A.V. Bereznikova, G.A. Posypanova, V.A. Makarov, O.V. Antipova, E.S. Severin

The subject of article is a study of the transport properties of a biologically active fragment of human alpha-fetoprotein: octapeptide GIP-8 (EMTPVNPG). GIP-8 displays biological antiproliferative activity. The mechanism of action of GIP-8 (and its analogues) is still unclear. In this work we studied the capability of the GIP-8 to penetrate into tumor cells and the specificity of this process; we also sought to make it clear whether one can use GIP-8 for delivery of antitumor drugs into cancer cells. The binding and endocytosis of FITC-GIP-8 to tumor cells (human ovarian carcinoma SKOV3) and normal cells (human peripheral blood lymphocytes) were investigated by flow cytometry. We found out that FITC-GIP-8 binds vigorously to SKOV3 cells, but only insignificantly to stimulated lymphocytes. The measurement of the endocytosis level of FITC-GIP-8 provided evidence of a high specificity of GIP-8 uptake by tumor cells. The study of and intracellular distribution of FITC-GIP-8 with fluorescent microscopy has shown that bright luminous clusters could be seen after a one-hour incubation of cells with FITC-GIP-8. After the lapse of 24 h the fluorescence was more intensive and besides the clusters one could see wide luminous zones in the cytoplasm of cells, for the most part in the perinuclear region. The dates obtained provide evidence of high binding and endocytosis of octapeptide by SKOV3 tumor cells and a significantly lower (by 5-10 times) level in case of peripheral blood lymphocytes. This finding allows to consider this peptide as a new potential vehicle for targeted delivery of drugs into tumor cells. The conjugate GIP-8 with the antitumor antibiotic doxorubicin (DOX) have been synthesized with the use of 4(4-N-maleimidomethyl)cyclohexane-1-carboxyl hydrazide crosslinker. The accumulation and distribution of the GIP-8-DOX conjugate in tumor cells (SKOV3) were studied by fluorescence microscopy. The distinctly expressed red fluorescence of DOX in cells treated with the GIP-8-DOX conjugate provides evidence that conjugated DOX is efficiently captured by tumor cells. As the time of incubation increased, the fluorescence intensity also increased. Cells treated with GIP-8-DOX displayed DOX distribution predominantly at the nuclei of cells, such phenomenon being typical for unbound (free) DOX; this means that DOX within conjugate keeps the ability to penetrate into nuclei of cells and interact with its target DNA. The uptake of GIP-DOX by several different tumor cells was detectable by flow cytometry and was observed to be a rapid process. After a 15-min incubation of cells with conjugate at 37оС one could see a significant increase in the intensity of fluorescence of DOX in tumor cells, and this fact provides evidence that the cells accumulated the conjugate. In contrast to tumor cells, in case of lymphocytes from peripheral blood the accumulation of GIP-8-DOX after 15 minutes of incubation virtually did not differ from that for DOX for the same time. The study of cytotoxic activity of the GIP-8-DOX conjugate showed that the in vitro toxicity of the conjugate for SKOV3 and MCF-7 cells approximated that of the unbound (free) DOX for the same cell lines. Concurrently, cytotoxicity of stimulated lymphocytes due to the GIP-8-DOX was markedly lower (by one order of magnitude at the minimum) than that for DOX. The results of the study of cytotoxicity are in good agreement with the data on accumulation and uptake of the GIP-8-DOX conjugate into cells. Thus the results obtained suggested GIP-8 peptide promising vector for targeted delivery of cytotoxic agents in tumor cells.