 Doxorubicin-treated cancer cells release HMGB1 when dying that can modify the expression of angiogenic factors in a MyD88 dependant and independent way

EMILIANO ROSELLI

Congreso; LXII REUNION ANUAL DE LA SOCIEDAD ARGENTINA DE IMNUNOLOGIA; 2014

HMGB1 (High mobility group box one) is a nuclear chromatin-binding protein that is released by necrotic cells, acting as a DAMP by binding to different receptors such as TLR4/TLR2/RAGE present on neighbour cells. HMGB1 participates in the ?immunogenic cell death? promoting the antitumoral immune response but also can be a pro-angiogenic molecule acting on endothelial cells. Little is known about the effect of HMGB1 on cancer cells themselves. Here, we investigate if HMGB1 released by dying doxorubicin-treated murine melanoma B16 cells induces the expression of angiopoietin 2 (Ang2) and other angiogenic factors 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. The release of HMGB1 was detected by western blotting in Doxotreated B16 cell conditioned medium (Doxo-CM) but not in non-treated cells (CM). Doxo-CM and CM were added to B16 and B16shMyD88 cells for 24h and the expression of Ang 2 transcript was evaluated by qRT-PCR. A TLR3 ligand was used as a positive control and glycyrrhizin (Gly) was used as a HMGB1 inhibitor. The expression of Ang2 increased robustly in Doxo-CM treated B16 and B16shMyD88 cells (x6.0 and x5.6 respectively). Intriguingly, when Gly is added to inhibit HMGB1 a 20% inhibition in the expression of Ang2 is observed only when MyD88 is silenced. In addition, recombinant HMGB1 (rHMGB1-1ug/mL) was used to stimulate B16 and B16shMyD88 cells and through a proteome array a set of 53 angiogenic proteins were evaluated in the supernatants. ADAMTS1, PDGF-AA, VEGF, Tissue Factor were up regulated on a MyD88 dependant manner whereas others such as CD105 increased in a MyD88-independent way. Therefore, HMGB1 released by dying tumor cells can act on neighbour cells in a MyD88 dependant and independent way and promote angiogenesis.