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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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Characterization of 599 peptide-mediated RNAi responses in oral cancer cells.(A) Western blot analyses of CIP2A protein expression levels in CAL 27 and SCC-25 oral cancer cells 1, 3, 5, 7, and 9 days post-treatment with 599 peptide complexed to either 100 nM of control non-targeting siRNA (siNT) or siRNA targeting CIP2A (siCIP2A) at a 50∶1 peptide-to-siRNA molar ratio. GAPDH protein levels were monitored to ensure equal loading of samples. (B) Western blot analysis of CIP2A protein knockdown in SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide alone (5 µM) or complexed at a 50∶1 peptide-to-siRNA molar ratio to either siNT (100 nM) or decreasing concentrations of siCIP2A. The CIP2A protein levels in untreated cells were also analyzed for comparison. GAPDH protein levels were monitored to ensure equal loading of samples. (C) Western blot analyses of CIP2A protein knockdown in SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide complexed to either 100 nM of siNT or siCIP2A at a 50∶1 peptide-to-siRNA molar ratio, in the presence (+) or absence (-) of serum, compared to untreated cells or cells treated with commercial transfection reagents Lipofectamine® RNAiMAX (RNAiMAX), Lipofectamine® 2000 (LF2000), INTERFERin™ (IFN), and HiPerFect®. GAPDH protein levels were monitored to ensure equal loading of samples.
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pone-0073348-g007: Characterization of 599 peptide-mediated RNAi responses in oral cancer cells.(A) Western blot analyses of CIP2A protein expression levels in CAL 27 and SCC-25 oral cancer cells 1, 3, 5, 7, and 9 days post-treatment with 599 peptide complexed to either 100 nM of control non-targeting siRNA (siNT) or siRNA targeting CIP2A (siCIP2A) at a 50∶1 peptide-to-siRNA molar ratio. GAPDH protein levels were monitored to ensure equal loading of samples. (B) Western blot analysis of CIP2A protein knockdown in SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide alone (5 µM) or complexed at a 50∶1 peptide-to-siRNA molar ratio to either siNT (100 nM) or decreasing concentrations of siCIP2A. The CIP2A protein levels in untreated cells were also analyzed for comparison. GAPDH protein levels were monitored to ensure equal loading of samples. (C) Western blot analyses of CIP2A protein knockdown in SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide complexed to either 100 nM of siNT or siCIP2A at a 50∶1 peptide-to-siRNA molar ratio, in the presence (+) or absence (-) of serum, compared to untreated cells or cells treated with commercial transfection reagents Lipofectamine® RNAiMAX (RNAiMAX), Lipofectamine® 2000 (LF2000), INTERFERin™ (IFN), and HiPerFect®. GAPDH protein levels were monitored to ensure equal loading of samples.

Mentions: In an effort to further characterize 599 peptide-mediated silencing of CIP2A in oral cancer cells the duration of gene silencing, dose-dependent gene silencing, and activity of silencing in serum were also assessed. The successful implementation of RNAi as a form of therapy depends on these three key features. Thus, in an effort to determine the persistence of 599 peptide-mediated silencing of CIP2A in oral cancer cells over time, a Western blot analyses of CIP2A protein expression levels in CAL 27 and SCC-25 oral cancer cells 1, 3, 5, 7, and 9 days post-treatment with 599 peptide complexed to either siNT or siCIP2A at a 50∶1 peptide-to-siRNA molar ratio were performed (Fig. 7A). After a single treatment with 599/siCIP2A, the silencing effect was observed to last for 5 days in CAL-27 cells and up to 9 days in SCC-25 cells with maximum silencing occurring for 3 and 7 days, respectively. In dose-response experiments in SCC-25 cells (Fig. 7B), the 599 peptide was observed to confer siCIP2A mediated silencing dose-dependently with siRNA concentrations ≥50 nM conferring ∼100% CIP2A protein knockdown and siRNA concentrations as low as 20 nM still having measurable silencing effects. Similar results were observed in CAL 27 cells (data not shown), except that higher siRNA concentrations (≥80 nM) were needed to confer at best ∼90% CIP2A protein silencing. Lastly, because the stability of siRNAs is an important concern in RNAi-therapy, 599 peptide-mediated CIP2A silencing was assessed in the absence and presence of serum and compared to several commercially available standard cationic lipid-based transfection reagents (Fig. 7C). Upon treating SCC-25 cells with 599/siCIP2A, ∼95% CIP2A protein was knocked down in the absence of an initial input of serum and was comparable to CIP2A silencing observed for all cationic lipid-based transfection reagents tested (>99%) except HiPerFect® which exhibited only a ∼30% reduction in protein levels. Of significance, however, is that even in the presence of serum, the 599/CIP2A complex could still produce a very effective RNAi response at ∼70% CIP2A protein knockdown. The quantitation of these data were based on the densitometric analyses of three independent Western blot experiments using Image J software [28].


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)

Characterization of 599 peptide-mediated RNAi responses in oral cancer cells.(A) Western blot analyses of CIP2A protein expression levels in CAL 27 and SCC-25 oral cancer cells 1, 3, 5, 7, and 9 days post-treatment with 599 peptide complexed to either 100 nM of control non-targeting siRNA (siNT) or siRNA targeting CIP2A (siCIP2A) at a 50∶1 peptide-to-siRNA molar ratio. GAPDH protein levels were monitored to ensure equal loading of samples. (B) Western blot analysis of CIP2A protein knockdown in SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide alone (5 µM) or complexed at a 50∶1 peptide-to-siRNA molar ratio to either siNT (100 nM) or decreasing concentrations of siCIP2A. The CIP2A protein levels in untreated cells were also analyzed for comparison. GAPDH protein levels were monitored to ensure equal loading of samples. (C) Western blot analyses of CIP2A protein knockdown in SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide complexed to either 100 nM of siNT or siCIP2A at a 50∶1 peptide-to-siRNA molar ratio, in the presence (+) or absence (-) of serum, compared to untreated cells or cells treated with commercial transfection reagents Lipofectamine® RNAiMAX (RNAiMAX), Lipofectamine® 2000 (LF2000), INTERFERin™ (IFN), and HiPerFect®. GAPDH protein levels were monitored to ensure equal loading of samples.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073348-g007: Characterization of 599 peptide-mediated RNAi responses in oral cancer cells.(A) Western blot analyses of CIP2A protein expression levels in CAL 27 and SCC-25 oral cancer cells 1, 3, 5, 7, and 9 days post-treatment with 599 peptide complexed to either 100 nM of control non-targeting siRNA (siNT) or siRNA targeting CIP2A (siCIP2A) at a 50∶1 peptide-to-siRNA molar ratio. GAPDH protein levels were monitored to ensure equal loading of samples. (B) Western blot analysis of CIP2A protein knockdown in SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide alone (5 µM) or complexed at a 50∶1 peptide-to-siRNA molar ratio to either siNT (100 nM) or decreasing concentrations of siCIP2A. The CIP2A protein levels in untreated cells were also analyzed for comparison. GAPDH protein levels were monitored to ensure equal loading of samples. (C) Western blot analyses of CIP2A protein knockdown in SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide complexed to either 100 nM of siNT or siCIP2A at a 50∶1 peptide-to-siRNA molar ratio, in the presence (+) or absence (-) of serum, compared to untreated cells or cells treated with commercial transfection reagents Lipofectamine® RNAiMAX (RNAiMAX), Lipofectamine® 2000 (LF2000), INTERFERin™ (IFN), and HiPerFect®. GAPDH protein levels were monitored to ensure equal loading of samples.
Mentions: In an effort to further characterize 599 peptide-mediated silencing of CIP2A in oral cancer cells the duration of gene silencing, dose-dependent gene silencing, and activity of silencing in serum were also assessed. The successful implementation of RNAi as a form of therapy depends on these three key features. Thus, in an effort to determine the persistence of 599 peptide-mediated silencing of CIP2A in oral cancer cells over time, a Western blot analyses of CIP2A protein expression levels in CAL 27 and SCC-25 oral cancer cells 1, 3, 5, 7, and 9 days post-treatment with 599 peptide complexed to either siNT or siCIP2A at a 50∶1 peptide-to-siRNA molar ratio were performed (Fig. 7A). After a single treatment with 599/siCIP2A, the silencing effect was observed to last for 5 days in CAL-27 cells and up to 9 days in SCC-25 cells with maximum silencing occurring for 3 and 7 days, respectively. In dose-response experiments in SCC-25 cells (Fig. 7B), the 599 peptide was observed to confer siCIP2A mediated silencing dose-dependently with siRNA concentrations ≥50 nM conferring ∼100% CIP2A protein knockdown and siRNA concentrations as low as 20 nM still having measurable silencing effects. Similar results were observed in CAL 27 cells (data not shown), except that higher siRNA concentrations (≥80 nM) were needed to confer at best ∼90% CIP2A protein silencing. Lastly, because the stability of siRNAs is an important concern in RNAi-therapy, 599 peptide-mediated CIP2A silencing was assessed in the absence and presence of serum and compared to several commercially available standard cationic lipid-based transfection reagents (Fig. 7C). Upon treating SCC-25 cells with 599/siCIP2A, ∼95% CIP2A protein was knocked down in the absence of an initial input of serum and was comparable to CIP2A silencing observed for all cationic lipid-based transfection reagents tested (>99%) except HiPerFect® which exhibited only a ∼30% reduction in protein levels. Of significance, however, is that even in the presence of serum, the 599/CIP2A complex could still produce a very effective RNAi response at ∼70% CIP2A protein knockdown. The quantitation of these data were based on the densitometric analyses of three independent Western blot experiments using Image J software [28].

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