Doxorubicin Induces Apoptosis and CD95 Gene Expression in Human Primary Endothelial Cells through a p53-dependent Mechanism

Abstract

Regulation of the homeostasis of vascular endothelium is critical for the processes of vascular remodeling and angiogenesis under physiological and pathological conditions. Here we show that doxorubicin (Dox), a drug used in antitumor therapy, triggered a marked accumulation of p53 and induced CD95 gene expression and apoptosis in proliferating human umbilical vein endothelial cells (HUVECs). Transfection and site-directed mutagenesis experiments using the CD95 promoter fused to an intronic enhancer indicated the requirement for a p53 site for Dox-induced promoter activation. Furthermore, the p53 inhibitor pifithrin-␣ (PFT-␣) blocked both promoter inducibility and protein up-regulation of CD95 in response to Dox. Up-regulated CD95 in Doxtreated cells was functional in eliciting apoptosis upon incubation of the cells with an agonistic CD95 antibody. However, Dox-mediated apoptosis was independent of CD95/CD95L interaction. The analysis of apoptosis in the presence of PFT-␣ and benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone revealed that both p53 and caspase activation are required for Dox-mediated apoptosis of HUVECs. Finally, Dox triggered Bcl-2 downregulation, cytochrome c release from mitochondria, and the activation of caspases 9 and 3, suggesting the involvement of a mitochondrially operated pathway of apoptosis. These results highlight the role of p53 in the response of primary endothelial cells to genotoxic drugs and may reveal a novel mechanism underlying the antitumoral properties of Dox, related to its ability to induce apoptosis in proliferating endothelial cells.

In vivo upregulation of CD95 and CD95L causes synergistic inhibition of angiogenesis by TSP1 peptide and metronomic doxorubicin treatment

“…Although this increase in the expression of CD95 was not involved in the signaling cascade that led to caspase activation and endothelial cell apoptosis by DXR, CD95 itself was functionally active and capable of inducing cell death upon stimulation with agonistic CD95 antibodies, showing that the required molecular machinery remained functional. 18 Here, we showed that DXR concentrations as low as 50 ng/ml still increased CD95, but caused no significant apoptosis in EC nor in cancer cells. These noncytotoxic concentrations augmented in vitro EC sensitivity to apoptosis by DI-TSP via CD95 cascade.…”

“…18 Interestingly, although at these doses, DXR induced apoptosis independent of CD95/CD95L interactions, the upregulated CD95 remains functional and CD95-dependent apoptotic cascade can be triggered with the activating monoclonal antibodies in DXRtreated cells but not in untreated control population. 18 Here we show that at low concentrations, DXR resulted in sustained upregulation of the endothelial CD95 without noticeable cytotoxic effects towards endothelial or cancer cells. The increase in the endothelial CD95 by low DXR doses significantly improved the antiangiogenic and antitumor efficacy of DI-TSP.…”

“…Thus, DXR significantly increased surface CD95 at doses at least 10 times lower than those required to induce endothelial cell apoptosis in vitro. 18 DI-TSP upregulated CD95L expression and induced HMVECs apoptosis in synergy with DXR TSP1 increases mRNA and surface CD95L in the endothelial cells via signaling cascade mediated by its antiangiogenic receptor, CD36. 10,11,25 Since HUVECs are CD36 negative, 6 we examined the ability of DI-TSP, a short peptide derivative of TSP1, to induce similar events only in the CD36-positive HMVECs.…”

“…18 Here, we tested whether DXR doses below MTD (50-200 ng/ml) were sufficient to upregulate endothelial CD95. We analyzed the effect of DXR between 50 and 500 ng/ml on apoptosis and CD95 presentation of HUVECs and microvascular endothelial cells (HMVECs) cultured in full serum.…”

“…10,11 The same antibody completely abrogated endothelial cell apoptosis and antiangiogenesis by DI-TSP/DXR combination (Figure 6a-c). Since CD95 upregulation by DXR is p53-dependent 18 while apoptosis by DI-TSP is not, 10 we used Pifithrin-a (PFT-a), an inhibitor of p53-dependent transcription, 28 in order to eliminate DXR induction of CD95 mRNA and protein. Predictably, PFT-a relieved the effect of DI-TSP/DXR on both microvessel counts and endothelial cell.…”