Oligodeoxyribozymes That Cleave β-Catenin Messenger RNA Inhibit Growth of Colon Cancer Cells via Reduction of β-Catenin Response Transcription

Abstract

Abnormal regulation of Wnt/β-catenin signaling followed by increased levels of the β-catenin protein have been identified in enhanced cellular proliferation and development of colon polyps and cancers. To inhibit β-catenin gene expression in colon cancer cells, RNA-cleaving oligodeoxyribozyme (DNAzyme) was employed to destroy the β-catenin mRNA. We designed a strategy to identify the cleavage sites in β-catenin RNA with a pool of random sequences from a DNAzyme library and identified four potential DNAzymeworking sites. DNAzymes were constructed for the selected target sites and were tested for the ability to cleave β-catenin RNA. When introduced into the cells, the selected DNAzymes decreased the expression of β-catenin significantly as well as its downstream gene, cyclin D1. Additionally, we designed short hairpin RNA that targets the same cleavage site for the selected DNAzyme. The designed short hairpin RNA also inhibited β-catenin gene expression in colon cancer cells. Our studies show that RNA-cleaving DNAzymes and RNA interference targeted to β-catenin significantly reduced β-catenin-dependent gene expression, resulting in inhibition of colon cancer cell growth. These results indicate that the functional antisense oligonucleotides directed against β-catenin might have potential as a therapeutic intervention to treat colon cancer.

Small interfering RNA-mediated downregulation of beta-catenin inhibits invasion and migration of colon cancer cells in vitro

“…Thus, inhibition of invasion and metastasis of cancer cells has been a feasible strategy for the treatment of malignancies [21]. Abnormal regulation of Wnt/β-catenin signaling and subsequent increase of β-catenin expression has been found to promote cell proliferation and be related to carcinogenesis in colon cancer [22]. Whether β-catenin down-regulation in LoVo cells can compromise the invasion and migration potentials in vitro remains to be determined.…”

DNA enzymes as potential therapeutics: towards clinical application of 10-23 DNAzymes

“…Stat3 was found to participate in the expression of genes that control critical cellular functions such as proliferation, survival, self-renewal, angiogenesis, metastasis, inflammation and immune response [104] IGF-II Fetal growth protein and important proangiogenic factor, reactivation and dysregulation of which is associated with attenuation of apoptosis and increased proliferation in liver cancers [105,106] Survivin Expressed in most human tumors but not in normal adult tissues, it has functions in regulation of cell division and the inhibition of apoptosis [107] Twist A transcription factor [108] Bcl-xL An apoptosis suppressor [109] b-Catenin Abnormal regulation of Wnt/b-catenin signaling and increased levels of the protein were found during enhanced cell proliferation and development of colon polyps and cancers [110] PKC-a A PKC isoform over-expressed in some cancer cell lines [47,111] Aurora kinase A Involved in malignant progression of many cancers including prostate cancer [18] AKT1 Involved in thyroid cancer metabolism, growth, proliferation and survival; it is overexpressed in thyroid tumors [112,113] Raf-1 kinase Protein kinase Raf-1 is downstream of Ras. A hyperactive function of the ras oncogene is a hallmark of Juvenile myelomonocytic leukaemia [114] BCR-ABL fusion gene Characteristic of cells from patients with chronic myelogenous leukaemia, the two types of fusion BCR--ABL mRNAs, b3a2 (composed of BCR exon 3 and ABL exon 2) and b2a2 (composed of BCR exon 2 and ABL exon 2) encode for p210 protein with tyrosine kinase activity that plays a significant role in the pathogenesis of the malignancies through suppression of apoptotic cell death, which leads to accumulation of myeloid cells [26,27,30] PML-RARa fusion gene Associated with acute promyelocytic leukaemia [28] K-Ras Point mutations of the Ras family are frequently found in human cancers at a prevalence rate of 30%, the most common mutation being K-Ras (G12V) required for tumor proliferation, survival and metastasis due to its GTPase activity [115] MMP: Matrix metalloproteinase.…”

“…To inhibit b-catenin gene expression in colon cancer cells, Dzs against b-catenin mRNA were constructed [110]. When introduced into cells, they decreased significantly the expression of b-catenin as well as its downstream gene cyclin D1 resulting in inhibition of colon cancer cell growth.…”

Anti-metastatic Treatment in Colorectal Cancer: Targeting Signaling Pathways

“…The most direct strategy for expression reduction of the key player -catenin is the employment of specific si/shRNA technologies [123] or of oligodeoxyribozymes that cleave its mRNA [124], all of them leading to growth inhibition. We demonstrated that, in addition to inhibition of cell proliferation, siRNA acting oncatenin also reduced cell migration and invasion [111].…”