The Synergistic Effect of Everolimus and Chloroquine on Endothelial Cell Number Reduction Is Paralleled by Increased Apoptosis and Reduced Autophagy Occurrence

Endothelial Progenitor Cells (EPCs), a minor subpopulation of the mononuclear cell fraction in peripheral blood, play a critical role in cancer development as they contribute to angiogenesis-mediated pathological neovascularization. In response to tumor cytokines, including VEGF, EPCs mobilize from the bone marrow into the peripheral circulation and move to the tumor bed where they incorporate into sprouting neovessels. In the present study, we evaluated the effects of everolimus (Afinitor, Novartis), a rapamycin analogue, alone or in combination with chloroquine, a 4-alkylamino substituted quinoline family member, one of the autophagy inhibitors, on EPCs biological functions. We found that either everolimus or chloroquine induce growth inhibition on EPCs in a dose-dependent manner after 72 h from the beginning of incubation. The combined administration of the two drugs to EPC was synergistic in inducing growth inhibition; in details, the maximal pharmacological synergism between everolimus and chloroquine in inducing growth inhibition on EPCs cells was recorded when chloroquine was administered 24 h before everolimus. Moreover, we have studied the mechanisms of cell death induced by the two agents alone or in combination on EPCs and we have found that the synergistic effect of combination on EPC growth inhibition was paralleled by increased apoptosis induction and reduced autophagy. These effects occurred together with biochemical features that are typical of reduced autophagic death such as increased co-immunoprecipitation between Beclin 1 and Bcl-2. Chloroquine antagonized the inhibition of the activity of Akt→4EBP1 axis mediated by everolimus and at the same time it blocked the feed-back activation of Erk-1/2 induced by RAD in EPCs. These data suggest a new strategy in order to block angiogenesis in tumours in which this process plays a key role in both the sustainment and spreading of cancer cells.