Abstract
Inhibition of integrins αvβ3/αvβ5 by the cyclic function-blocking peptide, RGDfV (Arg-Gly-Asp-Phe-Val) can induce apoptosis in both normal cells and tumor cells. We show that RGDfV induced apoptosis in ECV-304 carcinoma cells, increased activity and mRNA expression of acid sphingomyelinase (ASM), and increased ceramides C16, C18∶0, C24∶0 and C24∶1 while decreasing the corresponding sphingomyelins. siRNA to ASM decreased RGDfV-induced apoptosis as measured by TUNEL, PARP cleavage, mitochondrial depolarization, and caspase-3 and caspase-8 activities, as well as by annexinV in a 3D collagen model. These findings indicate a causal role for ASM in RGDfV-induced apoptosis in ECV-304. We have shown that c-Abl, a non-receptor tyrosine kinase, also mediates RGDfV-induced apoptosis. However, c-Abl, has not been previously linked to ASM in any system. Here we show that STI-571 (imatinib, inhibitor of c-Abl) inhibited RGDfV-induced ASM activity. Furthermore, STI-571 and c-Abl-siRNA both inhibited RGDfV-induced increase in ASM mRNA, but ASM-siRNA did not affect c-Abl phosphorylation or expression, supporting that c-Abl regulates the RGDfV-induced increase in ASM expression. These studies implicate ASM as a mediator of apoptosis induced by inhibition of integrins αvβ3/αvβ5, and for the first time place c-Abl as an upstream regulator of ASM expression and activity.
Highlights
Integrins, heterodimeric cell-surface receptors, are central regulators of cell functions such as proliferation, differentiation, growth factor secretion and protection from apoptosis [1,2,3]
Inhibition of integrins avb3 and avb5 by RGDfV resulted in apoptosis of ECV-304 cells seeded on vitronectin (Fig. 1A; supported by poly(adenosine diphosphate-ribose) polymerase (PARP) cleavage, caspase-3 and caspase-8 activation, mitochondrial depolarization and AnnexinV staining shown in the non-silencing siRNA controls of the third and fourth figures)
Using real time quantitative RT-PCR we consistently observed increase in acid sphingomyelinase (ASM) Messenger RNA (mRNA) in cells incubated with RGDfV, which persisted at least up to 96 hrs incubation (Fig. 1C and data not shown)
Summary
Heterodimeric cell-surface receptors, are central regulators of cell functions such as proliferation, differentiation, growth factor secretion and protection from apoptosis [1,2,3]. Integrins avb and avb are expressed on a variety of cell types including cancer cells and endothelial cells [3,4,5,6]. Inhibition of integrins avb and avb can induce cell death and affect tumor growth [7,8,9,10]. Integrins avb and avb bind to arginineglycine-aspartic acid (RGD)-containing matrix proteins such as vitronectin. Integrin avb3/avb signaling can be blocked by soluble function-blocking RGD peptides such as the cyclic RGDfV (Arg-Gly-Asp-Phe-Val) peptide, resulting in apoptosis [8,9,11]. In vivo RGDfV inhibits growth of cell line-derived tumors such as glioblastoma, medulloblastoma, and breast cancer in mice [3,12]. The clinical version of RGDfV, Cilengitide, is in clinical trials [12,13,14,15], underscoring the need to fully understand the molecular mechanism(s) that are affected by RGDfV
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