epidermal-growth-factor and cepharanthine

Receptor-mediated superoxide (O2.-)-generation and tyrosyl phosphorylation of neutrophil proteins, such as 58, 65, 84, 108 and 115 kDa, were enhanced by priming cells with granulocyte colony stimulating factor (G-CSF) [Akimura, K. et al. Arch. Biochem. Biophys. 298: 703-709, 1992]. To elucidate the possible involvement of tyrosyl phosphorylation of neutrophil proteins in the enhancing mechanism of O2.- generation, the effect of cepharanthine, a biscoclaurine alkaloid that inhibits phorbol 12-myristate 13-acetate (PMA)- and receptor-mediated O2.- generation, on the priming of human peripheral neutrophils (HPPMN) was studied. Both enhancement of formyl-methionyl-leucyl- phenylalanine (FMLP)-mediated O2.- generation and tyrosyl phosphorylation of some neutrophil proteins, i.e., 115, 108 and 84 kDa proteins, by HHPMN after treatment with G-CSF were strongly inhibited by cepharanthine in a concentration- and treatment-time-dependent manner. In contrast, inhibition of PMA-mediated O2.- generation by cepharanthine was weak and independent of treatment time. These results suggest that cepharanthine might inhibit the priming step of neutrophil activation concomitantly with its inhibition of the tyrosyl phosphorylation of some neutrophil proteins that might underlie the mechanism for priming of neutrophils with G-CSF.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Annexin A1; Anti-Inflammatory Agents, Non-Steroidal; Benzylisoquinolines; Epidermal Growth Factor; Granulocyte Colony-Stimulating Factor; Humans; Isoquinolines; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oxygen; Phosphorylation; Piperazines; Protein-Tyrosine Kinases; Respiratory Burst; Tumor Necrosis Factor-alpha

Cepharanthine, a biscoclaurine alkaloid, causes an 8-fold enhancement of the cytotoxic effect of a conjugate of epidermal growth factor (EGF) coupled with Pseudomonas exotoxin in HeLa cells. Cepharanthine also potentiates the effect of Pseudomonas exotoxin. Cepharanthine does not affect the binding and uptake of 125I-EGF by HeLa cells, but it delays the release of radioactivity associated with 125I-EGF into the medium. Analysis by colloidal silica gradients using cell homogenates suggests that 125I-EGF accumulates in the lysosomes of cells treated with cepharanthine and that [3H]cepharanthine accumulates in lysosomes. The pH in HeLa cell lysosomes is 5.2, and cepharanthine does not significantly increase the pH. Electron microscopy shows an increased number of electron-dense bodies and dilated Golgi apparatus after cepharanthine treatment. Cepharanthine appears to accumulate in lysosomes, and it may delay degradation of EGF-Pseudomonas exotoxin in the cells as does 125I-EGF.

Topics: ADP Ribose Transferases; Alkaloids; Bacterial Toxins; Benzylisoquinolines; Cell Survival; Drug Synergism; Epidermal Growth Factor; Exotoxins; HeLa Cells; Humans; Hydrogen-Ion Concentration; Lysosomes; Pseudomonas aeruginosa Exotoxin A; Tissue Distribution; Virulence Factors