Ribozyme-mediated cleavage of the human survivin mRNA and inhibition of antiapoptotic function of survivin in MCF-7 cells

Abstract

Survivin is a new member of the inhibitor of apoptosis protein ( IAP ) family that is implicated in the control of cell proliferation and the regulation of cell life span. This protein is selectively expressed in most human carcinomas but not in normal adult tissues. To down -regulate a human survivin expression as a strategy for cancer gene therapy, we designed two hammerhead ribozymes ( RZ -1, RZ -2 ) targeting human survivin mRNA. RZ -1 and RZ -2 efficiently cleaved the human survivin mRNA at nucleotide positions + 279 and + 289, which was identified by in vitro cleavage assay using in vitro transcribed ribozymes and truncated survivin mRNA substrate. To investigate the function of the ribozymes in cells, the sequences of the ribozymes were cloned into replication -deficient adenoviral vector and transferred to breast cancer cell, MCF -7. The infection with adenovirus encoding the ribozymes resulted in a significant reduction of survivin mRNA ( 74% and 73%, respectively ) and protein. As revealed by nuclear condensation / fragmentation and flow cytometry analysis, inhibition of survivin gene by ribozymes increased apoptosis and sensitivity induced by etoposide or serum starvation. Our results suggest that the designed hammerhead ribozymes against survivin mRNA are good candidates for feasible gene therapy in the treatment of cancer.

Knockdown of survivin expression by small interfering RNA reduces the clonogenic survival of human sarcoma cell lines independently of p53

“…9,11,13,34,35 In a preliminary investigation, we found that an exogenous apoptotic stimulus, for example, radiation, in combination with the knockdown of survivin expression increased the caspase-3 and caspase-7 activity 2.5-fold in the sarcoma cell line A-204, which expresses wild-type p53 (unpublished results). However, even after an apoptotic stimulus is applied, only a small percentage of cells from sarcoma cell lines undergoes apoptosis.…”

“…Furthermore, clonogenic survival of cells from each line was reduced by 65-86% (Fig 1c, Table 1). By using antisense oligonucleotides, 8,[10][11][12][34][35][36] ribozymes, 9,13,37 and siRNA (in a colon cancer cell line) 23 to knock down expression of survivin mRNA and protein, other investigators have observed a remarkable reduction in survivin mRNA that ranged from 70 to 90% and a reduction in survivin protein that ranged from 60 to 90%. The study conducted by Williams et al 23 has described the application of survivin-specific siRNA, which differs from the construct applied in our study, resulting in a reduction of survivin protein in colon cancer.…”

“…Treatment with either survivin-specific antisense oligonucleotides or ribozymes has resulted in reduced survivin expression, which has been correlated with a decrease in tumor growth in mice, with an increase in caspasedependent cell death (apoptosis), and with the formation of polyploid cells in vitro. [8][9][10][11][12][13][14][15][16] In addition to the use of antisense oligonucleotides and ribozymes, the application of small interfering RNA (siRNA) is an effective way to silence gene transcription. 17,18 Transcription silencing is thought to be a defense strategy that preserves genomes through evolution from attack by double-stranded RNA viruses and transposons.…”

Plasmid-based Survivin shRNA and GRIM-19 carried by attenuated Salmonella suppresses tumor cell growth

“…6 Because of its roles in reducing apoptosis, and stimulating cell division and proliferation, and its differential expression in cancers compared to normal tissues, Survivin is an attractive target for cancer gene therapy. 7 Thus far, different approaches have been taken to target Survivin, such as antisense oligonucleotides, 8 small interfering RNAs, 9 dominant negative mutants, 10 ribozymes 11 and triplex DNA-dependent kinase for cancer therapeutics. 12 However, none of these studies aiming at suppression of Survivin transcription can provide an ideal therapeutic approach.…”

Differential regulation of survivin expression and apoptosis by vitamin D3 compounds in two isogenic MCF-7 breast cancer cell sublines

“…The accumulated data from survivin studies on human cancers suggest that survivin expression in cancer is associated with cancer progression, poor prognosis, drug resistance, and shorter patient survival (Li, 2003). In vitro and in vivo studies targeting survivin with antisense oligonucleotides (Li et al, 1999;Chen et al, 2000;Olie et al, 2000;Xia et al, 2002), dominant-negative mutants (Li et al, , 1999Grossman et al, 1999a, b;O'Connor et al, 2000;Mesri et al, 2001), ribozymes (Pennati et al, 2002;Choi et al, 2003), triplex DNA-formation (Shen et al, 2003) and RNA interference (Ling and Li, 2004) have shown induction of apoptosis, reduction of tumorgrowth potential, and sensitization to chemotherapeutic drugs and other therapeutic approaches . These studies indicate that survivin is an excellent novel target for cancer therapeutics.…”