Tumor necrosis factor (TNF) has been shown to be cytotoxic to tumor cell lines in vitro, but the mechanism by which TNF exerts its cell growth-regulatory effects is not known. In this report, we used various inhibitors to investigate the sequence of events that lead to cytotoxic effects of TNF on L.P3 cells, a highly sensitive, murine fibroblast cell line. Our results indicate that mitochondrial respiration chains are damaged by a hydroxyl radical at an early stage of the cell lysis after TNF treatment. This event is followed by the activation of phospholipase A2, and finally leads to cell lysis.

Summary: We compared the tumoricidal activity of human monocytes cultured with interleukin-2 (IL-2) or human recombinant interferon-7 (IFN-7) alone, or IFN-7 in combination with a small amount of lipopolysaccharides (LPS). Human monocytes cultured with IL-2 for 7 days or longer, termed lymphokine-activated macrophages (LAMs), showed higher tumoricidal activity than those cultured for 1 day. In contrast, monocytes cultured with IFN-7 alone or in combination with LPS for 7 days or longer showed lower tumoricidal activity. LAMs were identified as macrophages by nonspecific esterase staining and immunofluorescence staining with anti-CD 14 antibody. LAMs were not induced in fetal calf serum-containing medium, but they were induced when colony-stimulating factor-1 was added to the medium. LAMs showed high tumoricidal activity against all human and murine tumor cell lines tested, although they showed no cytotoxic activity against human normal cells. During incubation with IL-2, tumoricidal activity of LAMs was maximal at days 8-16 and was sustained until day 28. The difference in tumoricidal mechanism between LAMs and lymphokine-activated killer (LAK) cells was also shown by using two kinds of cytotoxic assay systems. LAMs require a long incubation time to kill tumor cells, but LAK cells can kill them immediately. Furthermore, LAMs kill tumor cells with complete DNA degradation, whereas LAK cells can induce significant but not complete DNA degradation. These results indicate that LAMs and LAK cells have different tumoricidal mechanisms for killing target cells, although they were induced by incubation with the same lymphokine, IL-2.

We examined whether sulfated hyaluronan exerts inhibitory effects on enzymatic and biological actions of heparanase, a sole endo-beta-glucuronidase implicated in cancer malignancy and inflammation. Degradation of heparan sulfate by human and mouse heparanase was inhibited by sulfated hyaluronan. In particular, high-sulfated hyaluronan modified with approximately 2.5 sulfate groups per disaccharide unit effectively inhibited the enzymatic activity at a lower concentration than heparin. Human and mouse heparanase bound to immobilized sulfated hyaluronan. Invasion of heparanase-positive colon-26 cells and 4T1 cells under 3D culture conditions was significantly suppressed in the presence of high-sulfated hyaluronan. Heparanase-induced release of CCL2 from colon-26 cells was suppressed in the presence of sulfated hyaluronan via blocking of cell surface binding and subsequent intracellular NF-κB-dependent signaling. The inhibitory effect of sulfated hyaluronan is likely due to competitive binding to the heparanase molecule, which antagonizes the heparanase-substrate interaction. Fragment molecular orbital calculation revealed a strong binding of sulfated hyaluronan tetrasaccharide to the heparanase molecule based on electrostatic interactions, particularly characterized by interactions of (−1)- and (−2)-positioned sulfated sugar residues with basic amino acid residues composing the heparin-binding domain-1 of heparanase. These results propose a relevance for sulfated hyaluronan in the blocking of heparanase-mediated enzymatic and cellular actions.