Division of Hematology-Oncology, Department of Pediatrics, Keck School of Medicine, University of Southern California and the Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, California, United States of America.
PLoS One. 2012;7(8):e42291. doi: 10.1371/journal.pone.0042291. Epub 2012 Aug 3.
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 C(16), C(18:0), C(24:0) and C(24:1) 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.
整合素 αvβ3/αvβ5 的环肽抑制剂 RGDfV（精氨酸-甘氨酸-天冬氨酸-苯丙氨酸-缬氨酸）可诱导正常细胞和肿瘤细胞凋亡。我们表明，RGDfV 诱导 ECV-304 癌细胞凋亡，增加酸性鞘磷脂酶（ASM）的活性和 mRNA 表达，并增加神经酰胺 C（16）、C（18：0）、C（24：0）和 C（24：1），同时降低相应的神经鞘磷脂。ASM 的 siRNA 降低了 TUNEL、PARP 裂解、线粒体去极化以及 caspase-3 和 caspase-8 活性和 3D 胶原模型中的 annexinV 所测量的 RGDfV 诱导的细胞凋亡。这些发现表明 ASM 在 ECV-304 中 RGDfV 诱导的细胞凋亡中起因果作用。我们已经表明，非受体酪氨酸激酶 c-Abl 也介导 RGDfV 诱导的细胞凋亡。然而，c-Abl 以前从未在任何系统中与 ASM 相关联。在这里，我们表明 STI-571（伊马替尼，c-Abl 的抑制剂）抑制了 RGDfV 诱导的 ASM 活性。此外，STI-571 和 c-Abl-siRNA 都抑制了 RGDfV 诱导的 ASM mRNA 增加，但 ASM-siRNA 不影响 c-Abl 磷酸化或表达，支持 c-Abl 调节 RGDfV 诱导的 ASM 表达增加。这些研究表明 ASM 是抑制整合素 αvβ3/αvβ5 诱导的细胞凋亡的介体，并且首次将 c-Abl 作为 ASM 表达和活性的上游调节剂。
