herbimycin and Carcinoma--Squamous-Cell

Ultraviolet (UV) light is a strong apoptotic trigger that can induce a caspase-dependent biochemical change in cells. We previously showed that UV irradiation can elicit caspase-3 activation and the subsequent cleavage and activation of p21-activated kinase 2 (PAK2) in human epidermal carcinoma A431 cells. We report that genistein, an isoflavone compound with known inhibitory activities to protein tyrosine kinases (PTKs) and topoisomerase-II (topo-II), can prevent UV irradiation-induced apoptotic biochemical changes (DNA fragmentation, caspase-3 activation, and cleavage/activation of PAK2) in A431 cells. Surprisingly, two typical PTK inhibitors (tyrphostin A47 and herbimycin A) and three known topo-II inhibitors (etoposide, daunorubicin, and novomycin) had no effect on UV irradiation-induced apoptotic biochemical changes, suggesting that the inhibitory effect of genistein is not dependent on its property as a PTK/topo-II inhibitor. In contrast, azide, a reactive oxygen species (ROS) scavenger, could effectively block the UV irradiation-induced apoptotic cell responses. Flow cytometric analysis using the cell-permeable dye 2',7'-dichlorofluorescin diacetate as an indicator of the generation of ROS showed that UV irradiation caused increase of the intracellular oxidative stress and that this increase could be abolished by azide, suggesting that oxidative stress plays an important role in mediating the apoptotic effect of UV irradiation. Importantly, the UV irradiation-induced oxidative stress in cells could be significantly attenuated by genistein, suggesting that impairment of ROS formation during UV irradiation is responsible for the antiapoptotic effect of genistein. Collectively, our results demonstrate the involvement of oxidative stress in the UV irradiation-induced caspase activation and the subsequent apoptotic biochemical changes and show that genistein is a potent inhibitor for this process.

Topics: Antineoplastic Agents; Apoptosis; Benzoquinones; Carcinoma, Squamous Cell; Enzyme Inhibitors; Genistein; Humans; Lactams, Macrocyclic; Oxidative Stress; p21-Activated Kinases; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Quinones; Reactive Oxygen Species; Rifabutin; Sodium Azide; Topoisomerase II Inhibitors; Tumor Cells, Cultured; Tyrphostins; Ultraviolet Rays

Interleukin-6 (IL-6) is a multifunctional cytokine that is produced not only by a variety of normal cells but also by cancer cells. IL-6 produced by cancer cells stimulates the proliferation of these cancer cells in an autocrine/ paracrine manner and causes paraneoplastic syndromes including hypercalcemia, cachexia, and leukocytosis. We have reported previously that a human oral squamous cancer associated with hypercalcemia produces large amounts of IL-6, that animals bearing this cancer exhibit elevated levels of plasma IL-6, and that neutralizing antibodies to human IL-6 reverse hypercalcemia in tumor-bearing animals, indicating an important role of IL-6 in the hypercalcemia in this model. Because these cancer cells overexpress epidermal growth factor receptors (EGFR) with intrinsic tyrosine kinase (TK) activity similar to many other squamous cancers, we examined the effects of herbimycin A, a tyrosine kinase inhibitor, on IL-6 production and hypercalcemia in animals bearing this cancer to develop a new approach to treat the hypercalcemia associated with malignancy. Intraperitoneal administration (once a day for 2 days) of herbimycin A to cancer-bearing hypercalcemic mice reduced the plasma levels of human IL-6 and impaired the hypercalcemia. During 2-day treatment with herbimycin A, no changes were observed in tumor size. Of interest, plasma levels of mouse, but not human, soluble IL-6 receptors were also elevated. However, herbimycin A showed no effects on plasma levels of mouse soluble IL-6 receptors. Herbimycin A suppressed the tyrosine autophosphorylation of EGFR and IL-6 mRNA expression and production, all of which were stimulated by EGF. The data raise the possibility that TK inhibitors may be potential mechanism-based therapeutic agents for the treatment of hypercalcemia associated with squamous cancers which overexpress EGFR.

Topics: Animals; Antibiotics, Antineoplastic; Antigens, CD; Benzoquinones; Carcinoma, Squamous Cell; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Hypercalcemia; Interleukin-6; Lactams, Macrocyclic; Male; Mice; Mice, Nude; Neoplasm Transplantation; Protein-Tyrosine Kinases; Quinones; Receptors, Interleukin; Receptors, Interleukin-6; Rifabutin; Solubility; Tumor Cells, Cultured

Epidermal growth factor (EGF) mediates many pleiotrophic biological effects, one of which is alteration of cellular morphology. In the present study, we examine the possibility that this alteration in cell morphology is caused in part by the dysfunction of cadherin-mediated cell-cell adhesion using the human oesophageal cancer cell line TE-2R, which expresses E-cadherin and EGF receptor. In the presence of EGF, TE-2R changed its shape from round to fibroblastic and its colony formation from compact to sparse. Vanadate, a tyrosine phosphatase inhibitor, further potentiated the EGF response, whereas herbimycin A, a tyrosine kinase inhibitor, interfered with it. Moreover, EGF enabled the cells to invade in organotypic raft culture. These phenomena were accompanied not by decreased expression of the E-cadherin molecule but by a change in its localisation from the lateral adhesion site to the whole cell surface. Both alpha- and beta-catenin, cadherin-binding proteins, were also expressed at the same level throughout these morphological changes. Finally, we examined tyrosine phosphorylation of E-cadherin and alpha- and beta-catenin, and observed tyrosine phosphorylation of beta-catenin induced by EGF. These results suggest that EGF counteracts E-cadherin-mediated junctional assembly through phosphorylation of beta-catenin and modulates tumour cell behaviour to a more aggressive phenotype.

Topics: alpha Catenin; Benzoquinones; beta Catenin; Cadherins; Carcinoma, Squamous Cell; Cell Adhesion; Cell Aggregation; Cell Line; Culture Techniques; Cytoskeletal Proteins; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Fibroblasts; Fluorescent Antibody Technique; Gels; Humans; Lactams, Macrocyclic; Neoplasm Invasiveness; Neoplasm Proteins; Phenotype; Phosphorylation; Phosphotyrosine; Protein Processing, Post-Translational; Quinones; Rifabutin; Signal Transduction; Trans-Activators; Tumor Cells, Cultured; Tumor Stem Cell Assay; Tyrosine; Vanadates

Fibroblasts or their conditioned medium stimulated invasion by squamous cell carcinoma cells. The fibroblast-derived activity responsible for increased invasion is the hepatocyte growth factor/scatter factor (HGF/SF), a ligand for the c-Met receptor. HGF/SF stimulated migration of the cells on various extracellular matrix substrates but did not alter their adhesion efficiency nor integrin expression. HGF/SF stimulated motility in a two step process: initially cells spread rapidly and formed focal adhesions, and then they disassembled these condensations, which was followed by increased cell locomotion. The focal adhesions contained vinculin, p125FAK, beta 1 integrin, and phosphotyrosine. Within minutes after exposure of cells to HGF/SF, proteins of 125 and 145 kDa showed elevated tyrosine phosphorylation and were identified as p125FAK and c-Met, respectively. Gradual loss of tyrosine phosphorylation coincided with disruption of focal adhesions and conversion to a motile phenotype. HGF/SF-mediated tyrosine phosphorylation of p125FAK was inhibited by the tyrosine kinase inhibitor, herbimycin A, which also blocked spreading and the migratory response. These results indicate that fibroblast-derived HGF/SF triggers migration through the initial recruiting of integrins, cytoskeletal proteins, and p125FAK into focal adhesions that is dependent on tyrosine kinase activity.

Topics: Benzoquinones; Carcinoma, Squamous Cell; Cell Adhesion; Cell Adhesion Molecules; Cell Movement; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Hepatocyte Growth Factor; Humans; In Vitro Techniques; Integrins; Lactams, Macrocyclic; Mouth Neoplasms; Neoplasm Invasiveness; Phosphotyrosine; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-met; Quinones; Receptor Protein-Tyrosine Kinases; Rifabutin; Tumor Cells, Cultured; Tyrosine

We examined effect of the tyrosine kinase inhibitor herbimycin A on A431 human epidermoid carcinoma cells which over-express epidermal growth factor (EGF) receptor. Herbimycin A inhibited the autophosphorylation of EGF-stimulated receptors in intact cells both time- and dose-dependently. The inhibition was found to be due to a decrease in the level of expression of the receptor amount, because herbimycin A equally decreased the receptor quantity and EGF-stimulated receptor kinase activity in intact cells, but did not exhibit a direct inhibitory effect on EGF receptor kinase activity in vitro. The decrease of the level of EGF receptor was also confirmed by 125I-EGF binding to herbimycin A-treated cells, and Scatchard analysis showed that the decrease in the receptor number occurred in the major population of the low-affinity binding ones, whereas the number with high-affinity binding was unaffected. Interestingly, although the proliferation of A431 cells was inhibited by EGF, herbimycin A converted EGF into a stimulatory ligand for cell growth, as determined by both cell number and DNA synthesis. These findings indicated that herbimycin A decreased the level of expression of EGF receptor by a mechanism other than inactivation of the receptor kinase and reversed the transformed phenotype of A431 cells to a normal one in the proliferative response to EGF.

Topics: Benzoquinones; Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Lactams, Macrocyclic; Ligands; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tumor Cells, Cultured

The effect of herbimycin A on the biosynthesis of epidermal growth factor (EGF) receptor was examined in human epidermoid carcinoma A431 cells. Cells were pulse-labelled with [35S]methionine, and EGF receptor biosynthesis was quantified by immunoprecipitation using a monoclonal anti-(EGF receptor) antibody. In the presence of herbimycin A, an immature 160 kDa EGF receptor precursor accumulated in 1 h and disappeared completely in 4 h. Pulse-labelled 160 kDa receptor precursor in the absence of herbimycin A, however, was converted normally into a 170 kDa one by chase with herbimycin A. Herbimycin A affected neither the synthesis of the secreted form of EGF receptor devoid of cytoplasmic domain, nor that of the transferrin receptor in A431 cells. The herbimycin A-induced degradation of 160 kDa EGF receptor precursor was not inhibited by an inhibitor of lysosomal enzymes, NH4Cl. Endoglycosidase H digestion of the 160 kDa precursor converted it into the deglycosylated 130 kDa precursor peptide. These results suggested that herbimycin A selectively acted on the EGF receptor precursor during the synthesis of the 160 kDa form, probably on the cytoplasmic domain, to form an aberrant molecule which was subjected to rapid degradation in the endoplasmic reticulum.

Topics: Benzoquinones; Carcinoma, Squamous Cell; Endoplasmic Reticulum; ErbB Receptors; Humans; Kinetics; Lactams, Macrocyclic; Molecular Weight; Protein Precursors; Protein-Tyrosine Kinases; Quinones; Receptors, Transferrin; Rifabutin; Tumor Cells, Cultured

Accumulating evidence indicates that the activation of cellular oncogenes is a cause of some human cancers. ErbB-1, erbB-2 and abl oncogenes encoding tyrosine kinases, ras oncogenes encoding GTP binding proteins and myc oncogenes whose functions are not well understood are some examples. Therefore, agents which inhibit the activity of these oncogene products may provide new means to overcome certain human tumors. Herbimycin A and tyrphostins have been found and developed as inhibitors of tyrosine kinases and the effectiveness of these agents against tumors of Ph1-positive leukemia (CML, ALL) or squamous cell carcinomas has been reported. Although specific inhibitors of ras or myc oncogene products have not yet been described, recent studies on the processing of Ras proteins toward the cell membrane provide a strategy to search for inhibitors of ras functions.

Topics: Antibiotics, Antineoplastic; Benzoquinones; Carcinoma, Squamous Cell; Catechols; Cyclin D1; Female; Genes, ras; Humans; Lactams, Macrocyclic; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Neoplasms; Nitriles; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Quinones; Rifabutin; Tyrphostins