The anti-cancer peptide, PNC-27, induces tumor cell lysis as the intact peptide.
OBJECTIVE
PNC-27, a peptide that contains an HDM-2-binding domain from p53 attached to a membrane-penetrating peptide on its carboxyl terminal end, is cytotoxic to cancer, but not normal, cells. It forms transmembrane pores in the cancer cell membrane. Our purpose is to determine if the whole peptide or critical fragments induce pore formation in cancer cells.


METHODS
We have prepared PNC-27 with a green fluorescent label on its amino terminus and a red fluorescent label on its carboxyl terminus and treated MCF-7 breast cancer cells and untransformed MCF-10-2A breast epithelial cells with this double-labeled peptide to determine if combined yellow fluorescence occurs in the membrane of the cancer cells during cancer cell killing.


RESULTS
At 30 min, there is significant combined punctate yellow fluorescence, indicative of intact peptide, in the cell membrane of cancer cells that increases during cancer cell lysis. MCF-10-2A cells show initial (30 min) uniform combined yellow membrane fluorescence that subsequently disappears. Unlike the cancer cells, these untransformed cells remain viable.


CONCLUSIONS
PNC-27 induces cancer cell membrane lysis by acting as the whole peptide, not fragments. The punctate yellow fluorescence is due to interaction of PNC-27 with intramembrane targets of MCF-7 cells that do not exist in the membrane of the untransformed cell line. This interaction increases the lifetime of PNC-27. Absence of these targets in the membranes of the untransformed MCF-10-2A cells results in initial uniform fluorescence of the double-labeled peptide in their membranes after which the peptide is degraded.
Sookraj
Kelley A
KA
Department of Surgery, SUNY Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA.
Bowne
Wilbur B
WB
Adler
Victor
V
Sarafraz-Yazdi
Ehsan
E
Michl
Josef
J
Pincus
Matthew R
MR
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000473:pathology / Q000648:ultrastructure; D004396:Coloring Agents; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D018613:Microscopy, Confocal; D009336:Necrosis / Q000473:pathology; D009369:Neoplasms / Q000188:drug therapy* / Q000473:pathology; D012235:Rhodamines; D013778:Tetrazolium Salts; D013844:Thiazoles; D016159:Tumor Suppressor Protein p53 / Q000494:pharmacology*
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D000970:Antineoplastic Agents; D004396:Coloring Agents; D005456:Fluorescent Dyes; C549751:PNC-27; D012235:Rhodamines; D013778:Tetrazolium Salts; D013844:Thiazoles; D016159:Tumor Suppressor Protein p53; D049452:Green Fluorescent Proteins; D007770:L-Lactate Dehydrogenase; D020169:Caspases; C022616:thiazolyl blue; D016650:Fluorescein-5-isothiocyanate