No abstract

Neovascularization is often required for rapid growth of solid tumors and also limits vascular metastasis of tumor cells. Neovascularization-targeting agents are a recent innovation that may be a novel means of anticancer therapy. These antiangiogenic drugs have been developed by targeting cell proliferation of vascular endothelial cells, basement-membrane-degrading enzymes, angiogenic factors/receptors, extracellular matrix, angiogenesis signaling, and cell-cell/cell-matrix interactions. In this report, we describe how tumor angiogenesis occurs and how antiangiogenic agents are developed.

Human omental microvascular endothelial (HOME) cells seeded on Matrigel begin to migrate within 1 h, forming honeycomb-like structures and capillary-like networks within 18 h. Cross-sections of the capillary networks show them to be tube-like structures. Northern blot analysis showed that tissue-type plasminogen activator (t-PA) mRNA synthesis increased from the initial state at 0 h after seeding on Matrigel, reaching a steady state after 4 h. This elevated cellular t-PA mRNA level decreased markedly at 24 h. In contrast, the cellular plasminogen activator inhibitor-1 (PAI-1) mRNA level demonstrated biphasic curves during the 24 h after seeding on Matrigel: the PAI-1 mRNA level was increased eightfold initially at 4 h over that at 0 h, then declined, and again secondarily increased to greater than tenfold at 18 h. Cellular levels of both 72 kD type IV collagenase and tissue inhibitor of metalloproteinase (TIMP-2) mRNA were increased only a slightly within 2-4 h. These elevated mRNA levels were maintained for 18 h, while the TIMP-1 mRNA level increased up to 18 h, reaching around three times the level at 0 h. However, on collagen-coated dishes, cellular levels of t-PA, PAI-1, 72 kD type IV collagenase, TIMP-1, and TIMP-2 mRNA were not greatly changed during incubation for 24 h. On Matrigel, the cellular t-PA mRNA level at 18 h after seeding was greatly increased when treated with specific anti-transforming growth factor-beta (TGF-beta) antibody. In contrast, both PAI-1 and TIMP-1 mRNA levels at 18 h were reduced in the presence of anti-TGF-beta antibody. Development of the capillary network on Matrigel was inhibited in the presence of anti-t-PA antibody. Epidermal growth factor (EGF) enhanced t-PA gene expression and TGF-beta inhibited its expression in HOME cells cultured on collagen-coated dishes. On the other hand, TGF-beta enhanced cellular expression of the PAI-1 gene. The formation of a capillary network by HOME cells on Matrigel appears to be balanced by angiogenic EGF and anti-angiogenic TGF-beta through modulation of PA activity.

We previously described an in vitro invasion assay model, using a monolayer of vascular endothelial cells grown on collagen gel, that mimics the metastatic abilities of the highly metastatic human renal carcinoma cell lines, MM‐1,3 and 8 and their poorly metastatic counterparts, SN12C and Q‐8. MM‐1, 3 and 8 cells were observed to penetrate the monolayer of vascular endothelial cells and grew in a spreading or scattering manner with loose cell‐cell contact on collagen gel or on vascular endothelial cells. SN12C and Cl‐8 cells failed to penetrate and grew in a clustering manner with tight cell‐cell contact. Treatment with all‐trans‐retinoic acid (ATRA) at non‐toxic concentrations induced clustering or growth of MM‐1, 3 and 8 cells on collagen gel or on vascular endothelial cells with tight cell‐cell contact, and inhibited penetration. The clustering induced by ATRA was virtually blocked in the presence of anti‐E cadherin antibody. E‐Cadherin and β‐catenin were each localized mainly at the cell‐cell adherent junctions of colonizing cell populations that had been treated with ATRA. While the cellular levels of E‐cadherin and β‐catenin did not change significantly following ATRA treatment, the tyrosine residue of β‐catenin was rapidly dephosphorylated. The concomitant administration of Na vanadate, an inhibitor of tyrosine dephosphorylase, inhibited both the ATRA‐induced clustering and the dephosphorylation of β‐catenin tyrosine. ATRA‐induced clustering of MM‐3 cells may be linked to the state of tyrosine phosphorylation of β‐catenin.

No abstract