Doxorubicin-treated cancer cells release HMGB1 that can modify the expression of VEGF and Angiopoietin I in an autocrine way via MyD88

ROSELLI EMILIANO; DAVID ANDRÉS NOCERA; GERARDO GATTI; NICOLAS NUÑEZ; PAULA ARAYA; MARIANA MACCIONI

Los Cocos, Córdoba, Argentina

Congreso; Congreso Anual de la Sociedad Argentina de Inmunologia; 2013

Sociedad Argentina de Inmunologia

HMGB1 (High mobility group box one) is a nuclear chromatin-binding protein that is released by necrotic or stressed cells, acting as a DAMP (danger associated molecular pattern). Once released, HMGB1 can bind to different receptors such as TLR4, TLR2 or RAGE present on neighbour cells. HMGB1 participates in the ?immunogenic cell death?, activating dendritic cells and promoting the antitumoral immune response but also can be a pro-angiogenic molecule acting in several autocrine and/or paracrine feedback mechanisms on endothelial cells. Little is known about the effect of HMGB1 on cancer cells themselves. In this work, we investigate the capacity of HMGB1 released by Doxorubicin-treated murine melanoma B16 cells of inducing the expression of vascular endothelial growth factor (VEGF) and Angiopoietin I (Ang I) on other B16 cells in which the expression of MyD88 was silenced (B16shMyD88). B16 cells were treated with Doxorubicin (Doxo, 1-10uM) and its cytotoxic effect was monitored by MTT assay and flow cytometry. The release of HMGB1 was detected by western blotting in Doxo-treated B16 cell conditioned medium (Doxo-CM) but not in non-treated cells (CM). Doxo-CM and CM were then added to B16 and B16shMyD88 cells for 24h and the expression of VEGF and Ang I transcripts were evaluated by qRT-PCR, analyzing the data by the 2(-ΔΔC(T)) method. LPS was used as a positive control. Our results indicate that the expression of VEGF and AngI increased in LPS-activated B16, but not in LPS-B16shMyD88 (VEGF: x7,3 vs x0,5; Ang I: x250 vs x0,5 respectively). When the cells were incubated with CM and Doxo-CM similar results were obtained (VEGF in B16 cells: CM x2,3; Doxo-CM x3,8 vs CM x0,41; Doxo-CM x0,21 in B16shMyD88 B16 cells / AngI in B16 cells: CM x11,9; Doxo-CM x215 vs CM x0,9; Doxo-CM x4 in B16shMyD88 B16 cells. Our results indicate that Doxo-treated cancer cells release HMGB1 and that Doxo-CM can modify the expression of VEGF and Angiopoietin I in an autocrine way via MyD88