We have previously shown that two novel peptides, PNC-27 and PNC-28, that contain p53 hdm2 binding domain sequences 12-26 and 17-26, respectively, and are attached to penetratin, a leader sequence from the antennapedia protein, effectively kill a wide variety of cancer cells in vitro (Kanovsky, M et al, PNAS 98, 2001; Do, T et al, Oncogene 22, 2002) and in vivo (Michl, J et al, IJC 119, 2006). Remarkably, neither peptide affects growth of normal untransformed cells nor hematopoiesis. We further demonstrated that these peptides induce plasma membrane lysis resulting in cancer cell necrosis, not apoptosis. We now show that immunoblotting of cell lysates from treated cancer cells suggests a specific binding-dependent mechanism that is not present in normal cells. Moreover, a double fluorescent-labeled peptide (amino terminal FITC and carboxyl terminal TRITC) was found exclusively on cell membranes of PNC-27- and PNC-28-treated cancer but not normal cells. Furthermore, we identified the presence of mdm2/hdm2 in cancer cell plasma membranes otherwise not detected in normal cell plasma membranes. Confocal microscopy of peptide-treated and -untreated cancer cells demonstrated strong staining with a TRITC-labeled mAb to mdm2/hdm2. Cancer cells that had been treated (5-15min) with PNC-27 also showed strong membrane staining with FITC-anti-PNC-27 Ab which co-localized with the TRITC-mAb to mdm2/hdm2 and further suggested PNC-27 membrane interaction with mdm2/hdm2. In contrast, staining for PNC-27 was absent in normal and cancer cells treated with a control peptide, PNC-29. Ultrastructural studies confirmed these results: Immunostaining with gold-conjugated Ab demonstrated the co-localization of PNC-27 and mdm2/hdm2 on the plasma membranes of cancer cells whereas in untreated cancer cells only mdm2/hdm2 was observed. With longer PNC-27 treatment (30min) the disruption of the cells' plasma membranes and mitochondria was noted whereas the nuclear membranes remained intact, an indication of necrosis. In contrast, none of these morphological changes was observed in untreated cancer and cancer cells treated with control peptide. We further found in cancer cell plasma membranes oligomeric PNC-27 and hdm2 in ring-shaped structures similar to those seen in studies with Streptolysin-O (SLO), a well-known pore-forming toxin. The similarity of PNC-27-hdm2 "pores" to SLO pores suggests an important cause of rapid cancer cell death. The pore formation of PNC-27 in cancer cells is likely to be achieved by the direct interaction of PNC-27 via its hdm2/mdm2-specific binding site with the hdm2/mdm2 molecules that are expressed only in cancer cell plasma membrane. We propose PNC-27/-28 binds to the hdm2/mdm2 protein in cancer cell membrane enabling several PNC-molecules to associate to form membrane-penetrating pores leading to cell death.
99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA