HMGB1 Release by Urothelial Carcinoma Cells in Response to Bacillus Calmette-Guerin Functions as a Paracrine Factor to Potentiate the Direct Cellular Effects of Bacillus Calmette-Guerin

Purpose: Prior study demonstrated that HMGB1 release by urothelial carcinoma cells in response to bacillus Calmette-Guerin is required for an in vivo antitumor effect. We evaluated the direct effects of HMGB1 on the in vitro response of urothelial carcinoma cells to bacillus Calmette-Guerin. Materials and Methods: Two human urothelial carcinoma cell lines were used to study the effect of exogenous HMGB1 alone and combined with bacillus Calmette-Guerin on the tumor cell response to bacillus Calmette-Guerin. Antibody mediated blockade of receptors for HMGB1 or HMGB1 protein was used to determine the contribution of paracrine HMGB1 release to bacillus Calmette-Guerin biological effects. Response end points evaluated included the activation of intracellular signaling pathways, gene transactivation and cytotoxicity. Results: Urothelial carcinoma cells expressed the receptor for HMGB1 signaling. Antibody blockade of the RAGE receptor confirmed the dependence of signaling in response to HMGB1 on RAGE function. Exogenous HMGB1 activated cell signaling pathways for NF kappa B, NRF2 and CEBP. Quantitative reverse transcriptase-polymerase chain reaction on a panel of bacillus Calmette-Guerin responsive genes revealed peak expression resulting from the combination of bacillus Calmette-Guerin and HMGB1. Blockade of paracrine HMGB1 released in response to bacillus Calmette-Guerin using HMGB1 and/or RAGE receptor blocking antibodies showed a significant decrease in gene expression relative to that of bacillus Calmette-Guerin alone. HMGB1 potentiated the cytotoxic effects of bacillus Calmette-Guerin. Conclusions: HMGB1 released by urothelial carcinoma cells after bacillus Calmette-Guerin treatment functions as a paracrine factor to potentiate the urothelial carcinoma cell response to bacillus Calmette-Guerin. This paracrine activity likely contributes to the dependence of an in vivo tumor response on HMGB1 release.