Nucleolin is a receptor that mediates antiangiogenic and antitumor activity of endostatin

Abstract

The exact molecular mechanism of how endostatin inhibits angiogenesis and tumor growth remains uncharacterized. Here, we report that endostatin specifically binds to the cell surface nucleolin with high affinity. Blockage of nucleolin by a neutralizing antibody or knockdown of nucleolin by the RNA interference results in loss of antiendothelial activities of endostatin. Importantly, a neutralizing antinucleolin antibody abrogates the antiangiogenic and antitumor activities of endostatin in vivo. Nucleolin and endostatin are colocalized on the cell surface of endothelial cells of angiogenic blood vessels in the tumor environment. Finally, we found that endostatin is internalized and transported into cell nuclei of endothelial cell via nucleolin. In the nucleus, the phosphorylation of nucleolin, which is critical for cell proliferation, can be inhibited by endostatin. Our studies demonstrate that nucleolin is a novel functional receptor for endostatin, and mediates the antiangiogenic and antitumor activities of endostatin. These findings also provide mechanistic insights of how endostatin specifically inhibits proliferating endothelial cell growth and angiogenesis. IntroductionAngiogenesis, sprouting new blood vessels from existing capillaries, is critical for tumor growth. 1,2 Therefore, antiangiogenic molecules offer new promises as novel therapeutic modalities for the treatment of tumors. Endostatin (ES), a 20-kDa C-terminal globular domain of the collagen XVIII, was originally isolated from the supernatant of a cultured murine hemangioendothelioma cell line for its ability to inhibit tumor angiogenesis. 3 In animal models, tumor dormancy could be induced by repeated administration of ES for several cycles without causing drug resistance. 4 Moreover, low toxicity of ES has been reported in both animal studies and human trials. [4][5][6] ES exhibits potent anti-endothelial cell activities including inhibition of cell proliferation, migration, adhesion, and survival, which are all required for angiogenesis in vivo. 3,[7][8][9][10][11] The exact molecular mechanism of ES still remains an enigma, although a number of ES-binding proteins such as integrins, tropomyosin, glypicans, laminin, and MMP2 have been reported as ES receptors. 9,[12][13][14][15] However, whether these ES receptors are involved in the antitumor function of ES remains elusive. Recently, using DNA gene microarray and proteomic analysis, Huber and colleagues have identified a number of potential intracellular targets of ES (Abdollahi et al 16 ). Nevertheless, several antiangiogenic-related properties of ES remain uncharacterized, and they have raised several critical unexplored issues at the molecular level. These include (1) Why does ES specifically target angiogenic blood vessels but not quiescent blood vessels 17 ? (2) Why does ES specifically inhibit tumor growth and produce little if any toxicity in animal studies and clinical trials 4-6 ? (3) Why are heparin-binding sites required for the angiostatic activities of ES 18 ? To unravel these p...

ADAMTS-2 functions as anti-angiogenic and anti-tumoral molecule independently of its catalytic activity

“…The hypothesis that nucleolin is a receptor mediating the proapoptotic effect of ADAMTS-2 may seemed challenged by the observation that HEK 293-EBNA cells are not affected by ADAMTS-2 although they express nucleolin at their surface (not shown). Similar data have been obtained with endostatin, which induces apoptosis in endothelial cells and some transformed cells while other cell types, although expressing nucleolin, are not altered [43]. Cell-specific intracellular pathways or the requirement of specific membrane co-receptors may explain this apparent discrepancy.…”

“…It has also been shown that nucleolin can translocate to the plasma membrane in some specific cell types such as endothelial cells and many transformed cells [42]. When present at the cell surface, it has been shown that nucleolin can interact with Erb B receptors or serves as a receptor for L-selectin, midkine and, most interestingly, endostatin [43], a potent antiangiogenic fragment of collagen XVIII. The implication of nucleolin in the control of angiogenesis was strengthened by recent publications showing that an antibody targeting the acidic domain of nucleolin caused apoptosis of cultured endothelial cells and was anti-angiogenic in vitro and in vivo [44].…”

Endostatin Has ATPase Activity, Which Mediates Its Antiangiogenic and Antitumor Activities

“…Internalization of endostatin is important for its biological functions (14,17). Consistently, Lim and colleagues (18) reported that manipulation of endostatin by addition of an MTD significantly increased endostatin uptake and improved its antitumor effects.…”

“…It can initiate endothelial cell apoptosis through the induction of VDAC1 phosphorylation, which facilitates the mitochondrial permeability transition pore (mPTP) opening (10). Moreover, it also binds to cell surface receptors such as integrins (11), glypicans (12), laminin (13) and nucleolin (14), and thereby regulates a myriad of signaling cascades. For example, endostatin binding to integrin a5b1 results in the inhibition of the FAK-c-Raf-MEK1/2-p38-ERK1 MAPK pathway (15).…”

Isthmin targets cell-surface GRP78 and triggers apoptosis via induction of mitochondrial dysfunction

“…The plasma membrane fractions were isolated as described. 46 Ni-NTA beads (Promega, Madison, WI, USA) coupled with rISM were incubated with the plasma membrane fraction of HUVECs. Native beads were used as the control.…”