# Peptides That Block RAS-p21 Protein-Induced Cell Transformation
## 2 Anti-Onogenic RAS-p21 Peptides from RAS-p21 Itself
### Effects of PNC-2 and PNC-7 in Cancer and Normal Cells  
Both PNC-2 and PNC-7 were synthesized and attached on their carboxyl termini to a leader transmembrane-penetrating sequence (KKWKMRRNQFVKVQRG) from _Drosophila_ antennapedia protein that enables the peptides to be transported across cell membranes--referred to as cell-penetrating peptides or CPP [24]. As discussed in ref. [24] and references therein, CPPs are short peptides or, in some cases, whole proteins such as the drosophila _Antennapedia_ protein, that cause peptides or whole proteins or other types of molecules bound to them, usually covalently but sometimes non-covalently, to cross cell and nuclear membranes. After entry into the cell, the CPPs are often cleaved, allowing for their cargo to exert their effects intracellularly. Generally, CPPs contain a high percentage of positively charged amino acid residues, such as Lys, Arg and sometimes His. Other types of CPPs contain a predominance of non-polar, hydrophobic amino acids such as Leu, Ile,Val, Phe and Met or alternating segments of polar and non-polar residues. The mechanisms by which trans-membrane penetration occurs are largely unknown but are thought to involve endocytosis, using a variety of possible mechanisms such as pore formation from interactions of the positively charged amino acids with negatively charged membrane moieties such as phospholipids and heparan sulfates and formation of inverted micelles, among a number of other proposed mechanisms. These two modified peptides were incubated with a normal pancreatic acinar cell line (BMRPA1) and its k-ras (G12V) transformed counterpart cell line TUC-3. The transplantation of TUC-3 but not BMRPA1 cells in nude mice resulted in metastatic pancreatic cancer with peritoneal carcinomas [25]. Incubation of these peptides with TUC-3 cells resulted  
Figure 3: Ribbon representation of the superimposed average molecular dynamics RAF RBD structures bound to oncogenic (Val 12-) RAS-p21 (blue and red structure) and to wild-type RAS-p21 (pink and green). The exposed loop on the top surface of the figure of the 97–110 segment is colored green in the wild-type complex and red in the oncogenic complex (adapted from [15]). in complete phenotypic reversion. The same results were obtained on human fibrosarcoma (HT1080) cells [25; 26]. Neither peptide had any effect on the growth or viability of BMRPA1 normal acinar cells, indicating that the effects of these peptides were specific to cancer cells. Interestingly, both peptides induced the tumor cell necrosis of MIA-PaCa-2 cells, a human metastatic pancreatic cancer cell line, homozygous for the oncogenic G12C mutation (see below) [25]. These results were attributed to the zygosity of the different cell lines. Both TUC-3 and HT1080 cells are heterozygous for oncogenic RAS-p21. It was postulated that, if a blockade of the oncogenic pathway occurs, a normal RAS-p21 protein can utilize alternate pathways, resulting in reversion to the untransformed phenotype. Additionally, since MIA-PaCa-2 cells are homozygous for oncogenic RAS-p21 and do not have normally functioning RAS-p21, a blockade of the oncogenic pathway blocks cell growth, resulting in cell death. In all cancer cell lines treated with either peptide, JNK-P levels were continuously reduced as a function of time, as found in the oocyte studies [15; 25]. These results suggest that these peptides may be effective in treating RAS-p21-induced cancers with no off-target effects on normal cells. Peptides from Proteins That Interact Directly with RAS-p21 or Selectively Block Targets on the Oncogenic RAS-p21 Pathway  
A comparison of the average structures for G12V-RAS-p21-GppNH-p and wild-type RAS-p21-GppNHp bound to the ras-binding domain (RBD) of RAF revealed a major 90\({}^{\circ}\) flip in a surface loop involving in the RBD at its 97-110 sequence, as shown in Figure 3. The peptide corresponding to this domain (AVFRLLHEHKGKKA) was found to inhibit G12V-RAS-p21 selectively in the oocyte system.