects in SIV formation (W–Y,Aa–Ac, white dashed lines). Treatment with 10 μM JSH-23 for 48 h caused pericardial edema (red arrow). The complications were caused by vascular defects in the SIV (white arrow). (Ad,Ae) Quantification of the SIV area shows a significant decrease in the SIV area in JSH-23-treated embryos compared to control embryos. RFI, relative fluorescence intensity; dpi, days post-injection. Columns, mean; bars, SEM (n = 10; ANOVA; **p < 0.01, ***p < 0.001, ****p < 0.0001).