Affiliation: Universidad de Santiago de Compostela. Dept. Farmacologia, Facultad de Veterinaria, 27002 Lugo, Spain.
The human mast cell line HMC-1560,816 carries activating mutations in the proto-oncogene of c-kit that cause autophosphorylation and permanent c-kit receptor activation. The compound CCT129202 is a new and selective inhibitor of Aurora kinase A and B that decreases the viability of a variety of human tumor cell lines. The effect of Aurora kinase inhibition was assessed in the HMC-1560,816 line in order to find a suitable tool for mastocytosis treatment. CCT129202 treatment induces a significant decrease in cell viability in HMC-1560,816 cells after 48 hours of treatment. Moreover, caspase-3 and caspase-8 activation was induced after incubation of HMC-1560,816 cells in the presence of CCT129202. It has been demonstrated that Protein Kinase C (PKC) plays a crucial role in mast cell activation as well as cell migration, adhesion and apoptotic cell death. Co-treatment of Ca2+-independent PKCs (δ ε and θ) inhibitor GF109203X with CCT129202, reduces caspase-3 activation which controls cell levels. In contrast, Go6976, an inhibitor of Ca2+-dependent PKCs, increases caspase-3 activation. Oppositely, GF109203X does not modify CCT129202-induced apoptosis through the caspase-8 pathway whereas Go6976 treatment abolishes the increase on caspase-8 activity due to CCT129202. This implies that Ca2+-independent PKC isoforms seems to be related with CCT129202-induced apoptosis through the caspase- 3 pathway, whereas Ca2+-dependent PKC isoforms are related with the CCT129202 effect on the caspase-8 pathway. Interestingly, CCT129202 cytotoxic effect remains even though Ca2+-dependent PKCs are inhibited, which shows that the Aurora kinase inhibitor effect is acting through the caspase-3 pathway. On the other hand, Ca2+-independent PKCs inhibition does not affect the final apoptotic CCT129202 effect because this seems to be mediated by the caspase-8 pathway. Moreover, CCT129202 does not affect PKCδ and Ca2+-dependent PKC translocation, which indicates that PKC translocation pivots on its activation. This demonstrates that Aurora kinase inhibition is not related to this process. Finally, when PKC is silenced in HMC-1560,816 cells, the effect of CCT129202 on the caspase-3 pathway disappears, which indicates that the CCT129202 effect is clearly PKC-dependent.