Tumor necrosis factor-␣ (TNF) is initially expressed as a 26-kDa membrane-bound precusor protein (pro-TNF) that is shed proteolytically from the cell surface, releasing soluble 17-kDa TNF. We have identified human ADAM 10 (HuAD10) from THP-1 membrane extracts as a metalloprotease that specifically clips a peptide substrate spanning the authentic cleavage site between Ala 76 and Val 77 in pro-TNF. To confirm that HuAD10 has TNF processing activity, we cloned, expressed, and purified an active, truncated form of HuAD10. Characterization of recombinant HuAD10 (rHuAD10) suggests that this enzyme has many of the properties (i.e. substrate specificity, metalloprotease activity, cellular location) expected for a physiologically relevant TNF-processing enzyme. Tumor necrosis factor-␣ (TNF)1 is a cytokine that is produced primarily by activated monocytes and macrophages in response to a variety of physiological stresses such as infection or injury (1). Clinical and experimental evidence has also identified TNF as a mediator of chronic autoimmune diseases such as rheumatoid arthritis (2) and Crohn's disease (3), as well as being involved in the pathology associated with sepsis (1). Accordingly, TNF has become a primary target for therapeutic intervention of several inflammatory diseases.TNF is initially synthesized as a 26-kDa membrane-bound protein (pro-TNF) that is subsequently cleaved to release soluble 17-kDa TNF with an NH 2 terminus of Val 77 (4). The identity of the protease(s) responsible for TNF processing remains controversial. Robache-Gallea et al. (5) detected a serine protease activity (PR3) in monocyte membrane preparations which was able to generate a 17-kDa active TNF with an NH 2 terminus of Arg 78 . In 1994, the partial isolation and characterization of a membrane-bound activity capable of generating the 17-kDa form of TNF were reported (6). The TNF-processing enzyme was thought to be a non-matrix metalloprotease since it was not inhibited by TIMP-1,2 or phosphoramidon, and no calcium requirement was detected. More recently, two members of the ADAM family (TNF-␣ converting enzyme (TACE) and bovine ADAM 10 (BoAD10)) have been shown to possess pro-TNF processing activity (7-9).In this report we describe the isolation, cloning, and characterization of a TNF-processing enzyme from the human monocytic cell line THP-1. The purified recombinant enzyme, rHuAD10, specifically recognizes the authentic cleavage site in pro-TNF and is sensitive to metalloprotease inhibitors that block soluble TNF production (6). MATERIALS AND METHODSReagents-Dinitrophenol-labeled polypeptides were synthesized by the Fmoc (N-(9-fluorenyl)methoxycarbonyl)/t-butyl-based solid phase peptide chemistry method using an Applied Biosystems, Inc. 431A peptide synthesizer (10). All peptides were purified by reversed phase HPLC, and molecular weights were verified by mass spectrometry.HPLC Peptide Assay-TNF processing activity was measured as the ability to cleave a 12-residue peptide spanning the Ala 76 -Val 77 site in pro-TNF. The chromatopho...
Reticulol was isolated from the culture broth of the strain Streptoverticillium sp. NA-4803. Recticulol (M.W. 222.2) exhibited a potent in vitro cytotoxicity against A427, a human lung tumor cell line, and B16F10, a mouse melanoma cell line. In the trypan blue staining assay for B16F10 cells, the cell viability by reticulol treatment was significantly decreased in a dose-dependent manner. The in vivo assay for the lung metastasis-blocking effect showed that reticulol injected intravenously suppressed the increase in colonies on the lung in a dose-dependent manner. In addition, the survival rate of tumor-implanted mice treated with reticulol was closely associated with its antitumoral efficacy. Reticulol administered via the peritoneum of mice showed less metastasis inhibition than that injected intravenously. To demonstrate the mechanism for inhibition of metastasis, the inhibitory effect of reticulol for matrix metalloproteinase-2 or -9 involved in melanoma metastasis was investigated; however, they were not observed on zymogram gel. In addition, the antitumor efficacy of reticulol was not associated with cell cycle arrest or apoptosis. Therefore, it was inferred that reticulol known as a phosphodiesterase inhibitor directly inhibited the growth of B16F10 melanoma, showing necrotic response. These results suggest that reticulol protects its lung metastasis via the bloodstream by inhibiting the growth of B16F10 melanoma at the cellular level.
An important goal of photodynamic therapy (PDT) for treatment of various cancers is to shorten PDT-performing time and simultaneously enhance PDT efficacy. Here, we investigated the nontumor tissue distribution of and the tumor vascular damage caused by a new photosensitizer, DH-I-180-3, in mice with implanted EMT6 mammary tumor cells. In addition, we performed cell-based assays to evaluate the basic antitumor effect of DH-I-180-3/PDT in EMT6 cells. After administration of PDT, the type of cell death was characterized to be apoptosis, and a change in the mitochondrial membrane potential was also observed within minutes. On the other hand, tumor growth was remarkably retarded in vivo in mice that received DH-I-180-3/PDT, compared with mice in the control group, which were exposed to light irradiation alone. Finally, tumors in some mice nearly healed. The antitumor drug reached a maximum concentration approximately 3 h after administration. However, PDT was most effective when there was substantial accumulation of DH-I-180-3 in the tumor vasculature and in healthy tissue. The histological demonstration provided further evidence of tumor vascular damage. On the basis of these findings, we suggest that PDT with the photosensitizer DH-I-180-3 induces vascular damage with blood vessel shutdown, in addition to direct killing of tumor cells, in mice.
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