Src protein involvement in histamine - induced MDA-MB-231 cells migration

MOHAMAD N, PORRETTI J, GIRARDI P, COCCA C, RIVERA E, CRICCO G, MARTÍN G

Belfast

Congreso; XLI Annual Meeting of the European Histamine Research Society (EHRS); 2012

European Histamine Research Society (EHRS)

We have previously demonstrated that cell migration and invasion of human breast carcinoma MDA-MB-231 cells were stimulated by low doses (0.5-1 uM) of histamine (HA), while inhibited by concentrations over 10 uM. These opposite actions are mediated by different intracellular hydrogen peroxide (H202) levels. c-Src protein is activated by phosphorylation and this is critical for cell migration and cancer progression to metastasis. This protein phosphorylates substrates such as beta-catenin which translocates to nucleus and modulates the expression of genes related to the epithelial mesenchymal transition (EMT). The aim of this work was to study the role of the c-Src protein in HA-induced migratory response of MDA-MB-231 cells. We used a c-Src specific inhibitor, PP2, in a concentration which had no effect on cell proliferation. 1 uM PP2 blocked the stimulatory action of low doses of HA in cell migration and invasion as determined by transwells units coated or not with matrigel®. PP2 also blocked increase in other EMT markers (gelatinolytic activity and alpha smooth muscle actin expression) induced by 1 uM HA. An increase in c-Src phosphorylation was observed by western blot with 1 uM HA or exogenous 0.5 uM H202 while a decrease was registered when HA over 10 uM or 5 uM H202 were used. Catalase treatment reversed those effects. The expression of beta-catenin protein was evaluated by immunostaining and Western blot. There was a higher nuclear and perinuclear expression with 1 uM HA. Conversely, the combined treatment of 1 uM HA plus 1 uM PP2 produced a cytoplasmic and nuclear beta catenin expression similar to non treated cells. Low doses of HA also inactivated GSK-3beta, an enzyme involved in cytoplasmic degradation of beta catenin, favoring its nuclear translocation. In summary our results indicate that HA modulates the migratory and invasive capacity of MDA-MB 231 cells through a singular GPCR signaling pathway involving a H2O2-induced c-Src phosphorylation.