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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-mediated silencing of CIP2A decreases oral cancer cell invasiveness and anchorage-independent growth.(A) Quantitation of the percentage of invading SCC-25 oral cancer cells after 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). Data are mean ± SEM of four separate experiments, where *P<0.05 compared to siNT treated cells (Student’s t test). (B) Anchorage-independent growth of SCC-25 oral cancer cells after treatment with 599 peptide complexed at a 50∶1 peptide-to-siRNA molar ratio to 100 nM of siCIP2A compared with control siNT. Data are mean ± SEM of four separate experiments, where *P<0.05 compared to siNT treated cells (Student’s t test).
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pone-0073348-g008: The 599 peptide-mediated silencing of CIP2A decreases oral cancer cell invasiveness and anchorage-independent growth.(A) Quantitation of the percentage of invading SCC-25 oral cancer cells after 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). Data are mean ± SEM of four separate experiments, where *P<0.05 compared to siNT treated cells (Student’s t test). (B) Anchorage-independent growth of SCC-25 oral cancer cells after treatment with 599 peptide complexed at a 50∶1 peptide-to-siRNA molar ratio to 100 nM of siCIP2A compared with control siNT. Data are mean ± SEM of four separate experiments, where *P<0.05 compared to siNT treated cells (Student’s t test).

Mentions: Previously published literature has demonstrated that CIP2A silencing in cancer cells derived from different tissues, such as HNSCCs, renal cell carcinomas, and breast cancer can have effects on cancer cell invasion and anchorage-independent growth [25], [29], [30]. Thus, to further demonstrate the potential usefulness of the 599 peptide in siRNA-based therapeutics for oral cancer, we tested whether 599 peptide-mediated silencing of CIP2A could affect the invasiveness and anchorage-independent growth properties of oral cancer cells. Due to the fact that CAL 27 cells exhibited only a brief silencing effect and do not grow well in semi-solid medium [31], the 599 peptide-mediated CIP2A silencing effects on cell invasion and anchorage-independent growth were only tested in SCC-25 cells. Upon treatment of SCC-25 cells with the 599 peptide complexed to siCIP2A, we observed a significant ∼33% inhibition in cell invasiveness, compared to control 599/siNT treated cells (Fig. 8A). Moreover, 599 peptide-mediated silencing of CIP2A in SCC-25 cells significantly reduced anchorage-independent growth by ∼39% compared to control 599/siNT treated cells (Fig. 8B). Together, these results demonstrated the ability of the 599 peptide to function as an effective delivery vehicle for siRNA-based oral cancer therapeutics.


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-mediated silencing of CIP2A decreases oral cancer cell invasiveness and anchorage-independent growth.(A) Quantitation of the percentage of invading SCC-25 oral cancer cells after 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). Data are mean ± SEM of four separate experiments, where *P<0.05 compared to siNT treated cells (Student’s t test). (B) Anchorage-independent growth of SCC-25 oral cancer cells after treatment with 599 peptide complexed at a 50∶1 peptide-to-siRNA molar ratio to 100 nM of siCIP2A compared with control siNT. Data are mean ± SEM of four separate experiments, where *P<0.05 compared to siNT treated cells (Student’s t test).
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getmorefigures.php?uid=PMC3760901&req=5

pone-0073348-g008: The 599 peptide-mediated silencing of CIP2A decreases oral cancer cell invasiveness and anchorage-independent growth.(A) Quantitation of the percentage of invading SCC-25 oral cancer cells after 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). Data are mean ± SEM of four separate experiments, where *P<0.05 compared to siNT treated cells (Student’s t test). (B) Anchorage-independent growth of SCC-25 oral cancer cells after treatment with 599 peptide complexed at a 50∶1 peptide-to-siRNA molar ratio to 100 nM of siCIP2A compared with control siNT. Data are mean ± SEM of four separate experiments, where *P<0.05 compared to siNT treated cells (Student’s t test).
Mentions: Previously published literature has demonstrated that CIP2A silencing in cancer cells derived from different tissues, such as HNSCCs, renal cell carcinomas, and breast cancer can have effects on cancer cell invasion and anchorage-independent growth [25], [29], [30]. Thus, to further demonstrate the potential usefulness of the 599 peptide in siRNA-based therapeutics for oral cancer, we tested whether 599 peptide-mediated silencing of CIP2A could affect the invasiveness and anchorage-independent growth properties of oral cancer cells. Due to the fact that CAL 27 cells exhibited only a brief silencing effect and do not grow well in semi-solid medium [31], the 599 peptide-mediated CIP2A silencing effects on cell invasion and anchorage-independent growth were only tested in SCC-25 cells. Upon treatment of SCC-25 cells with the 599 peptide complexed to siCIP2A, we observed a significant ∼33% inhibition in cell invasiveness, compared to control 599/siNT treated cells (Fig. 8A). Moreover, 599 peptide-mediated silencing of CIP2A in SCC-25 cells significantly reduced anchorage-independent growth by ∼39% compared to control 599/siNT treated cells (Fig. 8B). Together, these results demonstrated the ability of the 599 peptide to function as an effective delivery vehicle for siRNA-based oral cancer therapeutics.

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