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.
Highlights
It is estimated that about 40,000 new cases and approximately 8,000 deaths related to cancer of the oral cavity and pharynx will occur annually in the USA in 2012 [1]
Using various amounts of the 599 peptide, ranging from 1 to 50-fold molar excess of small interfering RNAs (siRNAs), it was demonstrated that the 599 peptide could bind siRNA molecules designed to target CIP2A 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
Upon demonstrating that the 599 peptide could bind to siRNAs at specific P:N ratios, we examined the ability of the peptide to deliver fluorescently-labeled siRNAs into oral cancer cells by fluorescence microscopy analysis (Fig. 2A)
Summary
It is estimated that about 40,000 new cases and approximately 8,000 deaths related to cancer of the oral cavity and pharynx will occur annually in the USA in 2012 [1]. RNA interference (RNAi) is a highly conserved post-transcriptional gene regulatory mechanism triggered by small, non-coding double-stranded RNA molecules that can silence gene expression by either repressing translation and/or inducing mRNA degradation [4,5]. Short double-stranded RNA molecules, known as small interfering RNA (siRNA) are functional molecules that in association with the RNA-induced silencing complex (RISC) mediate sequence-specific mRNA target selection and cleavage [6,7,8,9]. The discovery that the introduction of chemically synthesized siRNAs into mammalian cells could efficiently induce sequence-specific inhibition of gene expression [6], made evident the therapeutic potential of harnessing RNAi as a means to target and silence disease-causing genes. Subsequent preclinical experiments in animals and more recent clinical trials have further validated siRNAs as potent inhibitors of an assortment of disease-causing genes and as a promising new class of therapeutics [8,10,11]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have