Abstract #884: Mechanism of action of PNC-27/-28 anti-cancer peptides

In vivo and in vitro studies have shown the efficiency of anti-cancer peptides PNC-27 and PNC-28 in killing cancer cells by necrosis without affecting untransformed normal cells (Kanovsky et al., PNAS 2001; Do T et al., Oncogene 2002; Michl et al, IJC, 2006). The peptides were derived from the HDM2-binding domain of p53 (PNC-27: aa12-26 and PNC-28: aa17-26) and linked to a leader sequence from the Drosophila antennapedia homeodomain protein. In contrast, over-expression intra-cellularly of the aa17-26 sequence kills tumor cells by apoptosis but not by necrosis (Bowne et al., Ann. Surg. Onc. 2008). The critical role of HDM2 as the target molecule for PNC-27 in the tumor cells\#8217; plasma membrane was confirmed by successful competition of a hdm2-specific antibody against the binding of PNC-27 to intact tumor cells and prevention of cytotoxicity as evident by the absence of lactate dehydrogenase (LDH) release. We now show that PNC-27-induced cytotoxicity is temperature sensitive, since LDH is released from cells treated with PNC-27 (LD50 at 0.035\#956;M) at 37°C, with reduced efficiency from cells treated at 25°C, but not at all by cells expsoed to PNC-27 at or below 17°C. The sequence of cellular events following the exposure of tumor cells to PNC-27 was established with spinning disc confocal microscopy of human pancreatic cancer MIA-Paca-2 cells that were preloaded with mitotracker dye: Within <3min upon addition of PNC-27 to the propidium iodide-containing (PI) medium the expansion of the cells\#8217; plasma membrane was followed within 3-6min by the disruption of the mitochondria and the disappearance of the dye, which occurred just prior (6-10min) to the appearance of PI in the cells' nuclei. Immuno-gold transmission electron microscopy (TEM) showed that gold-conjugated antibodies to HDM2 and PNC-27 decorated ring-like structures in the plasma membrane of tumor cells, but not normal cells, that had been exposed for 5min to PNC-27, further confirming the co-localization of HDM2 and PNC-27 in tumor cell plasma membrane. Scanning electron microscopy (SEM) demonstrated that within 3min of adding PNC-27 large numbers of deposits, many arranged in pore-forming aggregates, had formed on the tumor cell plasma membrane, while deposits were not present on the membrane of normal cells. The results strongly suggest, that PNC-27 (and by inference PNC-28) is inserted in the plasma membrane of cancer cells upon binding to HDM2, where, by random movement, the HDM2-PNC-27 complexes assemble into oligomers and eventually into membrane pores. Influx of free PNC-27 molecules through the newly formed membrane pores into the cells\#8217; cytoplasm leads to the assembly of pores in mitochondrial membranes and rapid cell death. Absence of HDM2 in the plasma membrane of normal untransformed cells prevents the formation of membrane pores and, thus, cell death.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 884.