Animal peptide antibiotics are thought to mediate their cytotoxic and growth inhibitory action on bacteria, fungi, and cancer cells through a membrane-targeted mechanism. Although the membrane interactions of the peptide antibiotics and their penetration through the membranes have been studied in several models, the precise chain of events leading to cell death or growth arrest is not established yet. In this study we used in vitro kinase assays followed by imaging analyses to examine the effect of human cationic antimicrobial peptide ECAP on the activity of the protein kinases. We report that HPLC-grade ECAP is responsible for inhibition of EGFR autophosphorylation in plasma membrane fractions obtained from A-431 cells. The activity of ECAP is concentration dependent with a half-inhibitory concentration in the range of 0.1-0.2 microM. Marked decrease in autophosphorylation of immunoprecipitated non-receptor protein kinases belonging to different families, namely PKCmu, Lyn and Syk, is observed in the presence of as little as 0.2 microM of the peptide. Among the examined non-receptor protein kinases PKCmu was the most sensitive to the inhibitory action of ECAP, whereas Syk was inhibited least of all. ECAP exerted no detectable cytotoxicity on non-nucleate animal cells at concentrations up to 3 microM. The capability of ECAP to inhibit protein kinases at concentrations, that are at least 10 fold lower than antibacterial and cytotoxic ones, suggests that the protein kinases are possible intracellular targets for antimicrobial peptides. We suppose that inhibition of the protein kinases may provide a mechanism for the action of cationic antimicrobial peptides on host cells including tumour cells.

To investigate the important role of triple-domain recombinant F N polypeptide in tumor therapy. Two Expressing plasmids have been constructed and used to express two tripledomain recombinant polypeptides of human F N in E.coli. They were CH62(Pro 1239-Ser 1515 of F N linked with Ala 1690-Val 2049 through Met) and CH82 (CH62 without Pro 1953-Glu 1978. CH82 is inclusion bodies in 37??,After denaturation and renaturation of the inclusion bodies with 8M urea, it was purified after the affinity chromatograph with Heparin-agarose. The expression level of CH62 in E.coli was very low, but that CH82 was very high, it suggested that N terminal sequence of Cell I1 in F N is the key sequence which influenced the expression of tripledomain polypeptide in E.coli. The purified product was capable of binding heparin and cells, and it has a better binding activity than bifunctional-domain F N polypeptides. The expression and purification of CH82 provides a fundermental basis for further study of recombinant product with better function of antimetastasis and immune regulation.