Abstract:The mechanisms by which tumor cells extravasate to form metastasis remain controversial. Previous studies performed in vivo and in vitro demonstrate that the contact between tumor cells and the vascular wall impairs endothelium integrity. Here, we investigated the effect of breast adenocarcinoma MCF-7 cells on the apoptosis of human umbilical vein endothelial cells (HUVEC). TUNEL labeling, nuclear morphology, and DNA electrophoresis indicated that MCF-7 cells induced a two-to fourfold increase in HUVEC apoptosis. Caspase-3 activity was significantly enhanced. Neither normal cells tested (mammary epithelial cells, fibroblasts, leukocytes) nor transformed hematopoietic cells tested (HL60, Jurkat) induced HUVEC apoptosis. On the contrary, cells derived from solid tumors (breast adenocarcinoma, MDA-MB-231 and T47D; fibrosarcoma, HT 1080) had an effect similar to that of MCF-7 cells. The induction of apoptosis requires cell-to-cell contact, since it could not be reproduced by media conditioned by MCF-7 cells cultured alone or cocultured with HUVEC. Our results suggest that cells derived from solid tumors may alter the endothelium integrity by inducing endothelial cell apoptosis. On the contrary, normal or malignant leukocytes appear to extravasate by distinct mechanisms and do not damage the endothelium. Our data may lead to a better understanding of the steps involved in tumor cell extravasation.

SPARC/osteonectin/BM-40 is a matricellular protein that is thought to be involved in angiogenesis and endothelial barrier function. Previously, we have detected high levels of SPARC expression in endothelial cells (ECs) adjacent to carcinomas of kidney and tongue. Although SPARC-derived peptide showed an angiogenic effect, intact SPARC itself inhibited the mitogenic activity of vascular endothelial growth factor (VEGF) for ECs by the inhibiting phosphorylation of flt-1 (VEGF receptor 1) and subsequent ERK activation. Thus, the role of SPARC in tumor angiogenesis, stimulation or inhibition, is still unclear. To clarify the role of SPARC in tumor growth and progression, we determined the effect of VEGF on the expression of SPARC in human microvascular EC line, HMEC-1, and human umbilical vein ECs. VEGF increased the levels of SPARC protein and steady-state levels of SPARC mRNA in serum-starved HMEC-1 cells. Inhibitors (SB202190 and SB203580) of p38, a mitogen-activated protein (MAP) kinase, attenuated VEGF-stimulated SPARC production in ECs. Since intact SPARC inhibits phosphorylation ERK MAP kinase in VEGF signaling, it was suggested that SPARC plays a dual role in the VEGF functions, tumor angiogenesis, and extravasation of tumors mediated by the increased permeability of endothelial barrier function.Key Words: endothelial cells; SPARC/osteonectin/ BM-40; angiogenesis; VEGF; MAP kinase. SPARC/osteonectin/BM-40 is a matricellular protein with a molecular mass of 43 kDa and is known to have a variety of functions. For example, SPARC inhibits the formation of focal adhesion (1) and the production of basement membrane components (2, 3), increases the permeability of endothelial barriers (4), and induces the production and activation of matrix metallo-proteinases (5, 6). In addition, SPARC promotes the motility of tumor cells such as renal cell carcinoma (7) and malignant prostatic cancer cell lines (8). A high level of SPARC expression has been frequently observed in variety of malignant tumors (9-15), and its fragment, the (K)GHK motif, induces angiogenesis in vivo (16). These data suggest that SPARC contributes tumor progression.Angiogenesis is an essential process for tumor growth, and an increase in it raises the risk of tumor metastasis incidence. Since vascular endothelial growth factor (VEGF), which is a potent angiogenic factor, effectively stimulates tube/cord formation by vascular endothelial cells (ECs), a high productive ability for VEGF secretion is thought to be an important malignant phenotype of tumors. Paley et al. (17) determined the localization of VEGF and SPARC in clinical specimens of malignant ovarian carcinomas, and showed that VEGF and SPARC were expressed in the tumor cells and adjacent stromal cells, respectively. We also detected SPARC expression in ECs adjacent to carcinomas of the kidney (18) and tongue (Kato et al. unpublished data) which were producing VEGF. Although SPARC-derived (K)GHK peptide induces angiogenesis in vivo, SPARC was reported to inhibit the mitogenic activ...

The initiation of the angiogenic process requires a locally confined and time-limited proteolysis of the basement membrane (BM) components at the site of new vessel sprout. Gelatinase A, a member of the matrix metalloproteinase family, degrades BM type IV collagen and is involved in the BM breakdown by migrating tumor cells and endothelial cells (EC). Gelatinase A is synthesized as latent proenzyme and must be activated in order to express its proteolytic activity. A plasma membrane-dependent mechanism of activation has been described for several tumor and transformed cells lines. In the present study, we show that latent (72 kD) and mature (62-59 kD) forms of gelatinase A are present in EC membrane fraction from Triton X-114 extract while only latent form is found in the cytosolic fraction. The incubation of EC membrane fraction with exogenous latent gelatinase A resulted in a significant activation giving rise to 62-59 kD mature forms. 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong potentiator of angiogenesis in vitro and in vivo, increases the amount of both latent and activated forms of gelatinase A in EC membrane fraction as well as the ability of this latter fraction to activate exogenous latent gelatinase A. We show that the mRNA transcript coding for the membrane-integrated MMP, the MT-MMP, previously described as a potential gelatinase A activator in invasive tumor cells is also expressed in vascular EC and is regulated through a TPA sensitive process. This enzyme may be responsible for membrane-dependent gelatinase A activation in normal vascular EC and may therefore be a determinant in the control of BM proteolysis during angiogenesis.

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Co‐injection of fibroblasts with human epithelial breast‐tumor MCF7 cells in the presence of Matrigel enhances tumor growth in nude mice. While most of the matrix metalloproteinases (MMPs) have been shown to be produced by stromal cells, tumor cells such as MCF7 cells are unable to produce MMPs. We therefore, hypothesized that the tumor‐promoting effect of fibroblasts could be related to their production of MMPs. In order to inhibit stromal proteases, over‐production of TIMP‐2 was induced in MCF7 cells by in vitro retroviral‐mediated gene transfer. TIMP‐2‐producing MCF7 cells were then co‐injected with fibroblasts into nude mice. Alternatively, we evaluated the effect of Batimastat, a synthetic inhibitor of MMPs, on the tumorigenicity of MCF7 cells co‐inoculated with fibroblasts into nude mice. Both physiological (TIMP‐2) and synthetic (Batimastat) inhibitors of MMPs were able to abolish the tumor‐promoting effect of fibroblasts. On the contrary, they failed to modulate the tumorigenicity of MCF7 cells injected alone. Interestingly, Matrigel from which low‐molecular‐weight proteins or growth factors had been removed failed to favor the tumorigenicity of MCF7 cells inoculated with fibroblasts. These findings emphasize the importance of fibroblasts in cancer progression, and suggest that their role could be related at least in part to production of proteases which can induce the release of factors from the extracellular matrix. Int. J. Cancer 76:267–273, 1998.© 1998 Wiley‐Liss, Inc.