Fig. 6.

Glycyrrhizin suppresses tumor angiogenesis in HMGB1-overexpressing tumor/B-cell co-culture. a Cell supernatants collected from tumor cells treated in the presence ( +) or absence (-) of glycyrrhizin (GL, 0.5 mM) were subjected to western blotting for detecting HMGB1 soluble protein. Coomassie brilliant blue-stained gels were used to confirm equal loading. b Analysis of endothelial HUVECs cells migration and tube formation from conditioned medium (CM) from tumor cells overexpressing HMGB1 (EC18H, K510H) or vectors control (EC18pc and K510pc). HMGB1-expressing cells do not show any potentiate effect on HUVEC cell migration and differentiation (tube formation) when compared to vector cells (control). c Incubation of HUVECs with CM collected from co-cultures (EC18pc/B and K510pc/B vs. EC18H/B and K510H/B) in the presence ( +) or absence ( −) of GL (0.5 mM). HUVEC migration and tube formation were counted by ImageJ. Scale bar: 200 µm. d qRT-PCR analysis of the relative expression of VEGFR, CD31, and EDN1 in HUVECs after 24-h incubation with CM from the co-culture with ( +) or without ( −) GL. e The representative images of cytokine antibody array (left) and comparison of mean pixel density measurement among cytokines in collected CM of tumor/B-cell co-cultures in the presence or absence of GL (right). The arrays were quantified using ImageJ. Values are normalized to reference spots on the membranes (top left, top right, and bottom left). d Results are representative of three different experiments. *p < 0.05, **p < 0.01, ***p < 0.001 relative to control