batimastat and Carcinoma--Squamous-Cell

Collagenase-3 (MMP-13) is characterized by an exceptionally wide substrate specificity and restricted expression. MMP-13 is 1 of the few MMPs primarily expressed by tumor cells in malignant tumors, e.g., squamous cell carcinomas and its expression correlates with their invasion capacity. In this work, we have constructed an expression vector and a recombinant adenovirus harboring human MMP-13 cDNA to investigate the role of MMP-13 in cancer cell invasion. Our results show that constitutive expression of MMP-13 by HT-1080 cells stably transfected with MMP-13 expression vector or transduced with MMP-13 adenovirus markedly increased their invasion both through type I collagen and reconstituted basement membrane (Matrigel) with no alterations in expression or activation of collagenase-1 (MMP-1), gelatinase-A (MMP-2), or gelatinase-B (MMP-9). The enhanced invasion capacity of MMP-13 expressing HT-1080 cells was dependent on MMP activity, as it was blocked by MMP inhibitor Batimastat (BB-94) and tissue inhibitor of metalloproteinases-3 (TIMP-3). Our data provide direct evidence for the role of MMP-13 as a potent invasion proteinase, which alone can enhance the ability of malignant cells to penetrate through both basement membrane and fibrillar collagen.

Topics: Adenoviridae; Basement Membrane; Carcinoma, Squamous Cell; Collagen; Collagen Type I; Collagenases; DNA, Complementary; Drug Combinations; Fibrosarcoma; Genetic Vectors; Humans; Laminin; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Models, Genetic; Neoplasm Invasiveness; Neoplasm Metastasis; Phenylalanine; Proteoglycans; Thiophenes; Tissue Inhibitor of Metalloproteinase-3; Transfection; Tumor Cells, Cultured

The expression of selected metalloproteinases and tissue inhibitors of metalloproteinases (TIMP) was examined in three squamous cell carcinoma (SCC) cell lines (FaDu, SiHa, A431) and a keratinocyte cell line (HaCaT) to determine which metalloproteinases function in SCC invasiveness. A Matrigel invasion assay was used to assess invasiveness of the cell lines. Only the FaDu cell line showed invasiveness in this assay, and invasion of Matrigel by FaDu cells was inhibited by treatment with the metalloproteinase inhibitor, batimastat. No correlation was found between mRNA expression for matrilysin, stromelysins 1-3, TIMP-1, or TIMP-3 and secretion of these proteins, indicating that the extracellular activity of these molecules is regulated post-transcriptionally. The SCC cell lines differed from the HaCaT line in that matrilysin and TIMP-1 proteins were detected in conditioned medium from all SCC cell lines, but not in medium from HaCaT cells. Only the invasive cell line, FaDu, released active stromelysin-1 into the culture medium. These results indicate that while matrilysin contributes to the invasive phenotype, activation of stromelysin-1 is a key regulatory step for invasiveness in SCC cells.

Topics: Biocompatible Materials; Carcinoma, Squamous Cell; Cell Movement; Collagen; Drug Combinations; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Keratinocytes; Laminin; Matrix Metalloproteinase 3; Neoplasm Invasiveness; Phenylalanine; Protease Inhibitors; Proteoglycans; Skin Neoplasms; Thiophenes; Tissue Inhibitor of Metalloproteinase-1; Tumor Cells, Cultured

Recent reports have variously described expression of Fas ligand (FasL) or its absence in human tumors. The importance of the Fas-FasL mechanism for the immune evasion by tumors provided a strong rationale for the examination of FasL expression and function in squamous cell carcinoma of the head and neck (SCCHN), which is one of the most immunosuppressive human cancers. Using immunostaining or immunoblotting, SCCHN cell lines and tumor biopsies were examined for the presence of the components of the Fas-FasL pathway and found to express Fas, as well as both the full-length and cleaved forms of FasL. By reverse transcription-PCR, mRNA for FasL and Fas were detected in all SCCHN tested, and cross-hybridization to radioactive Fas and FasL cDNA probes confirmed the specificity of amplification. To demonstrate that FasL expressed on cell surface of SCCHN cells was biologically active, various SCCHN lines were coincubated with the Fas-sensitive Jurkat T-cell lines or activated peripheral blood mononuclear cells. Tumor-induced apoptosis of T cells was dependent on the ratio of tumor cells: lymphocytes. It was significantly but only partially inhibited by neutralizing antibodies to FasL and antagonistic antibodies to FasR. Tumor-induced apoptosis was enhanced by the pretreatment of tumor cells with metalloproteinase inhibitors, which increased expression of FasL on tumor cells. Supernatants of tumor cells transduced with FasL also induced apoptosis of Jurkat cells. Thus, coincubation of SCCHN with Fas-sensitive lymphocytes can induce apoptosis of these lymphocytes, and the Fas/FasL pathway appears to be responsible, at least in part, for tumor-induced lymphocyte death. The data suggest that the Fas/FasL pathway is potentially immunosuppressive and may be involved in the escape of human carcinoma cells from immune destruction.

Topics: Apoptosis; Carcinoma, Squamous Cell; Fas Ligand Protein; fas Receptor; Head and Neck Neoplasms; Humans; Immunoblotting; Jurkat Cells; Lymphocytes, Tumor-Infiltrating; Membrane Glycoproteins; Phenylalanine; RNA, Messenger; Thiophenes; Tumor Cells, Cultured