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Journal article
C-Terminal p53 Palindromic Tetrapeptide Restores Full Apoptotic Function to Mutant p53 Cancer Cells In Vitro and In Vivo
Biomedicines, v 11(1), 137
05 Jan 2023
PMID: 36672645
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
We previously demonstrated that a synthetic monomer peptide derived from the C-terminus of p53 (aa 361-382) induced preferential apoptosis in mutant p53 malignant cells, but not normal cells. The major problem with the peptide was its short half-life (half-life < 10 min.) due to a random coil topology found in 3D proton NMR spectroscopy studies. To induce secondary/tertiary structures to produce more stability, we developed a peptide modelled after the tetrameric structure of p53 essential for activation of target genes. Starting with the above monomer peptide (aa 361-382), we added the nuclear localization sequence of p53 (aa 353-360) and the end of the C-terminal sequence (aa 383-393), resulting in a monomer spanning aa 353-393. Four monomers were linked by glycine to maximize flexibility and in a palindromic order that mimics p53 tetramer formation with four orthogonal alpha helices, which is required for p53 transactivation of target genes. This is now known as the 4 repeat-palindromic-p53 peptide or (4R-Pal-p53p). We explored two methods for testing the activity of the palindromic tetrapeptide: (1) exogenous peptide with a truncated antennapedia carrier (Ant) and (2) a doxycycline (Dox) inducer for endogenous expression. The exogenous peptide, 4R-Pal-p53p-Ant, contained a His tag at the N-terminal and a truncated 17aa Ant at the C-terminal. Exposure of human breast cancer MB-468 cells and human skin squamous cell cancer cells (both with mutant p53, 273 Arg->His) with purified peptide at 7 mu M and 15 mu M produced 52% and 75%, cell death, respectively. Comparatively, the monomeric p53 C-terminal peptide-Ant (aa 361-382, termed p53p-Ant), at 15 mu M and 30 mu M induced 15% and 24% cell death, respectively. Compared to the p53p-Ant, the exogenous 4R-pal-p53p-Ant was over five-fold more potent for inducing apoptosis at an equimolar concentration (15 mu M). Endogenous 4R-Pal-p53p expression (without Ant), induced by Dox, resulted in 43% cell death in an engineered MB468 breast cancer stable cell line, while endogenous p53 C-terminal monomeric peptide expression produced no cell death due to rapid peptide degradation. The mechanism of apoptosis from 4R-Pal-p53p involved the extrinsic and intrinsic pathways (FAS, caspase-8, Bax, PUMA) for apoptosis, as well as increasing reactive oxygen species (ROS). All three death pathways were induced from transcriptional/translational activation of pro-apoptotic genes. Additionally, mRNA of p53 target genes (Bax and Fas) increased 14-fold and 18-fold, respectively, implying that the 4R-Pal-p53p restored full apoptotic potential to mutant p53. Monomeric p53p only increased Fas expression without a transcriptional or translational increase in Fas, and other genes and human marrow stem cell studies revealed no toxicity to normal stem cells for granulocytes, erythrocytes, monocytes, and macrophages (CFU-GEMM). Additionally, the peptide specifically targeted pre-malignant and malignant cells with mutant p53 and was not toxic to normal cells with basal levels of WT p53.
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Details
- Title
- C-Terminal p53 Palindromic Tetrapeptide Restores Full Apoptotic Function to Mutant p53 Cancer Cells In Vitro and In Vivo
- Creators
- Robert L. Fine - Columbia University Irving Medical CenterYuehua Mao - Columbia University Irving Medical CenterRichard Dinnen - Columbia UniversityRamon V. Rosal - Columbia UniversityAnthony Raffo - Columbia UniversityUri Hochfeld - Columbia UniversityPatrick Senatus - Columbia University Irving Medical CenterJeffrey N. Bruce - Columbia UniversityGwen Nichols - Columbia UniversityHsin Wang - Columbia UniversityYongliang Li - Columbia UniversityPaul W. Brandt-Rauf - Drexel University, School of Biomedical Engineering, Science, and Health Systems
- Publication Details
- Biomedicines, v 11(1), 137
- Publisher
- MDPI
- Number of pages
- 19
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- School of Biomedical Engineering, Science, and Health Systems
- Web of Science ID
- WOS:000914389600001
- Scopus ID
- 2-s2.0-85146804362
- Other Identifier
- 991021860717004721
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Biochemistry & Molecular Biology
- Medicine, Research & Experimental
- Pharmacology & Pharmacy