| Cancer is a critical worldwide problem that needs to be solved.In recent years,with the development of technology and the improvement of people’s health awareness,the incidence of cancers has declined slightly,but the overall situation is not too optimistic.More and more studies have shown that early diagnosis of tumors and effective targeted therapy play an important role in anti-tumor therapies.In general,early diagnosis and targeted treatment of tumors most rely on specific tumor probes.Using differences of molecular signatures between tumors and normal tissues,screening of specific tumor probes is of great significance in the early diagnosis and targeted therapy of tumors.Currently,there are variety types of specific tumor probes,such as antibodies,peptides,mimetic peptides,nucleic acids,and so on.Widely use of antibodies in the early diagnosis and targeted therapy of tumors is self-evident.Antibodies are coupled with radionuclides for early imaging of tumors,or coupled with anti-tumor drugs to achieve targeted therapy.However,after decades of clinical validation and investigations by FDA and other drug approval agencies,there are many limitations in the clinical use of antibodies.For example,antibody heavy chains and other regions are easily captured by the reticuloendothelial system,resulting in obvious liver,spleen,and bone marrow tissue toxicity;with more than 160 k D molecular weight,antibodies are difficult to enter the solid tumor tissue;in addition,the high cost of production brings huge economic burdens to patients and society.Compared to these defects in antibodies,targeted polypeptides have many advantages such as low immunogenicity,rapid plasma clearance,and high tumor permeability due to their small size.Moreover,the production cost of small peptides is much lower than that of antibodies,making it easier to produce on a large scale.Therefore,tumor-targeting small peptides have always been one of the hot spots for the development of new drugs at home and abroad.In the previous study,we obtained a heptapeptide(SNF*)that specifically binds to esophageal adenocarcinoma tissues by phage peptide library screening.After conjugated with a fluorophore,the small peptide,specifically targeting to esophageal adenocarcinoma tissue,was used for endoscopic screening of Barrett’s esophagus in EA patients by confocal endoscopy(Gastroenterology 2010 Nov;139(5):1472-80).However,the targeted molecule of SNF* is unknown for using the esophageal adenocarcinoma cells and immortalized precancerous cells in the previous research.In this research,we have identified and analyzed the specific molecules binded by SNF*.We hope to provide a theoretical foundation for the application of the small peptide-based tumor probes through acknowledging the binding molecule of this small peptide.At the same time,the application values of this small peptide will be further expanded.In order to accomplish the above goals,we conducted the following research work: 1)The targetd molecule was analyzed by Pull Down test and mass spectrometry,and finally determined by Western Blot.2)We used computer molecular docking techniques to simulate the spatial association between SNF* and target molecules,and predict binding sites.ELISA assays and fluorescence polarization assays were used to detect the interaction between SNF* and targeted molecules in vitro.RNAi and cellular immunofluorescence assays verified the specific binding of SNF* in esophageal adenocarcinoma cells OE33.3)We analyzed the expression of targeted molecules in variety types of tumors by TCGA and tissue microarray.4)The specific binding of SNF* polypeptides to targeted molecule was verified in colon and gastric tumor cells,clinical tumor samples and tumor-bearing mouse models.At the same time,CCK-8 and tumor-bearing mouse models treated with SNF* polypeptide drugs were used to identified anti-tumor biological activity of SNF* and effects on Wnt pathway.5)SNF*-Cy5.5 molecular probes were constructed to identify the specifically targeting ability of SNF*-Cy5.5 to CDX2P-NLS Cre;Apc+/lox P mice(with spontaneous colon cancer)by fluorescence endoscopy.6)SNF*-PHIS-DSPE-PTX were constructed and used in tumor-bearing mices to detect the tumor targeting ability,anti-tumor effects and toxic side effects in vivo.7)The specific binding of SNF* polypeptides to targeted molecule was verified in esophageal squamous,breast and pancreatic carcinoma tumor cells,clinical tumor samplesThrough the above work study,we have obtained the following results: 1)Ep CAM was the potential targeted molecule of SNF*.FITC labeled SNF*(SNF*-FITC)co-localized with Ep CAM antibody on the surface of esophageal adenocarcinoma cells OE33.Few SNF*-FITC binded to OE33 cells with Ep CAM knockdown,and binding of SNF*-FITC to Ep CAM significantly increased after exogenous Ep CAM transfection in normal esophageal epithelium cells Q-h TERT.2)Molecular Docking predicted the binding sites of Ep EX were amino acids 173,174,233,and 241.Transfection of Ep CAM with no binding site did not change the binding of SNF*-FITC to Q-h TERT cells.3)The TCGA analysis showed that Ep CAM was overexpressed in 17 types of epithelialderived cancers.The tissue microarray results confirmed that Ep CAM was highly expressed in colon cancer and gastric cancer,except esophageal adenocarcinoma.4)The detection of immunofluorescence assay and xenografts’ mice models confirmed that SNF*-FITC specifically binded to colon cancer,gastric cancer cells and tissues with high expression of Ep CAM.SNF* had no influence on tumor proliferation and activation of Wnt signaling pathway.5)Observed by mouse fluorescence endoscopy,the Cy5.5-conjugated SNF* peptide clearly binded to spontaneous colon tumors in CDX2P-NLS Cre;Apc+/lox P gene knockout mices.6)SNF* improved the targeted binding ability of SNF*-PHIS-DSPE-PTX to colon and gastric adenocarcinoma cells.SNF* also enhanced drug utilization and antitumor activity,and reduced the toxic side effects of PTX chemotherapy.7)The tissue microarray results confirmed that Ep CAM was highly expressed in esophageal squamous,breast and pancreatic cancer.The detection of immunofluorescence assay confirmed that SNF*-FITC specifically binded to esophageal squamous,breast and pancreatic cancer cells and tissues with high expression of Ep CAM.In summary,this study confirms that the targeted molecule of the previously developed esophageal adenocarcinoma-specific binding polypeptide SNF* was the epithelial cell adhesion molecule-Ep CAM;in addition to esophageal adenocarcinoma,Ep CAM was highly expressed on surface of variety epithelium cancers,like colon cancer,gastric cancer,breast cancer,etc.SNF* had specifically binding abilities to many kinds of tumor cells and tissues such as colon cancer and gastric cancer,and had potentials as a tumor-specific probe for endoscopic detection and tumor-targeted treatment of colon cancer.This study not only lays a theoretical foundation for the application and development of SNF*,but also greatly expands the application of SNF* small peptides.In addition,this study also provides references and accumulates information for the developments and researches of tumorspecific probes based on small peptides. |
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