Kelley A. Sookraj

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

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.

The Penetratin Sequence in the Anticancer PNC-28 Peptide Causes Tumor Cell Necrosis Rather Than Apoptosis of Human Pancreatic Cancer Cells

BackgroundPNC-27 and PNC-28 are p53-derived peptides from the human double minute (hdm-2) binding domain attached to penetratin. These peptides induce tumor cell necrosis of cancer cells, but not normal cells. The anticancer activity and mechanism of PNC-28 (p53 aa17–26-penetratin) was specifically studied against human pancreatic cancer.MethodsMiaPaCa-2 cells were treated with PNC-28. Necrosis was determined by measuring lactate dehydrogenase (LDH) and apoptosis as assayed for measuring elevation of proapoptotic proteins. PNC-29, an unrelated peptide, and hdm-2-binding domain p53 aa12-26 without penetratin (PNC-26) were used as controls. Since there is evidence that penetratin is required for tumor cell necrosis, we tested “naked” p53 peptide without penetratin by transfecting a plasmid that encodes p53 aa17–26 segment of PNC-28 into MiaPaCa-2 and an untransformed rat pancreatic acinar cell line, BMRPA1. Time-lapse electron microscopy was employed to further elucidate anticancer mechanism.ResultsTreatment with PNC-28 does not result in the elevation of proapoptotic proteins found in p53-induced apoptosis, but elicits rapid release of LDH, indicative of tumor cell necrosis. Accordingly, we observed membrane pore formation and dose-dependent killing. In direct contrast, transfected MiaPaCa-2 cells underwent apoptosis, and not necrosis, as evidenced by expression of high levels of caspases-3 and 7 and annexin V with background levels of LDH.ConclusionThese results suggest that PNC-28 may be effective in treating human pancreatic cancer. The penetratin sequence appears to be responsible for the fundamental change in the mechanism of action, inducing rapid necrosis initiated by membrane pore formation. Cancer cell death by apoptosis was observed in the absence of penetratin.

The Role of the Assistant in Laparoscopic Surgery: Important Considerations for the Apprentice-in-Training

Laparoscopic surgery is a dynamic and integral component of surgical training. In many surgical programs, the surgeon-in-training gradually incorporates the knowledge and skill-sets through a variable spectrum of assistant/ apprentice instruction with different surgical mentors. As a result, this lack of formal and/or standardized instruction may be inconsistent with a structured educational process. In the year 2008, with widespread applications for minimally invasive techniques and technology, contributions from skilled assistants are now increasingly more important for effective and safe operative conduct. Incorporating these challenges into a balanced educational process remains no easy matter. The authors believe the assistants role is vital to all aspects of laparoscopic surgery, no matter how routine or complex. Laparoscopic assistants should participate and contribute directly in the (a) preoperative evaluation and preparation, ( b) patient positioning, (c) operative suite arrangement, ( d) trocar placement, plus important (e) intraoperative maneuvers contingent upon acquired mastery of laparoscopic skills. Understanding these principles plus effective administration of various duties allows for the apprentice in training to progress to more complex procedures and eventual primary surgeon responsibility. In this report, the role of the laparoscopic assistant/apprentice is reviewed, with particular attention focused on requisite fundamentals for evolving laparoscopic surgeons. To date, there are few publications within the world literature that directly address these observations. Important considerations delineating the expectations and goals for the assistant/apprentice, as well as the mentor, during laparoscopic training are provided.

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

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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.

Abstract 5770: MDM2 protein variants expression in the plasma membrane of cancer cells: A target for anticancer peptide PNC-27

PNC-27 anti-cancer peptide, derived from the MDM2 binding site of p53 and linked to a membrane residency peptide (MRP), has been shown to cause necrosis of cancer cells without affecting untransformed cells. PNC-27 was also able to eradicate pancreatic tumor xenografts in mice (Michl et al, Int. J of Cancer, 2006). Recently, we have identified the mechanism of action of this peptide as due to formation of oligomeric pores in the plasma membrane (PM) of cancer cells but not in the PM of untransformed cells. The mechanism of pore-formation by PNC-27 closely resembles the pore-formation process by streptolysin-O, melittin and similar pore-forming agents. We have shown MDM2 as a targeting molecule that leads to PNC-27 selectivity towards cancer cells by its mis-localization to cancer cell PM (Sarafraz-Yazdi et al, PNAS, in press). Examining purified PM of a variety of cancer cells by immunoblotting, we now provide evidence for multiple MDM2 protein variants, and that were absent in the PM of normal untransformed cells. To confirm the purity of the isolated PM, fractions were also immunoblotted for the PM markers Na+/K+-ATPase, E-Cadherin and s-Catenin, all of which were enriched in the PM fractions. In contrast, Abs against membrane markers specific for intracellular organelles, COX IV and Cytochrome C for mitochondrial membrane, showed no reaction in the PM fractions while they reacted with total membrane fractions. Three protein variants of MDM2 with MW of 27kD, 40kD and 57kD were consistently expressed in the PM of human and rat pancreatic cancer cells, human melanoma, breast cancer. We also confirmed these observations in isolated PM from freshly obtained primary ovarian tumors from human patients with aggressive tumor. Remarkably, no MDM2 variant was detectable in the PM fractions of primary human fibroblasts and untransformed pancreatic HPNE cells. Our finding of differently sized MDM2 variants complies with other, previously reported MDM2 protein variants. Of the different MDM2 mRNA splice variants published, 5 have been shown to be translated into protein including proteins of 27kD, 40kD and 57kD. Our present study demonstrates the localization of the 3 variant proteins in the PM of cancer cells, suggesting a possible role for these membrane-localized MDM2 variant in the action of PNC-27. The notion of this specific MDM2-PNC-27 interaction at the PM level was strongly supported by the effective competition by a monoclonal MDM2-specific Ab, reducing PNC-27-mediated cytotoxicity by >80% as measured by LDH cytotoxicity assay and propidium iodide staining of the nuclei of dead cells in real-time spinning disc confocal microscopy. These findings for the first time demonstrate not only the novel localization of different MDM2 variants in PM of different cancer cells but they also suggest a potentially wider applicability of PNC-27 as a novel selective anti-cancer drug for clinical trials. 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 5770.