Inhibition of ovarian cancer cell proliferation by a cell cycle inhibitory peptide fused to a thermally responsive polypeptide carrier.
Current treatment of solid tumors is limited by normal tissue tolerance, resulting in a narrow therapeutic index. To increase drug specificity and efficacy and to reduce toxicity in normal tissues, we have developed a polypeptide carrier for a cell cycle inhibitory peptide, which has the potential to be thermally targeted to the tumor site. The design of this polypeptide is based on elastin-like polypeptide (ELP). The coding sequence of ELP was modified by the addition of the cell penetrating peptide Bac-7 at the N-terminus and a 23 amino acid peptide derived from p21 at the C-terminus (Bac-ELP1-p21). Bac-ELP1-p21 is soluble in aqueous solutions below physiological temperature (37 degrees C) but aggregates when the temperature is raised above 39 degrees C, making it a promising thermally responsive therapeutic carrier that may be actively targeted to solid tumors by application of focused hyperthermia. While Bac-ELP1-p21 at 37 degrees C did not have any effect on SKOV-3 cell proliferation, the use of hyperthermia increased the antiproliferative effect of Bac-ELP1-p21 compared with a thermally unresponsive control polypeptide. Bac-ELP1-p21 displayed both a cytoplasmic and nuclear distribution in the SKOV-3 cells, with nuclear-localized polypeptide enriched in the heated cells, as revealed by confocal microscopy. Using Western blotting, we show that Bac-ELP1-p21 caused a decrease in Rb phosphorylation levels in cells treated at 42 degrees C. The polypeptide also induced caspase activation, PARP cleavage, and cell cycle arrest in S-phase and G2/M-phase. These studies indicate that ELP is a promising macromolecular carrier for the delivery of cell cycle inhibitory peptides to solid tumors.
Massodi
Iqbal
I
Department of Biochemistry, University of Mississippi Medical Center, Jackson, 39216, USA.
Moktan
Shama
S
Rawat
Aruna
A
Bidwell
Gene L
GL
Raucher
Drazen
D
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000737:chemistry / Q000235:genetics; D004549:Elastin / Q000737:chemistry / Q000235:genetics; D005260:Female; D006358:Hot Temperature; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D016160:Retinoblastoma Protein / Q000378:metabolism; D044366:Transition Temperature
D050759:Cyclin-Dependent Kinase Inhibitor p21; D010455:Peptides; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D004549:Elastin; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases