DOXORUBICIN-TREATED CANCER CELLS RELEASE HMGB1 WHEN DYING THAT CAN MODIFY THE EXPRESSION OF ANGIOGENIC FACTORS IN A MYD88 DEPENDANT AND INDEPENDENT WAY

E. ROSELLI, P. ARAYA, D.A.NOCERA, G.GATTI, N.G. NUÑEZ, M. MACCIONI

Mar del Plata

Congreso; LXI Reunión Anual de la Sociedad Argentina de Inmunología; 2014

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 ability of HMGB1 released by dying doxorubicin-treated murine melanoma B16 cells of inducing the expression of angiopoietin 2 (Ang2) 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 Ang 2 transcript was evaluated by qRT-PCR, analyzing the data by the 2(-ΔΔC(T)) method. Poli-AU was used as a positive control and glycyrrhizin (Gly) was used as a HMGB1 inhibitor. Our results indicate that the expression of Ang2 increased in Poli-AU treated B16 and B16shMyD88 cells (x1.5 and x3.0 respectively), and even more robustly in Doxo-CM treated B16 cells (x6.0 and x5.6 respectively). This effect is reduced when Gly is added to inhibit HMGB1 in B16shMyD88 (x6.1 to x4.9 fold) but unaltered in B16 cells (x5.7 ? x6.0). In addition, recombinant HMGB1 (HMGB1r) was used at 1ug/mL to stimulate B16 and B16shMyD88 cells and through a proteome array we evaluated a set of 53 angiogenic proteins in a 24h supernatant. Obtaining the induction of different angiogenic factors such as ADAMTS1, PDGF-AA, VEGF, Tissue Factor on a MyD88 dependant manner, and other such as CD105 on an independent manner.