Anticancer peptide PNC-27 adopts an HDM-2-binding conformation and kills cancer cells by binding to HDM-2 in their membranes

Abstract

The anticancer peptide PNC-27, which contains an HDM-2-binding domain corresponding to residues 12-26 of p53 and a transmembrane-penetrating domain, has been found to kill cancer cells (but not normal cells) by inducing membranolysis. We find that our previously determined 3D structure of the p53 residues of PNC-27 is directly superimposable on the structure for the same residues bound to HDM-2, suggesting that the peptide may target HDM-2 in the membranes of cancer cells. We now find significant levels of HDM-2 in the membranes of a variety of cancer cells but not in the membranes of several untransformed cell lines. In colocalization experiments, we find that PNC-27 binds to cell membrane-bound HDM-2. We further transfected a plasmid expressing full-length HDM-2 with a membrane-localization signal into untransformed MCF-10-2A cells not susceptible to PNC-27 and found that these cells expressing full-length HDM-2 on their cell surface became susceptible to PNC-27. We conclude that PNC-27 targets HDM-2 in the membranes of cancer cells, allowing it to induce membranolysis of these cells selectively.

Anti-cancer Peptides from Ras-P21 and P53 Proteins

“…Also, there is a prominent red fluorescence in the membrane of these cells showing that HDM-2 is present in these membranes. Combined yellow fluorescence (green and red) is prominent, indicating that PNC-27 colocalizes with HDM-2 in the cancer cell membrane [79]. Very similar results were obtained using MIA-PaCa-2 human pancreatic cancer cells, MCF-7 human breast cancer cells [79] and a primary human ovarian cancer (E Yazdi, MR Pincus and J Michl, manuscript in preparation).…”

“…We have found that each of our structures for residues 17-26 of PNC-27 superimpose closely on the structure for the corresponding residues in the x-ray structure [79]. This superposition occurs despite the fact that the peptide in the x-ray structure is bound to MDM-2, and PNC-27 is present free in solution and the fact that the peptide in the x-ray structure contains three extra p53 residues while PNC-27 instead contains the MRP which the peptide in the x-ray structure does not contain.…”

“…Shown as controls is the expression of HDM-2 in whole cell lysates. Expression of HDM-2 in whole cell lysates is significant for both cell types although lower in the untransformed cell lines [79]. Thus cancer cells express HDM-2 in their cell membranes while normal cells express this protein at much lower or, in some cases, at undetectable levels in their membranes.…”

“…A B Figure 18 shows the results of membrane fraction blotting of cancer and normal cells [79]. It may at once be seen that HDM-2 is expressed at many-fold greater levels in cancer cell than in normal cell membranes.…”

“…Thus cancer cells express HDM-2 in their cell membranes while normal cells express this protein at much lower or, in some cases, at undetectable levels in their membranes. Figure 19 shows that PNC-27 binds to the plasma cell membranes of cancer cells (green fluorescence) using the A-2058 human melanoma cell line as an example [79]. Also, there is a prominent red fluorescence in the membrane of these cells showing that HDM-2 is present in these membranes.…”