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Creation of a novel peptide with enhanced nuclear localization in prostate and pancreatic cancer cell lines
H Dan Lewis1, Ali Husain, Robert J Donnelly
1Department of Surgery, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 S, Orange Avenue, Newark, NJ 07103, USA.
Background:
For improved uptake of oligonucleotide-based therapy, the oligonucleotides often are coupled to peptides that facilitate entry into cells. To this end, novel cell-penetrating peptides (CPPs) were designed for mediating intracellular uptake of oligonucleotide-based therapeutics. The novel peptides were based on taking advantage of the nuclear localization properties of transcription factors in combination with a peptide that would bind putatively to cell surfaces. It was observed that adding a glutamate peptide to the N-terminus of the nuclear localization signal (NLS) of the Oct6 transcription factor resulted in a novel CPP with better uptake and better nuclear colocalization than any other peptide tested.
Results:
Uptake of the novel peptide Glu-Oct6 by cancer cell lines was rapid (in less than 1 hr, more than 60% of DU-145 cells were positive for FITC), complete (by 4 hr, 99% of cells were positive for FITC), concentration-dependent, temperature-dependent, and inhibited by sodium azide (NaN3). Substitution of Phe, Tyr, or Asn moieties for the glutamate portion of the novel peptide resulted in abrogation of novel CPP uptake; however none of the substituted peptides inhibited uptake of the novel CPP when coincubated with cells. Live-cell imaging and analysis by imaging flow cytometry revealed that the novel CPP accumulated in nuclei. Finally, the novel CPP was coupled to a carboxyfluorescein-labeled synthetic oligonucleotide, to see if the peptide could ferry a therapeutic payload into cells.
Conclusions:
These studies document the creation of a novel CPP consisting of a glutamate peptide coupled to the N-terminus of the Oct6 NLS; the novel CPP exhibited nuclear colocalization as well as uptake by prostate and pancreatic cancer cell lines.
Insights
Researchers developed a novel cell-penetrating peptide (CPP) by modifying the Oct6 transcription factor. This new CPP, Glu-Oct6, shows enhanced cellular uptake and nuclear delivery for oligonucleotide therapeutics.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Delivery
Background:
- Oligonucleotide-based therapies require efficient cellular uptake, often facilitated by cell-penetrating peptides (CPPs).
- Novel CPPs were designed by combining nuclear localization signals (NLS) with cell-surface binding peptides.
- Modification of the Oct6 transcription factor's NLS with a glutamate peptide yielded a promising new CPP.
Purpose of the Study:
- To design and characterize a novel CPP for enhanced intracellular delivery of oligonucleotide therapeutics.
- To evaluate the uptake efficiency and intracellular localization of the novel CPP.
Main Methods:
- Synthesis of a novel CPP (Glu-Oct6) by conjugating a glutamate peptide to the Oct6 NLS.
- In vitro assessment of CPP uptake in cancer cell lines using fluorescence microscopy and flow cytometry.
- Analysis of uptake kinetics, temperature dependence, and inhibition by sodium azide.
- Live-cell imaging and imaging flow cytometry for nuclear accumulation studies.
- Coupling of the CPP to a labeled oligonucleotide to test therapeutic payload delivery.
Main Results:
- The novel CPP, Glu-Oct6, demonstrated rapid and complete cellular uptake in cancer cell lines (e.g., DU-145).
- Uptake was concentration-dependent, temperature-dependent, and inhibited by sodium azide, indicating an active process.
- Substitution of the glutamate moiety abrogated CPP uptake, confirming its critical role.
- Live-cell imaging confirmed nuclear accumulation of the novel CPP.
- The CPP successfully delivered a labeled oligonucleotide into cells.
Conclusions:
- A novel CPP was successfully created by linking a glutamate peptide to the Oct6 NLS.
- The novel CPP exhibits efficient cellular uptake and nuclear colocalization in prostate and pancreatic cancer cell lines.
- This CPP holds potential for delivering oligonucleotide-based therapeutics into target cells.
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