| Autor: |
Fine RL; Experimental Therapeutics Program, Division of Medical Oncology, College of Physicians and Surgeons of Columbia University, New York, NY 10314, USA., Mao Y; Experimental Therapeutics Program, Division of Medical Oncology, College of Physicians and Surgeons of Columbia University, New York, NY 10314, USA., Dinnen R; Experimental Therapeutics Program, Division of Medical Oncology, College of Physicians and Surgeons of Columbia University, New York, NY 10314, USA., Rosal RV; Department of Environmental Health Sciences, Mailman School of Public Health of Columbia University, New York, NY 10314, USA., Raffo A; Experimental Therapeutics Program, Division of Medical Oncology, College of Physicians and Surgeons of Columbia University, New York, NY 10314, USA., Hochfeld U; Experimental Therapeutics Program, Division of Medical Oncology, College of Physicians and Surgeons of Columbia University, New York, NY 10314, USA., Senatus P; Department of Neurosurgery, Neurologic Institute of New York, Columbia University Medical Center, New York, NY 10032, USA., Bruce JN; Department of Neurosurgery, Neurologic Institute of New York, Columbia University Medical Center, New York, NY 10032, USA., Nichols G; Experimental Therapeutics Program, Division of Medical Oncology, College of Physicians and Surgeons of Columbia University, New York, NY 10314, USA., Wang H; Department of Chemistry, College of Staten Island, 2800 Victory Boulevard, New York, NY 10314, USA., Li Y; Department of Environmental Health Sciences, Mailman School of Public Health of Columbia University, New York, NY 10314, USA., Brandt-Rauf PW; Department of Environmental Health Sciences, Mailman School of Public Health of Columbia University, New York, NY 10314, USA.; School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA 19104, USA. |
| Abstrakt: |
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 µM and 15 µM produced 52% and 75%, cell death, respectively. Comparatively, the monomeric p53 C-terminal peptide-Ant (aa 361−382, termed p53p-Ant), at 15 µM and 30 µM 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 µM). 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. |
