Establishment Of A New Human Endometrial Adenocarcinoma Cell Line And Selection Of Its Specific Targeted Cell-binding Mimicry Peptides Though Phage Display

Objective: To establish a new human endometrial adenocarcinoma cell line, named EAC, and to detect the biological characteristics of EAC cell line for basic and clinical study. To isolate targeted mimicry peptides that bound and internalized into EAC. Through these obtained peptides, ligands or proteins binding specially with EAC will be found. Further, to identified the sites of amino acid epitopes when ligands binding on the cell surface, and to present some basic data for study on targeted anticancer drug and idiosyncratic tumor diagnostic markers. Methods: The tumor tissues were inoculated subcutaneously into the back of nude mice. After forming solid tumor, the mice were killed and the implant was taken out for primary culture. The established cell line was maintained by serial passages. The biological characteristics of EAC cell line were deteced, such as morphological observation, growth curve, chromosome analysis, tumorigenicity. PTEN mutation and VEGF, ER, PR expression were also detected by RT-PCR, Western blot and immunohistochemistry. The tumor cells were screened five rounds through the Ph.D.-12 phage display library. Pick out random phage monoclones from peptide libraries screened, and analyzed the monoclones' specific binding efficiency compared with the wild type phage VcsM13 and specificity to six cell lines of differrent human tissues(HepG-2, MCF-7, JEC, NCI-H460, SWO38, HLO2). The DNA of phages was extracted, sequenced and translated to the sequences of amino acid and compared with the international protein database. From the sequence results, the peptides with high consistency were found and possible protein structure was predicted.Results: EAC cell line grew rapidly, steadily, and showed similar microscopic morphology to adenocarcinoma cell. Chromosome analysis revealed EAC chromosome counts ranging in hyperdiploid. ER, PR were negtive by SP immunohistochemistry. Tumorigenicity of EAC in nude mice was 100%. VEGFexpressed in high level and no PTEN mutation was detected. After five rounds of binding-eluting-amplifying-rebinding's biopanning, the displayed peptide phages had high specificity and strong affinity for their cognate cell type relative to wild type phage VcsM13 and different cell lines. The quantity of output phages reached 105 ~ 106, not only the cell surface-bound phages but also the internalized portion. Through sequencing, one sequence of displayed peptides having high consistency was selected. The sequence -SNPTTPDHLWRG- was both found in the surface-bound and internalized phages.Conclusions: A new human endometrial adenocarcinoma cell line, EAC, was established successfully. It will be helpful for basic and clinical study of endometrial carcinoma. Whole cell screening against tumor cells through random phage peptide library can find phage peptides with a highly tissue specific binding and internalizing ability. These obtained targeted mimicry peptides may be the epitopes in ligand proteins that be able to bind specially and tightly with some receptors on tumor cell surface, and can be taken up into cells by receptor-mediated endocytosis. The peptides provide basis for tumor targeted therapy.