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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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Both the endosome-disruptive and cationic cell-penetrating nona(D-arginine) residue portions of the 599 peptide are required for siRNA delivery into oral cancer cells.Fluorescence microscopy analyses of CAL 27 oral cancer cells incubated for 2 hours with DY547-conjugated siRNA targeting CIP2A (D-siCIP2A; red) complexed to peptides 599, 616, and 9R at a 50∶1 peptide-to-siRNA molar ratio. Nuclei (blue) were counterstained with DAPI. Scale bar: 50 µm.
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pone-0073348-g005: Both the endosome-disruptive and cationic cell-penetrating nona(D-arginine) residue portions of the 599 peptide are required for siRNA delivery into oral cancer cells.Fluorescence microscopy analyses of CAL 27 oral cancer cells incubated for 2 hours with DY547-conjugated siRNA targeting CIP2A (D-siCIP2A; red) complexed to peptides 599, 616, and 9R at a 50∶1 peptide-to-siRNA molar ratio. Nuclei (blue) were counterstained with DAPI. Scale bar: 50 µm.

Mentions: To test the importance of the two core components (i.e. the endosome-disruptive and cationic cell-penetrating nona(D-arginine) residue portions) in conferring the ability of the 599 peptide to deliver siRNAs into cells, two additional peptides were synthesized consisting of either the endosome-disruptive sequence (616) or the cationic cell-penetrating nona(D-arginine) residues alone (9R). Upon treatment of CAL 27 cells with either the 599, 616, or 9R peptides in complex with D-siCIP2A at a 50∶1 P:N ratio, fluorescence microscopy analyses revealed that only the intact 599 peptide with both the endosome-disruptive and cationic cell-penetrating nona(D-arginine) residue portions could mediate delivery of siRNAs into cells, whereas both the 616 and 9R peptides had no apparent effect on siRNA delivery 2 hours post-treatment (Fig. 5).


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)

Both the endosome-disruptive and cationic cell-penetrating nona(D-arginine) residue portions of the 599 peptide are required for siRNA delivery into oral cancer cells.Fluorescence microscopy analyses of CAL 27 oral cancer cells incubated for 2 hours with DY547-conjugated siRNA targeting CIP2A (D-siCIP2A; red) complexed to peptides 599, 616, and 9R at a 50∶1 peptide-to-siRNA molar ratio. Nuclei (blue) were counterstained with DAPI. Scale bar: 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073348-g005: Both the endosome-disruptive and cationic cell-penetrating nona(D-arginine) residue portions of the 599 peptide are required for siRNA delivery into oral cancer cells.Fluorescence microscopy analyses of CAL 27 oral cancer cells incubated for 2 hours with DY547-conjugated siRNA targeting CIP2A (D-siCIP2A; red) complexed to peptides 599, 616, and 9R at a 50∶1 peptide-to-siRNA molar ratio. Nuclei (blue) were counterstained with DAPI. Scale bar: 50 µm.
Mentions: To test the importance of the two core components (i.e. the endosome-disruptive and cationic cell-penetrating nona(D-arginine) residue portions) in conferring the ability of the 599 peptide to deliver siRNAs into cells, two additional peptides were synthesized consisting of either the endosome-disruptive sequence (616) or the cationic cell-penetrating nona(D-arginine) residues alone (9R). Upon treatment of CAL 27 cells with either the 599, 616, or 9R peptides in complex with D-siCIP2A at a 50∶1 P:N ratio, fluorescence microscopy analyses revealed that only the intact 599 peptide with both the endosome-disruptive and cationic cell-penetrating nona(D-arginine) residue portions could mediate delivery of siRNAs into cells, whereas both the 616 and 9R peptides had no apparent effect on siRNA delivery 2 hours post-treatment (Fig. 5).

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