Proteomics of human umbilical vein endothelial cells applied to etoposide‐induced apoptosis

Abstract

We have undertaken to continue the proteomic study of human umbilical vein endothelial cells (HUVECs) using the combination of 2-DE, automated trypsin digestion, and PMF analysis after MALDI-TOF MS and peptide sequencing using nano LC-ESI-MS/MS. The overall functional characterization of the 162 identified proteins from primary cultures of HUVECs confirms the metabolic capabilities of endothelium and illustrates various cellular functions more related to cell motility and angiogenesis, protein folding, anti-oxidant defenses, signal transduction, proteasome pathway and resistance to apoptosis. In comparison with controls cells, the differential proteomic analysis of HUVECs treated by the pro-apoptotic topoisomerase inhibitor etoposide further revealed the variation of eight proteins, namely, GRP78, GRP94, valosin-containing protein, proteinase inhibitor 9, cofilin, 37-kDa laminin receptor protein, bovine apolipoprotein, and tropomyosin. These data suggest that etoposide-induced apoptosis of human vascular endothelial cells results from the intricate involvement of multiple apoptosis processes including at least the mitochondrial and the ER stress pathways. The presented 2-D pattern and protein database, as well as the data related to apoptosis of HUVECs, are available at http://www.huvec.com.

Retrospective Proteomic Screening of 100 Breast Cancer Tissues

“…Concurrently, it is involved in the regulation of apoposis. However in this context, it appeares to have ambivalent roles, since in many cases it showed to play pro-apoptotic activity and in others anti-apoptotic [70][71][72]. In our samples we have identified 4 cofilin isoforms, one of these was ubiquitous, displaying a rather higher expression level (14x) with respect to the non-tumoral tissues.…”

DAP kinase mediates the phosphorylation of tropomyosin-1 downstream of the ERK pathway, which regulates the formation of stress fibers in response to oxidative stress

“…In this study, we investigated further the signaling mechanisms that lead to phosphorylation of tropomysoin-1 and regulate its functions. Given that tropomyosin has several isoforms that share sequence homology with tropomyosin-1 (Bruneel et al, 2005;Gunning et al, 2005), we first designed a set of experiments to demonstrate even more comprehensively that the tropomyosin-1 isoform is phosphorylated downstream of ERK in response to H 2 O 2 . We expressed exogenous human tropomyosin-1 in HEK293 cells as these cells have a very low basal level of tropomyosin-1 expression in comparison with human umbilical vein endothelial cells (HUVECs) and other cell types, including COS cells (Fig.…”

“…Moreover, in both cases, the phosphorylation is mediated by ERK, being blocked by inhibiting MEK1/2, and thereby ERK activation, with PD098059 or UO126. Nevertheless, given that HUVECs express diverse isoforms of tropomyosins, namely tropomyosin-3 and tropomyosin-4 that are closely related to tropomyosin-1 (Bruneel et al, 2005), one cannot exclude the possibility that, in addition to tropomyosin-1, these forms of tropomyosin could also be phosphorylated after H 2 O 2 treatment. In this context, the well-characterized TM311 antibody against tropomyosin that we used to immunoprecipitate phosphorylated tropomyosin-1 recognizes up to three phosphorylated bands that presumably correspond to different tropomyosin isoforms.…”

Critical Role of the Stress Chaperone GRP78/BiP in Tumor Proliferation, Survival, and Tumor Angiogenesis in Transgene-Induced Mammary Tumor Development

“…In contrast to normal endothelial cells, primary tumor endothelial cells proliferate more slowly and are much more resistant to cytotoxic drugs. Proteomics of human endothelial cells suggests that GRP78 inhibits endothelial cell apoptosis induced by topoisomerase inhibitors (42). Thus, it is possible that the antiapoptotic properties of GRP78 protect tumor as well as tumor-associated endothelial cells against stress in the tumor microenvironment.…”