Thrombospondin-1 Induces Apoptosis in Megakaryocytic Leukemia Via CD36 and Caspase-3 Signaling

Objective: Thrombospondin-1 (TSP-1) is a multi-modular glycoprotein synthesized by megakaryocytes, endothelial cells, smooth muscle cells, fibroblasts and some tumor cells, found in the platelet a-granules and extracellular matrix (ECM) of numerous tissues. TSP-1 has been identified as one of the few naturally occurring anti-angiogenic agents. It inhibited endothelial cells in vitro by the activation of the CD36-p59fyn-Caspase-3-p38MAPK apoptotic cascade. TSP-1 induced in vitro apoptosis of micro-vascular endothelial cells as well as tumor cells. The objectives of our study were to investigate the in vitro effects of TSP-1 on the apoptosis of megakaryocytic leukemia cells and the possible mechanism involving CD36. Methods: We investigated CD36 expression and the effect of TSP-1 on megakaryocytic leukemia cells (Meg-01 and CHRF-288-11), with and without thrombopoietin (TPO), and with and without blocking TSP-1 binding with receptor CD36 on megakaryocytic leukemia cells. The determination of apoptotic markers were used the Annexin V-FITC/Propidium Iodide (PI) and Caspase-3 detection kit by flow cytometry.Results: Our data showed that TSP-1 induced a dose dependent growth inhibition in human CFU-MK assays and significantly counteracted the mitogenic effect from TPO. Moreover, the growth suppression induced by TSP-1 was correlated with CD36 expression in megakaryocytic leukemia cells, where growth inhibition was demonstrated in CD36 positive (Meg-01 and CHRF-288-11) but not in CD36 negative (K562) cells. More importantly, the inhibitory effect of TSP-1 on both human CFU-MK and Meg-01 cells was partially but significantly reversed by the addition of FA6-152 (anti-CD36), a functionally blocking antibody which blocks the access of TSP-1 to CD36 receptor, suggesting that the TSP-1 induced inhibition of megakaryocytopoiesis is probably mediated in part by the binding of TSP-1 to CD36 expressed on the megakaryocytic progenitors. Thus, our findings represent the demonstration that TSP-1 inhibits in vitro megakaryocytopoiesis via interaction with CD36. Our results further demonstrated that TSP-1 induced apoptosis in CD36 positive cells CHRF-288-11 and Meg-01, but not CD36 negative K562 cells, at a dose-dependent manner as demonstrated by DNA Annexin V and propidium iodide (PI) stainings. The addition of anti-CD36 antibody FA6-152 or TPO significantly nullified the effects of TSP-1. TSP-1-mediated apoptosis was consistently associated with the up-regulation of active Caspase-3. Responses of CD36 positive primary AML samples (n=3) to TSP-1 and FA6-152 were similar with those of leukemia cell lines. CD36 negative AML cells appeared less susceptible to TSP-1 and FA6-152.Conclusions: Our data provided evidence that TSP-1 exerted direct apoptotic effects on megakaryocytic leukemia cells and could be developed as an adjunct to conventional therapy, particularly for leukemia cells that express CD36 or other TSP-1 receptors. Thus, our findings represent the demonstration that TSP-1 induces apoptosis in megakaryocytic leukemia cells via CD36 and Caspase-3.