Targeted Inhibition of Colorectal Carcinoma Using a Designed CEA-Binding Protein to Deliver p53 Protein and TCF/LEF Transcription Factor Decoy DNA
Abstract
1. Introduction
2. Results
2.1. Delivery of Purified p28-p14ARF Protein Prolonged the Effective Duration of Delivered p53 Protein and Increased Its Antitumor Effect
2.2. De Novo Design of Binding Proteins to Specifically Recognize the CRC Biomarker CEA
2.3. Specifically Targeting CRC Cells with p28-p53-CEABP1 Resulted in Considerably Higher Suppression of CRC Cell Proliferation and Xenograft Tumor Growth
2.4. Delivery of TCF/LEF TFD DNA by the CPP-Max Protein Suppressed CRC Cell Proliferation and Xenograft Tumor Growth
2.5. Targeting CEA-Expressing CRC Cells with a Designed CEA-Binding Protein Enhanced the Antitumor Effect of TCF/LEF TFD DNA
3. Discussion
4. Materials and Methods
4.1. Protein Expression and Purification
4.2. Cell Lines and Cell Culture
4.3. Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
4.4. CCK-8 Cell Proliferation Assay
4.5. Colony Formation Assay
4.6. Ethynyl Deoxyuridine (EdU) Incorporation Assay
4.7. Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick End Labeling (TUNEL) Assay
4.8. Cell Cycle Analysis
4.9. Xenograft Tumor Model and Administration of the p28-p53 Protein or TCF/LEF TFD DNA and pep1-Max Protein in the Presence or Absence of CEA-Binding Protein
4.10. De Novo Design of the CEA-Binding Proteins CEABP1 and CEABP2
4.11. Molecular Dynamics (MD) Simulation
4.12. Immunofluorescence Staining
4.13. Hematoxylin & Eosin (H&E) Staining
4.14. Antibodies
4.15. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Forward Primer | Reverse Primer |
---|---|---|
gapdh | ATGTGGCCGAGGACTTTGATT | AGTGGGGTGGCTTTTAGGATG |
cdkn1a | CTTTCTGGCCGTCAGGAACA | CTTCTATGCCAGAGCTCAACATGT |
bax | TCAGGATGCGTCCACCAAGAAG | TGTGTCCACGGCGGCAATCATC |
axin2 | AGCCAAAGCGATCTACAAAAG | AAGTCAAAAACATCTGGTAGGCA |
cyclin D1 | CAGAGTGATCAAGTGTGACCC | CGTCGGTGGGTGTGCAAGC |
c-myc | CTCCTACGTTGCGGTCACAC | CGGGTCGCAGATGAAACTCT |
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Wang, W.; Sun, X.; Wu, G. Targeted Inhibition of Colorectal Carcinoma Using a Designed CEA-Binding Protein to Deliver p53 Protein and TCF/LEF Transcription Factor Decoy DNA. Int. J. Mol. Sci. 2025, 26, 9846. https://doi.org/10.3390/ijms26209846
Wang W, Sun X, Wu G. Targeted Inhibition of Colorectal Carcinoma Using a Designed CEA-Binding Protein to Deliver p53 Protein and TCF/LEF Transcription Factor Decoy DNA. International Journal of Molecular Sciences. 2025; 26(20):9846. https://doi.org/10.3390/ijms26209846
Chicago/Turabian StyleWang, Wen, Xuan Sun, and Geng Wu. 2025. "Targeted Inhibition of Colorectal Carcinoma Using a Designed CEA-Binding Protein to Deliver p53 Protein and TCF/LEF Transcription Factor Decoy DNA" International Journal of Molecular Sciences 26, no. 20: 9846. https://doi.org/10.3390/ijms26209846
APA StyleWang, W., Sun, X., & Wu, G. (2025). Targeted Inhibition of Colorectal Carcinoma Using a Designed CEA-Binding Protein to Deliver p53 Protein and TCF/LEF Transcription Factor Decoy DNA. International Journal of Molecular Sciences, 26(20), 9846. https://doi.org/10.3390/ijms26209846