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Fusogenic-oligoarginine peptide-mediated delivery of siRNAs targeting the CIP2A oncogene into oral cancer cells.

Cantini L, Attaway CC, Butler B, Andino LM, Sokolosky ML, Jakymiw A - PLoS ONE (2013)

Bottom Line: However, an obstacle for RNAi-mediated therapies has been delivery, in particular, the retention of small interfering RNAs (siRNAs) in endosomes and their subsequent degradation in lysosomes, resulting in inefficient gene silencing.In fact, quantitative measurements of siRNA delivery into cells demonstrated that a 50∶1 peptide-to-siRNA molar ratio could deliver 18-fold higher amounts of siRNAs compared to cells treated with siRNA alone with no significant long-term cytotoxic effects.Most importantly, the 599 peptide-mediated siRNA delivery promoted significant CIP2A mRNA and protein silencing which resulted in decreased oral cancer cell invasiveness and anchorage-independent growth.

View Article: PubMed Central - PubMed

Affiliation: Depatment of Craniofacial Biology and Center for Oral Health Research, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina, United States of America.

ABSTRACT
Despite a better understanding of the pathogenesis of oral cancer, its treatment outcome remains poor. Thus, there is a need for new therapeutic strategies to improve the prognosis of this disease. RNA interference (RNAi) appears to be a promising therapeutic tool for the treatment of many diseases, including oral cancer. However, an obstacle for RNAi-mediated therapies has been delivery, in particular, the retention of small interfering RNAs (siRNAs) in endosomes and their subsequent degradation in lysosomes, resulting in inefficient gene silencing. Thus, the current study examined the feasibility of designing and utilizing a peptide, termed 599, consisting of a synthetic influenza virus-derived endosome-disruptive fusogenic peptide sequence and a stretch of cationic cell-penetrating nona(D-arginine) residues, to deliver siRNAs into oral cancer cells and induce silencing of the therapeutic target, CIP2A, an oncoprotein overexpressed in various human malignancies including oral cancer. Increasing the 599 peptide-to-siRNA molar ratio demonstrated a higher binding capacity for siRNA molecules and enhanced siRNA delivery into the cytoplasm of oral cancer cells. In fact, quantitative measurements of siRNA delivery into cells demonstrated that a 50∶1 peptide-to-siRNA molar ratio could deliver 18-fold higher amounts of siRNAs compared to cells treated with siRNA alone with no significant long-term cytotoxic effects. Most importantly, the 599 peptide-mediated siRNA delivery promoted significant CIP2A mRNA and protein silencing which resulted in decreased oral cancer cell invasiveness and anchorage-independent growth. Together, these data demonstrate that a chimeric peptide consisting of a fusogenic sequence, in combination with cell-penetrating residues, can be used to effectively deliver siRNAs into oral cancer cells and induce the silencing of its target gene, potentially offering a new therapeutic strategy in combating oral cancer.

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Optimization of 599 peptide binding to siRNAs.An ethidium bromide stained 4% agarose gel shift assay examining the ability of various amounts of the 599 peptide (ranging from 1 to 50-fold molar excess of siRNAs) to form complexes with siCIP2A. siCIP2A, siRNA targeting the CIP2A oncogene; MWM, molecular weight marker (the number of base pairs for each DNA fragment are shown).
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pone-0073348-g001: Optimization of 599 peptide binding to siRNAs.An ethidium bromide stained 4% agarose gel shift assay examining the ability of various amounts of the 599 peptide (ranging from 1 to 50-fold molar excess of siRNAs) to form complexes with siCIP2A. siCIP2A, siRNA targeting the CIP2A oncogene; MWM, molecular weight marker (the number of base pairs for each DNA fragment are shown).

Mentions: In order to test whether the 599 peptide via its cationic nona(D-arginine) residues could effectively bind negatively charged siRNAs based on electrostatic interactions, an agarose gel shift assay was performed (Fig. 1). Using various amounts of the 599 peptide, ranging from 1 to 50-fold molar excess of siRNAs, it was demonstrated that the 599 peptide could indeed bind siRNA molecules designed to target CIP2A (siCIP2A) by retarding the siCIP2A starting at a peptide-to-siRNA molar (P:N) ratio of 20∶1, with no free siRNAs detectable in the gel at 50∶1. At P:N ratios of 10∶1, the peptide only had moderate effects on siRNA binding, and at 1∶1 it was completely ineffective.


Fusogenic-oligoarginine peptide-mediated delivery of siRNAs targeting the CIP2A oncogene into oral cancer cells.

Cantini L, Attaway CC, Butler B, Andino LM, Sokolosky ML, Jakymiw A - PLoS ONE (2013)

Optimization of 599 peptide binding to siRNAs.An ethidium bromide stained 4% agarose gel shift assay examining the ability of various amounts of the 599 peptide (ranging from 1 to 50-fold molar excess of siRNAs) to form complexes with siCIP2A. siCIP2A, siRNA targeting the CIP2A oncogene; MWM, molecular weight marker (the number of base pairs for each DNA fragment are shown).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760901&req=5

pone-0073348-g001: Optimization of 599 peptide binding to siRNAs.An ethidium bromide stained 4% agarose gel shift assay examining the ability of various amounts of the 599 peptide (ranging from 1 to 50-fold molar excess of siRNAs) to form complexes with siCIP2A. siCIP2A, siRNA targeting the CIP2A oncogene; MWM, molecular weight marker (the number of base pairs for each DNA fragment are shown).
Mentions: In order to test whether the 599 peptide via its cationic nona(D-arginine) residues could effectively bind negatively charged siRNAs based on electrostatic interactions, an agarose gel shift assay was performed (Fig. 1). Using various amounts of the 599 peptide, ranging from 1 to 50-fold molar excess of siRNAs, it was demonstrated that the 599 peptide could indeed bind siRNA molecules designed to target CIP2A (siCIP2A) by retarding the siCIP2A starting at a peptide-to-siRNA molar (P:N) ratio of 20∶1, with no free siRNAs detectable in the gel at 50∶1. At P:N ratios of 10∶1, the peptide only had moderate effects on siRNA binding, and at 1∶1 it was completely ineffective.

Bottom Line: However, an obstacle for RNAi-mediated therapies has been delivery, in particular, the retention of small interfering RNAs (siRNAs) in endosomes and their subsequent degradation in lysosomes, resulting in inefficient gene silencing.In fact, quantitative measurements of siRNA delivery into cells demonstrated that a 50∶1 peptide-to-siRNA molar ratio could deliver 18-fold higher amounts of siRNAs compared to cells treated with siRNA alone with no significant long-term cytotoxic effects.Most importantly, the 599 peptide-mediated siRNA delivery promoted significant CIP2A mRNA and protein silencing which resulted in decreased oral cancer cell invasiveness and anchorage-independent growth.

View Article: PubMed Central - PubMed

Affiliation: Depatment of Craniofacial Biology and Center for Oral Health Research, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina, United States of America.

ABSTRACT
Despite a better understanding of the pathogenesis of oral cancer, its treatment outcome remains poor. Thus, there is a need for new therapeutic strategies to improve the prognosis of this disease. RNA interference (RNAi) appears to be a promising therapeutic tool for the treatment of many diseases, including oral cancer. However, an obstacle for RNAi-mediated therapies has been delivery, in particular, the retention of small interfering RNAs (siRNAs) in endosomes and their subsequent degradation in lysosomes, resulting in inefficient gene silencing. Thus, the current study examined the feasibility of designing and utilizing a peptide, termed 599, consisting of a synthetic influenza virus-derived endosome-disruptive fusogenic peptide sequence and a stretch of cationic cell-penetrating nona(D-arginine) residues, to deliver siRNAs into oral cancer cells and induce silencing of the therapeutic target, CIP2A, an oncoprotein overexpressed in various human malignancies including oral cancer. Increasing the 599 peptide-to-siRNA molar ratio demonstrated a higher binding capacity for siRNA molecules and enhanced siRNA delivery into the cytoplasm of oral cancer cells. In fact, quantitative measurements of siRNA delivery into cells demonstrated that a 50∶1 peptide-to-siRNA molar ratio could deliver 18-fold higher amounts of siRNAs compared to cells treated with siRNA alone with no significant long-term cytotoxic effects. Most importantly, the 599 peptide-mediated siRNA delivery promoted significant CIP2A mRNA and protein silencing which resulted in decreased oral cancer cell invasiveness and anchorage-independent growth. Together, these data demonstrate that a chimeric peptide consisting of a fusogenic sequence, in combination with cell-penetrating residues, can be used to effectively deliver siRNAs into oral cancer cells and induce the silencing of its target gene, potentially offering a new therapeutic strategy in combating oral cancer.

Show MeSH
Related in: MedlinePlus