Journal: International Journal of Molecular Sciences

Article Title: Targeted Inhibition of Colorectal Carcinoma Using a Designed CEA-Binding Protein to Deliver p53 Protein and TCF/LEF Transcription Factor Decoy DNA

doi: 10.3390/ijms26209846

Figure Lengend Snippet: Delivery of purified p28-p53 protein inhibited CRC cell proliferation and xenograft tumor growth. ( A ) Confocal microscopy images showed that after delivery into HCT116 cells, the internalized p53 protein tagged with GFP and the cell-penetrating peptide p28 escaped from lysosomes and were localized in the nuclei. ( B ) Delivery of purified p28-p53 protein into HCT116 CRC cells inhibited cell proliferation, as revealed by the ethynyl deoxyuridine (EdU) incorporation assay. The error bars represent the standard error of the mean (SEM), which was determined via two-way ANOVA. ( C ) Delivery of the p28-p53 protein did not cause as much inhibition on the cell proliferation rate of NCM460 normal colonic epithelial cells as on that of HCT116 and LS174T CRC cells, as revealed by the CCK-8 cell proliferation assay. ( D ) Treatment of HCT116 cell subcutaneous xenograft tumor-bearing mice with purified p28-p53 protein decreased tumor growth. Representative images of HCT116 cell xenograft tumors at the endpoint of the experiment were shown; the experiment included the following: physiological saline ( n = 6), 5 mg of p28-p53 protein per kg of mice ( n = 6), 10 mg of p28-p53 protein per kg of mice ( n = 5), 5 mg of p28-GFP protein per kg of mice ( n = 6), 10 mg of p28-GFP protein per kg of mice ( n = 6), and 5 mg cisplatin per kg of mice ( n = 5). Administration started when the tumors grew to 70 mm 3 in volume. Statistical significance is denoted as follows: *** p < 0.001, **** p < 0.0001.

Article Snippet: The cells used in this study included (i) the human colorectal cancer cell line HCT116 (ATCC#CCL-247 TM ), with wild-type p53; (ii) the human colorectal cancer cell line SW480 (ATCC#CCL-228 TM ), with two point mutations of p53 (R273H/P309S); (iii) the human colorectal cancer cell line LS174T (ATCC#CCL-188 TM ), with wild-type p53; (iv) the normal human colonic epithelial cell line NCM460; and (v) the human cervical cancer cell line HeLa (ATCC#CCL-2 TM ).

Techniques: Purification, Confocal Microscopy, Inhibition, CCK-8 Assay, Proliferation Assay, Saline

Journal: International Journal of Molecular Sciences

Article Title: Targeted Inhibition of Colorectal Carcinoma Using a Designed CEA-Binding Protein to Deliver p53 Protein and TCF/LEF Transcription Factor Decoy DNA

doi: 10.3390/ijms26209846

Figure Lengend Snippet: The de novo-designed CEABP1 and CEABP2, which specifically recognize the A3 and B2 domains of the CRC marker CEA, respectively. ( A ) The structure of CEABP1, the de novo-designed binding protein for CEA-A3, in complex with the extracellular A3 domain of CEA, as predicted by Alphafold 3. ( B ) The binding site of CEABP1 on the full-length CEA protein. ( C ) The structure of CEABP2, the de novo-designed binding protein for CEA-B2, in complex with the extracellular B2 domain of CEA, as predicted by Alphafold 3. ( D ) The binding site of CEABP2 on the full-length CEA protein. ( E ) CEABP1 colocalized with CEA on the cell membrane of the CEA-expressing CRC cell line LS174T. LS174T cells were incubated with purified GFP-tagged CEABP1 protein for 6 h before immunofluorescence staining. Scale bars, 20 μm. ( F ) CEABP2 colocalized with CEA on the cell membrane of LS174T cells.

Article Snippet: The cells used in this study included (i) the human colorectal cancer cell line HCT116 (ATCC#CCL-247 TM ), with wild-type p53; (ii) the human colorectal cancer cell line SW480 (ATCC#CCL-228 TM ), with two point mutations of p53 (R273H/P309S); (iii) the human colorectal cancer cell line LS174T (ATCC#CCL-188 TM ), with wild-type p53; (iv) the normal human colonic epithelial cell line NCM460; and (v) the human cervical cancer cell line HeLa (ATCC#CCL-2 TM ).

Techniques: Marker, Binding Assay, Membrane, Expressing, Incubation, Purification, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Targeted Inhibition of Colorectal Carcinoma Using a Designed CEA-Binding Protein to Deliver p53 Protein and TCF/LEF Transcription Factor Decoy DNA

doi: 10.3390/ijms26209846

Figure Lengend Snippet: Specific targeting of CRC cells by fusing CEABP1 to the C-terminus of p28-p53 enhanced its ability to suppress CRC cell proliferation. ( A ) qRT-PCR analysis revealed that fusing CEABP1 to the C-terminus of p28-p53 was most effective at increasing the ability of the delivered p53 protein to promote cdkn1a transcription in LS174T cells. The relative cdkn1a expression levels are indicated above the columns. ( B ) The p28-p53-CEABP1 protein was most effective at increasing the transcription of bax when it was delivered into LS174T cells. The relative bax expression levels are indicated above the columns. ( C ) Compared with delivery of p28-p53, delivery of the purified p28-p53-CEABP1 protein more strongly inhibited the proliferation of LS174T cells, as shown by the CCK-8 assay. Statistical significance is denoted as follows: ** p < 0.01, **** p < 0.0001.

Article Snippet: The cells used in this study included (i) the human colorectal cancer cell line HCT116 (ATCC#CCL-247 TM ), with wild-type p53; (ii) the human colorectal cancer cell line SW480 (ATCC#CCL-228 TM ), with two point mutations of p53 (R273H/P309S); (iii) the human colorectal cancer cell line LS174T (ATCC#CCL-188 TM ), with wild-type p53; (iv) the normal human colonic epithelial cell line NCM460; and (v) the human cervical cancer cell line HeLa (ATCC#CCL-2 TM ).

Techniques: Quantitative RT-PCR, Expressing, Purification, CCK-8 Assay

Journal: International Journal of Molecular Sciences

Article Title: Targeted Inhibition of Colorectal Carcinoma Using a Designed CEA-Binding Protein to Deliver p53 Protein and TCF/LEF Transcription Factor Decoy DNA

doi: 10.3390/ijms26209846

Figure Lengend Snippet: Specific targeting of CRC cells by fusing CEABP1 to the C-terminus of p28-p53 considerably increased its inhibition of xenograft tumor growth in mice. ( A ) Treatment with the purified p28-p53-CEABP1 protein inhibited LS174T cell subcutaneous xenograft tumor growth more effectively than p28-p53. The tumor growth curves of the following groups receiving different treatments are shown: physiological saline ( n = 6), 5 mg of p28-p53 protein per kg of mice ( n = 6), 5 mg of p28-p53 and p28-p14ARF each per kg of mice ( n = 5), 5 mg of p28-p53-CEABP1 per kg of mice ( n = 6), 8.28 mg of p28-p53-CEABP1 and TCF/LEF TFD DNA (molar ratio 3:1) per kg of mice ( n = 6), and 5 mg cisplatin per kg of mice ( n = 6). Administration started when the tumors grew to 100 mm 3 . ( B ) Representative images of LS174T cell xenograft tumors at the experimental endpoint (day 18). ( C ) LS174T tumor weights at the endpoint. The color scheme for the different groups is the same as that in ( A ). ( D ) Treatment with p28-p53-CEABP1 did not cause perceptible changes in mouse body weight during the experiment. The color scheme for the different groups is the same as that described in ( A ). ( E ) qRT-PCR analysis revealed that, compared with the absence of CEABP1, the injection of purified p28-p53-CEABP1 substantially increased the cdkn1a transcript level in the tumor tissue. ( F ) qRT-PCR analysis revealed that p28-p53-CEABP1 drastically increased bax mRNA expression compared with p28-p53 in tumors. The color scheme for the different groups is the same as that in ( E ). Statistical significance is denoted as follows: * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: The cells used in this study included (i) the human colorectal cancer cell line HCT116 (ATCC#CCL-247 TM ), with wild-type p53; (ii) the human colorectal cancer cell line SW480 (ATCC#CCL-228 TM ), with two point mutations of p53 (R273H/P309S); (iii) the human colorectal cancer cell line LS174T (ATCC#CCL-188 TM ), with wild-type p53; (iv) the normal human colonic epithelial cell line NCM460; and (v) the human cervical cancer cell line HeLa (ATCC#CCL-2 TM ).

Techniques: Inhibition, Purification, Saline, Quantitative RT-PCR, Injection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Targeted Inhibition of Colorectal Carcinoma Using a Designed CEA-Binding Protein to Deliver p53 Protein and TCF/LEF Transcription Factor Decoy DNA

doi: 10.3390/ijms26209846

Figure Lengend Snippet: Delivery of TCF/LEF TFD DNA into CRC cells inhibited CRC cell proliferation and xenograft tumor growth. ( A ) The delivery scheme of TCF/LEF TFD DNA. The TCF/LEF-binding DNA was linked with the Max-binding DNA to function as TCF/LEF TFD DNA. The human Max DNA-binding domain was fused with the cell-penetrating peptide pep1, and the fusion protein was employed to deliver the TCF/LEF TFD DNA into CRC cells, which competitively inhibited endogenous TCF/LEF from binding to its target gene promoters and prevented the transcription of Wnt signaling-responsive genes. ( B ) TCF/LEF TFD DNA could be efficiently delivered into LS174T CRC cells and was located in the nuclei. Fluorescence microscopy images of LS174T cells delivered with FAM-labeled TCF/LEF TFD DNA and purified pep1-Max protein are shown. Scale bars: 130 μm. ( C ) Delivery of TCF/LEF TFD DNA suppressed the expression of the Wnt signaling target genes axin2 , cyclin D1 , and c-myc . TCF/LEF TFD DNA and/or purified pep1-Max protein were delivered into HCT116 or LS174T CRC cells, and qRT-PCR analysis was performed to assess the relative expression levels of axin2 , cyclin D1 , and c-myc mRNA transcripts. ( D ) Delivery of TCF/LEF TFD DNA by purified pep1-Max protein suppressed HCT116 cell proliferation in the colony formation assay. A quantification of the colony formation assay results is shown. ( E ) Treatment of HCT116 cell xenograft mice via tail vein injection of TCF/LEF TFD DNA and purified pep1-Max protein decelerated tumor growth. Tumor growth curves of HCT116 cell subcutaneous xenograft mice treated with physiological saline ( n = 6), 2.76 mg of pep1-Max and TCF TFD DNA (molar ratio 3:1) per kg of mice ( n = 8), or 8.28 mg of pep1-Max and TCF TFD DNA (molar ratio 3:1) per kg of mice ( n = 7) are shown. Administration started when the tumors grew to 100 mm 3 in volume. ( F ) Treating mice with TCF/LEF TFD DNA and pep1-Max protein decreased axin2 and cyclin D1 mRNA expression in tumors, as revealed by qRT-PCR analysis of tumor samples. Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The cells used in this study included (i) the human colorectal cancer cell line HCT116 (ATCC#CCL-247 TM ), with wild-type p53; (ii) the human colorectal cancer cell line SW480 (ATCC#CCL-228 TM ), with two point mutations of p53 (R273H/P309S); (iii) the human colorectal cancer cell line LS174T (ATCC#CCL-188 TM ), with wild-type p53; (iv) the normal human colonic epithelial cell line NCM460; and (v) the human cervical cancer cell line HeLa (ATCC#CCL-2 TM ).

Techniques: Binding Assay, Fluorescence, Microscopy, Labeling, Purification, Expressing, Quantitative RT-PCR, Colony Assay, Injection, Saline

Journal: International Journal of Molecular Sciences

Article Title: Targeted Inhibition of Colorectal Carcinoma Using a Designed CEA-Binding Protein to Deliver p53 Protein and TCF/LEF Transcription Factor Decoy DNA

doi: 10.3390/ijms26209846

Figure Lengend Snippet: Targeting CEA-expressing CRC cells with a designed CEA-binding protein enhanced the antitumor effect of TCF/LEF TFD DNA. ( A ) Fusion of CEABP1 to the C-terminus of pep1-Max enhanced the suppression of LS174T cell proliferation by the delivered TCF/LEF TFD DNA, which was examined via a CCK-8 assay. The statistics in the figure are for comparison between the pep1-Max-CEABP1 protein plus TCF/LEF TFD DNA group and the pep1-Max protein plus the same DNA group. ( B ) Colony formation assays verified that fusing CEABP1 to pep1-Max caused the delivery of TCF/LEF TFD DNA to inhibit LS174T cell growth more strongly than in the absence of CEABP1. ( C ) Targeting LS174T cell subcutaneous xenograft tumors with CEABP1 enhanced the ability of TCF/LEF TFD DNA to decelerate tumor growth in mice. The tumor growth curves of the following groups receiving different treatments are shown: physiological saline ( n = 6), 2.76 mg of pep1-Max protein and TCF/LEF TFD DNA per kg of mice ( n = 6), 2.76 mg of CEABP2-pep1-Max protein and TCF/LEF TFD DNA per kg of mice ( n = 6), 2.76 mg of pep1-Max-CEABP2 protein and TCF/LEF TFD DNA per kg of mice ( n = 6), 2.76 mg of pep1-Max-CEABP1 protein and TCF/LEF TFD DNA per kg of mice ( n = 6), 8.28 mg of pep1-Max-CEABP2 protein and TCF/LEF TFD DNA per kg of mice ( n = 6), 5 mg cisplatin per kg of mice ( n = 6), 7.34 mg of pep1-Max protein per kg of mice ( n = 6), and 0.94 mg of TCF/LEF TFD DNA per kg of mice ( n = 5). For the above protein and DNA mixtures, the protein-to-DNA molar ratio was 3:1. Administration started when the tumors grew to 100 mm 3 in volume. ( D ) Fusing CEABP1 or CEABP2 to pep1-Max did not cause any body weight change in the mice during the experiment. ( E ) Specific targeting of CEA-expressing LS174T cells with CEABP1 further decreased the mRNA expression of Wnt-responsive genes in tumors. qRT-PCR analysis of axin2 and c-myc mRNA transcript levels in tumor samples from the indicated groups was performed. Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The cells used in this study included (i) the human colorectal cancer cell line HCT116 (ATCC#CCL-247 TM ), with wild-type p53; (ii) the human colorectal cancer cell line SW480 (ATCC#CCL-228 TM ), with two point mutations of p53 (R273H/P309S); (iii) the human colorectal cancer cell line LS174T (ATCC#CCL-188 TM ), with wild-type p53; (iv) the normal human colonic epithelial cell line NCM460; and (v) the human cervical cancer cell line HeLa (ATCC#CCL-2 TM ).

Techniques: Expressing, Binding Assay, CCK-8 Assay, Comparison, Saline, Quantitative RT-PCR