Abstract 729: Mechanism of action of a p53-derived peptide that targets a novel death pathway inducing selective necrosis in cancer cells

Abstract

Abstract In a series of preliminary in vitro and in vivo studies we have shown that p53-derived peptides consisting of residues 12-26 and 17-26 from the murine double minute binding domain (MDM-2) possess anti-cancer activity. These peptides are linked to a trans-membrane penetrating sequence (penetratin) derived from the antennapedia homeodomain of Drosophila and are designated PNC-27 and PNC-28, respectively. We also found that the previously determined 3D structure of these PNC residues are directly superimposeable on the same residues bound to MDM-2, suggesting that these peptide may target MDM-2 in cancer cells. These peptide constructs appear to promote cancer cell death through a novel pathway that leads to necrosis rather than apoptosis while sparing their untransformed counterparts. In our initial experiments, the critical role of the MDM-2 oncoprotein as a potential target molecule for these PNC peptides was elucidated when a sequence specific MDM-2 antibody blocked PNC-27 activity on cancer cells. To further elucidate this anti-cancer mechanism we then tested parent molecule PNC-27 against selected cancer cell lines (Panc-02, pancreatic; HT1080, fibrosarcoma; and MCF-7, breast) plus respective untransformed counterparts (BMRPA1, BJ, and MCF-10) which were treated with PNC-27 and control peptide. Subsequently, treatment with fluorescent labeled peptide, immunoprecipitation (IP), confocal, and time-lapse electron microscopy (EM) was employed as well as transfection of untransformed cells with the MDM-2 gene to study the anti-cancer mechanism. PNC-27 did not cause elevation of pro-apoptotic proteins but induced rapid dose-dependent tumor cell death with release of lactate dehydrogenase (LDH). This specific anti-cancer mechanism, in part, may be due to observed increased expression of MDM-2 found in cancer cells compared to their normal cellular counterparts which is consistent with abrogation of MDM-2 and stabilization of p53 protein levels after PNC-27 treatment. Moreover, following IP of fluorescent labeled PNC-27 from treated cancer cells; labeled MDM-2 was detected. Furthermore, confocal microscopy and EM revealed co-localization of PNC-27 and MDM-2 along with cancer cell membrane pore formation, respectively. Remarkably, transfection of the MDM-2 gene resulted with increased expression of MDM-2 and now untransformed cell susceptibility to PNC-27. These results suggest a mechanism of PNC-27 binding to MDM-2 in the cancer cell membrane, leading to pore formation and membrane disruption; a p53-independent pathway consistent with rapid selective cancer cell necrosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 729.