HMGB1 Mediates Endogenous TLR2 Activation and Brain Tumor Regression
Figure 7
HMGB1 Is Secreted from Dying Tumor Cells and Is Required for Ad-Flt3L and Ad-TK Mediated Brain Tumor Regression
(A) GL26 cells were infected with Ad-TK and incubated with 25 μM GCV. A western blot was performed using an antibody specific for HMGB1. The graph displays a quantification of the total amount of HMGB1 in the media of cells 24 h (yellow bars), 48 h (orange bars), or 72 h (black bars) after GCV treatment.
(B) GL26 cells were incubated with Ad-TK (with and without GCV and glycyrrhizin), and media was overlaid on HEK293 reporter cells transfected with either a plasmid encoding TLR2 (+pTLR2; black) or with a control plasmid (-pTLR2; red). NFκB activity was determined by quantifying the activity of Firefly Luciferase (under the control of the NFκB promoter), *, p < 0.05 (Kruskal-Wallis followed by Dunn's test). Inset: cells were incubated with 100 ng/ml PAM3 (PAM3CSK4) as a positive control.
(C) The levels of HMGB1 in mouse serum were quantified by ELISA 7 d after treatment of brain tumors with Ad-Flt3L and Ad-TK. *, p < 0.05 versus saline (Mann-Whitney U-test).
(D) GL26 cells were implanted into C57BL/6 mice (n = 5 mice/treatment group) and 17 d later were treated with saline, or with AdFlt3L and Ad-TK (F/T). Glycyrrhizin (Glyc), HMGB1-depleting (αHMGB1), or rabbit IgG isotype control antibodies were administered IP 2 d, 5 d, and 10 d after treatment, *, p < 0.05 Ad-Flt3L and Ad-TK versus Ad-Flt3L and Ad-TK + glycyrrhizin; ^p < 0.05 Ad-Flt3L and Ad-TK + isotype versus Ad-Flt3L and Ad-TK + αHMGB1; Mantel log-rank test.