title: The anti-cancer peptide, PNC-27, induces tumor cell necrosis of a poorly differentiated non-solid tissue human leukemia cell line that depends on expression of HDM-2 in the plasma membrane of these cells.
abstract: OBJECTIVE
  We have developed the anti-cancer peptide, PNC-27, which is a membrane-active peptide that binds to the HDM-2 protein expressed in the cancer cell membranes of solid tissue tumor cells and induces transmembrane pore formation in cancer, but not in normal cells, resulting in tumor cell necrosis that is independent of p53 activity in these cells. We now extend our study to non-solid tissue tumor cells, in this case, a primitive, possible stem cell human leukemia cell line (K562) that is also p53-homozygously deleted. Our purpose was twofold: to investigate if these cells likewise express HDM-2 in their plasma membranes and to determine if our anti-cancer peptide induces tumor cell necrosis in these non-solid tissue tumor cells in a manner that depends on the interaction between the peptide and membrane-bound HDM-2.
  
  
  METHODS
  The anti-cancer activity and mechanism of PNC-27, which carries a p53 aa12-26-leader sequence connected on its carboxyl terminal end to a trans-membrane-penetrating sequence or membrane residency peptide (MRP), was studied against p53-null K562 leukemia cells. Murine leukocytes were used as a non-cancer cell control. Necrosis was determined by measuring the lactate dehydrogenase (LDH) release and apoptosis was determined by the detection of Caspases 3 and 7. Membrane colocalization of PNC-27 with HDM-2 was analyzed microscopically using fluorescently labeled antibodies against HDM-2 and PNC-27 peptides.
  
  
  RESULTS
  We found that K562 cells strongly express HDM-2 protein in their membranes and that PNC-27 co-localizes with this protein in the membranes of these cells. PNC-27, but not the negative control peptide PNC-29, is selectively cytotoxic to K562 cells, inducing nearly 100 percent cell killing with LDH release. In contrast, this peptide had no effect on the lymphocyte control cells.
  
  
  CONCLUSIONS
  The results suggest that HDM-2 is expressed in the membranes of non-solid tissue tumor cells in addition to the membranes of solid tissue tumor cells. Since K-562 cells appear to be in the stem cell family, the results suggest that early developing tumor cells also express HDM-2 protein in their membranes. Since PNC-27 induces necrosis of K-562 leukemia cells and co-localizes with HDM-2 in the tumor cell membrane as an early event, we conclude that the association of PNC-27 with HDM-2 in the cancer cell membrane results in trans-membrane pore formation which results in cancer cell death, as previously discovered in a number of different solid tissue tumor cells. Since K562 cells lack p53 expression, these effects of PNC-27 on this leukemia cell line occur by a p53-independent pathway.
authors.affiliation: Department of Surgery, Drexel University College of Medicine, Philadelphia, PA.
authors.forename: Katlin
authors.initials: K
authors.lastname: Davitt
authors.affiliation: Department of Surgery, Drexel University College of Medicine, Philadelphia, PA.
authors.forename: Blake D
authors.initials: BD
authors.lastname: Babcock
authors.affiliation: Department of Surgery, SUNY Downstate Medical Center, Brooklyn, NY.
authors.forename: Maly
authors.initials: M
authors.lastname: Fenelus
authors.affiliation: Department of Surgery, SUNY Downstate Medical Center, Brooklyn, NY.
authors.forename: Chi Kong
authors.initials: CK
authors.lastname: Poon
authors.affiliation: Department of Surgery, SUNY Downstate Medical Center, Brooklyn, NY.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Sarkar
authors.affiliation: Department of Surgery, SUNY Downstate Medical Center, Brooklyn, NY.
authors.forename: Vincent
authors.initials: V
authors.lastname: Trivigno
authors.affiliation: Department of Surgery, SUNY Downstate Medical Center, Brooklyn, NY.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Zolkind
authors.affiliation: Department of Surgery, SUNY Downstate Medical Center, Brooklyn, NY.
authors.forename: Sheena M
authors.initials: SM
authors.lastname: Matthew
authors.affiliation: Division of Pharmacology and Physiology, Department of Microbiology and Anatomy and Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY.
authors.forename: Natalia
authors.initials: N
authors.lastname: Grin'kina
authors.affiliation: Department of Surgery, Drexel University College of Medicine, Philadelphia, PA.
authors.forename: Zulfiya
authors.initials: Z
authors.lastname: Orynbayeva
authors.affiliation: Department of Surgery, Drexel University College of Medicine, Philadelphia, PA.
authors.forename: Mohammad F
authors.initials: MF
authors.lastname: Shaikh
authors.affiliation: Department of Surgery, Drexel University College of Medicine, Philadelphia, PA.
authors.forename: Victor
authors.initials: V
authors.lastname: Adler
authors.affiliation: Department of Pathology, SUNY Downstate Medical Center, Brooklyn, NY Department of Microbiology and Anatomy and Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY.
authors.forename: Josef
authors.initials: J
authors.lastname: Michl
authors.affiliation: Department of Pathology, SUNY Downstate Medical Center, Brooklyn, NY.
authors.forename: Ehsan
authors.initials: E
authors.lastname: Sarafraz-Yazdi
authors.affiliation: Department of Pathology, SUNY Downstate Medical Center, Brooklyn, NY Department of Pathology and Laboratory Medicine, New York Harbor VA Medical Center, Brooklyn, NY and New York, NY, USA.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Pincus
authors.affiliation: Department of Surgery, Drexel University College of Medicine, Philadelphia, PA Department of Surgery, SUNY Downstate Medical Center, Brooklyn, NY wilbur.bowne@drexelmed.edu matthew.pincus2@med.va.gov.
authors.forename: Wilbur B
authors.initials: WB
authors.lastname: Bowne
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D020014:K562 Cells; D051379:Mice; D018613:Microscopy, Confocal; D011485:Protein Binding / Q000187:drug effects; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics* / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; C549751:PNC-27; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2
keywords: PNC-27; apoptosis; cancer; leukemia; necrosis; p53-derived peptide