sq-23377 and Urinary-Bladder-Neoplasms

T24 human bladder carcinoma cells reveal a high locomotor activity (70% locomoting cells) within a 3-dimensional collagen matrix. This high migratory activity is induced by an autocrine engagement of the interleukin-8 receptor A, as was shown by antibodies neutralizing the secreted interleukin-8. Treatment of the cells with these specific antibodies reduced the locomotor activity by half. The intracellular signal transduction underlying the interleukin-8-induced T24 locomotion involves the activity of protein tyrosine kinases (PTKs), the phospholipase Cgamma (PLCgamma) and the protein kinase C (PKC), as proven by the use of specific enzyme inhibitors. These results suggest the following model for the regulatory signal transduction of interleukin-8-induced human T24 bladder carcinoma cell migration: The engagement of the interleukin-8-receptor, a receptor of the serpentine family, leads to the beta-arrestin-mediated activation of PTKs. These kinases phosphorylate the PLCgamma, which generates the second messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP(3)). DAG activates the PKC, whereas IP(3) mediates the release of calcium from the endoplasmatic reticulum. By means of confocal laser microscopy, we observed an oscillation of the cytosolic calcium concentration in migrating T24 cells, which were loaded with the calcium-dye fluo-3/AM. Here, we report on a new autocrine function of secreted interleukin-8 and the intracellular signal transduction leading to the regulation of cytosolic calcium and to a migratory tumor cell phenotype.

Topics: Antibodies; Arrestins; beta-Arrestins; Calcium; Cell Movement; Diglycerides; Enzyme Activation; Humans; Inositol 1,4,5-Trisphosphate; Interleukin-8; Ionomycin; Isoenzymes; Kinetics; Microscopy, Confocal; Phospholipase C gamma; Protein Kinase C; Protein-Tyrosine Kinases; Receptors, Interleukin-8A; Second Messenger Systems; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Type C Phospholipases; Urinary Bladder Neoplasms

The objective of this study was to characterize the antiproliferative effects of the calcium ionophore, ionomycin on the human bladder cancer cell line HT1376 both in vitro and in vivo.. The in vitro growth rate of HT1376 after treatment with ionomycin was measured by using the MTT assay. The apoptotic features in HT1376 after ionomycin treatment were evaluated by flow cytometric analysis and DNA degradation assay. Bcl-2 and Bax expression levels in HT1376 after ionomycin treatment were examined by Northern and Western blot analyses. The effects of intratumoral injection of ionomycin on the growth of subcutaneous HT1376 tumors established in athymic nude mice were then tested. The efficacy of combined treatment with ionomycin and cisplatin against HT1376 growth was also examined.. The in vitro growth rate of HT1376 was suppressed by ionomycin in a dose- and time-dependent manner, and characteristic apoptotic DNA degradation was observed in HT1376. Ionomycin treatment caused a marked decrease in the ratios of Bcl-2 to Bax mRNA and protein in HT1376 cells. Intratumoral injection of ionomycin into subcutaneous HT1376 tumors reduced the tumorigenicity in nude mice. Furthermore, these in vivo growth-inhibitory effects of ionomycin were significantly enhanced by pretreatment with cisplatin.. These findings suggest that ionomycin-based therapy could be used as a novel therapeutic strategy for advanced bladder cancer through the effective induction of apoptosis by decreasing the ratio of Bcl-2 to Bax.

Topics: Animals; Antineoplastic Agents; bcl-2-Associated X Protein; Cell Division; Cisplatin; Drug Screening Assays, Antitumor; Drug Synergism; Gene Expression Regulation, Neoplastic; Humans; Ionomycin; Ionophores; Mice; Mice, Nude; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Urinary Bladder Neoplasms

We describe here the involvement of calcium-activated neutral protease (CANP or calpain, EC 3.4.22.17) in calcium-dependent proteolytic processing of the precursor of human interleukin 1 alpha (IL-1 alpha) into mature IL-1 alpha. Calcium ionophore ionomycin enhanced proteolytic processing of pre-IL-1 alpha and the release of mature IL-1 alpha either from lipopolysaccharide (LPS)-activated human adherent mononuclear cells or from a human bladder carcinoma cell line (HTB9 5637) that constitutively produces human IL-1 alpha and -beta. The proteolytic processing of pre-IL-1 alpha was completely inhibited by EGTA. Similar calcium-dependent proteolytic processing of pre-IL-1 alpha was also observed with lysates of either LPS-activated human adherent mononuclear cells or HTB9 5637 cells. Since the optimal pH for processing was between 7 and 8, and E-64 (a cysteine protease inhibitor) and leupeptin (a serine and cysteine protease inhibitor) both inhibited this processing by cell lysates, we hypothesized that a calcium-activated neutral protease, CANP, might be responsible for this processing. This hypothesis was supported by data showing that the specific CANP inhibitor peptide inhibited this proteolysis in cell lysates in a dose-dependent fashion (IC50 = 0.05 microM) and that treatment of pre-IL-1 alpha with purified CANP yielded the 17-kDa mature form of IL-1 alpha, which has an amino terminus identical with that reported for mature human IL-1 alpha. Taken together, these findings indicate that calcium-dependent proteolytic processing of pre-IL-1 alpha is selectively mediated by CANP.

Topics: Calcium; Calpain; Cell Line; Egtazic Acid; Humans; Interleukin-1; Ionomycin; Kinetics; Lipopolysaccharides; Methionine; Monocytes; Protein Processing, Post-Translational; Tumor Cells, Cultured; Urinary Bladder Neoplasms