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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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The 599 peptide mediates CIP2A gene silencing and subsequent destabilization of the c-Myc oncoprotein in oral cancer cells.(A) Real-time PCR analysis of CIP2A mRNA levels in CAL 27 and SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide complexed at a 50∶1 peptide-to-siRNA molar ratio to 100 nM of siRNA targeting CIP2A (siCIP2A) compared with control non-targeting siRNA (siNT). The CIP2A mRNA levels were normalized to 18S rRNA. Data are mean ± SEM of three separate experiments performed in triplicate, where **P<0.01 compared to siNT treated cells (Student’s t test). (B) Western blot analyses of CIP2A and c-Myc protein expression levels in CAL 27 and 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. GAPDH protein levels were monitored to ensure equal loading of samples.
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pone-0073348-g006: The 599 peptide mediates CIP2A gene silencing and subsequent destabilization of the c-Myc oncoprotein in oral cancer cells.(A) Real-time PCR analysis of CIP2A mRNA levels in CAL 27 and SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide complexed at a 50∶1 peptide-to-siRNA molar ratio to 100 nM of siRNA targeting CIP2A (siCIP2A) compared with control non-targeting siRNA (siNT). The CIP2A mRNA levels were normalized to 18S rRNA. Data are mean ± SEM of three separate experiments performed in triplicate, where **P<0.01 compared to siNT treated cells (Student’s t test). (B) Western blot analyses of CIP2A and c-Myc protein expression levels in CAL 27 and 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. GAPDH protein levels were monitored to ensure equal loading of samples.

Mentions: For the 599 peptide to be considered an effective delivery vehicle for siRNA-based therapeutics, it must be capable of delivering functional siRNAs into cells that can silence their intended gene target. To assess the functionality of the 599/siRNA complex, two oral cancer cell lines, CAL 27 and SCC-25, were treated with the 599 peptide complexed to either siCIP2A or a siNT control at a 50∶1 P:N ratio, after which both the CIP2A mRNA and protein levels were assessed 48 hours post-treatment by real-time PCR and Western blot analyses, respectively. Quantitation by real-time PCR demonstrated a significant knockdown in CIP2A mRNA levels in both oral cancer cell lines treated with the 599/siCIP2A complex compared to control 599/siNT treated cells (Fig. 6A). More specifically, a ∼60% and ∼85% reduction in CIP2A mRNA levels was observed in CAL 27 and SCC-25 oral cancer cell lines, respectively. Furthermore, Western blot analyses confirmed the ability of the 599 peptide to mediate delivery of functional siRNAs in both cell lines by demonstrating the suppression of CIP2A protein levels 48 hours post-treatment with the 599/siCIP2A complex compared to control 599/siNT treated cells (Fig. 6B). Of note, the protein levels of the oncogenic transcription factor c-Myc were also assessed because CIP2A is known to regulate the stability of this protein [25]. Western blot analyses confirmed that silencing of CIP2A caused destabilization of c-Myc in both cell lines (Fig. 6B).


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)

The 599 peptide mediates CIP2A gene silencing and subsequent destabilization of the c-Myc oncoprotein in oral cancer cells.(A) Real-time PCR analysis of CIP2A mRNA levels in CAL 27 and SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide complexed at a 50∶1 peptide-to-siRNA molar ratio to 100 nM of siRNA targeting CIP2A (siCIP2A) compared with control non-targeting siRNA (siNT). The CIP2A mRNA levels were normalized to 18S rRNA. Data are mean ± SEM of three separate experiments performed in triplicate, where **P<0.01 compared to siNT treated cells (Student’s t test). (B) Western blot analyses of CIP2A and c-Myc protein expression levels in CAL 27 and 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. GAPDH protein levels were monitored to ensure equal loading of samples.
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pone-0073348-g006: The 599 peptide mediates CIP2A gene silencing and subsequent destabilization of the c-Myc oncoprotein in oral cancer cells.(A) Real-time PCR analysis of CIP2A mRNA levels in CAL 27 and SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide complexed at a 50∶1 peptide-to-siRNA molar ratio to 100 nM of siRNA targeting CIP2A (siCIP2A) compared with control non-targeting siRNA (siNT). The CIP2A mRNA levels were normalized to 18S rRNA. Data are mean ± SEM of three separate experiments performed in triplicate, where **P<0.01 compared to siNT treated cells (Student’s t test). (B) Western blot analyses of CIP2A and c-Myc protein expression levels in CAL 27 and 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. GAPDH protein levels were monitored to ensure equal loading of samples.
Mentions: For the 599 peptide to be considered an effective delivery vehicle for siRNA-based therapeutics, it must be capable of delivering functional siRNAs into cells that can silence their intended gene target. To assess the functionality of the 599/siRNA complex, two oral cancer cell lines, CAL 27 and SCC-25, were treated with the 599 peptide complexed to either siCIP2A or a siNT control at a 50∶1 P:N ratio, after which both the CIP2A mRNA and protein levels were assessed 48 hours post-treatment by real-time PCR and Western blot analyses, respectively. Quantitation by real-time PCR demonstrated a significant knockdown in CIP2A mRNA levels in both oral cancer cell lines treated with the 599/siCIP2A complex compared to control 599/siNT treated cells (Fig. 6A). More specifically, a ∼60% and ∼85% reduction in CIP2A mRNA levels was observed in CAL 27 and SCC-25 oral cancer cell lines, respectively. Furthermore, Western blot analyses confirmed the ability of the 599 peptide to mediate delivery of functional siRNAs in both cell lines by demonstrating the suppression of CIP2A protein levels 48 hours post-treatment with the 599/siCIP2A complex compared to control 599/siNT treated cells (Fig. 6B). Of note, the protein levels of the oncogenic transcription factor c-Myc were also assessed because CIP2A is known to regulate the stability of this protein [25]. Western blot analyses confirmed that silencing of CIP2A caused destabilization of c-Myc in both cell lines (Fig. 6B).

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