Study Of Screening Homing Peptide To Tumor And Its Metastases And Tumor Targeting Therapy

Purpose: Our study was aimed to screen the homing peptides to tumor and itsmetastases.Methods: FliTrx^TM is a bacterial peptide display library containing the entirerepertoire of possible random dodecapeptides expressed on the flagella rip of E.coli. Two prostate cancer cell lines, invasive and highly-metastatic PC-3M-1E8 cells and poorly-metastatic PC-3M-2B4 cells were used for the screening process. Flitrx cells were incubated with PC-3M-2B4 at room temperature for lh to remove any potential clones displaying peptides that would bind to low or none metastatic potential prostate cancer cells. The unbinding Flitrx cells were then incubated with PC-3M-1E8 cells, allowing for clones displaying potential peptides to bind high metastatic potential tumor and its metastases. The bacterial population was then expanded and this "panning" process was carried out a total of four times. Peptide insert sequences from 100 bacterial colonies were analyzed for potential repetitive peptide motifs. Immunofluorescence assay was carried out to determine the specific binding capacities of the colonies and the potential repetitive peptide motifs recognizing PC-3M-1E8 cells. The motif with highest binding capacity to PC-3M-1E8 cells was named as "TMP1" with five amino acids and synthesized with the sequence GCGABDEFGC. This peptide contained flanking cysteines, which reformed a disulfide loop. Glycine residues provide a "space" function, permitting the physical formation of a loop structure. And this cyclic peptide was conjugated with FITC at the N-terminus, which aimed to be detected easily. Another peptide GCGBAFEDGC which had the same amino acid with different order was synthesized as the negative control. Immunofluorescence assay and competitive test were carried out to determine the specific binding capacity of TMP1 to PC-3M-1E8 cells. And the in vivo tumor and its metastases targeting capacity of TMP1 was measured in PC-3M-1E8 prostate cancer xenografts. Results: Of the recurring peptide sequences detected, two 5-mers repeated 2 times,one 4-mers repeated 3 times and three 3-mers repeated four times. Two colonies showed PC-3M-1E8 cells binding specificity, which both contained an five amino acid sequence ABDEF("TMP1'). The synthesized TMPl could specific binding to highly-metastatic prostate cancer cells PC-3M-1E8 and breast cancer cells MDA-MB-435S and MDA-MB-231, whereas it could not recognize poorly-metastatic prostate cancer cells PC-3M-2B4 and breast cancer cells MCF-7. In vivo study also showed that the TMPl could specific target MDA-MB-435S xenografts, PC-3M-1E8 xenografts and the liver metastases even if only 1mm in diameter, but not PC-3M-2B4 xenografts.Conclusion: These results suggest that the TMPl peptide can target bothhighly-metastatic tumors and their metastases. Consequently, it should be possible to design an early diagnosis and combination therapy approach both targets. Purpose: TMP1 was the peptide selected from a Flitrx^TM peptide library thatspecifically bound to prostate tumors and their metastases. Our study was aimed to study its direct anti tumor capacity.Methods: The binding and internalization of TMP1 to prostate cancer PC-3M-1E8cells were measured with immunofluorescence assay. Cell Proliferation Assay was used to determine the cytotoxic effect of TMPl to PC-3M-1E8. The subpopulation of apoptosis and cell cycles of PC-3M-1E8 treated with TMPl were calculated with FACS analyses. The express of procaspase-8 and caspase-8 in PC-3M-1E8 treated with 100μ M TMPl for 72h was analyzed with western blot.Results: Immunofluorescence assay indicated that TMPl could only bind to themembrane of PC-3M-1E8 cells in vitro, whereas not be internalized into the cells, which was quite different with other reported homing peptides. We found that this TMPl peptide, GCGABDEFGC, inhibited tumor cell proliferation in a dose-dependent manner with IC₅₀≈50μM, induced apoptosis and G2/M-phase cell cycle arrest, and increased the expression of caspase-8 in PC-3M-1E8 cells.Conclusion: This study suggests that the TMPl peptide not only processes tumortargeting capacity, but also has direct tumor cytotoxic effect which probably initiated by the death receptor pathway.