siRNA-Lipoplex-Mediated Bcl-2 and Bcl-xL Gene Silencing Induces Apoptosis in MCF-7 Human Breast Carcinoma Cells

Abstract

Bcl-2 family genes play a central role in cell apoptosis and cell proliferation, and are implicated in the pathology of many malignancies. We explored different ways of introducing siRNA duplexes into cells, comparing "naked" with lipoplex-and polyplex-based formulations in order to decrease the level of the Bcl-2 and Bcl-xL proteins. Our results show that siRNA binds efficiently to all cationic liposomes used. Upon binding, siRNA reduces the zeta potential of the particles, although in most cases they remain positively charged. 70% of MCF-7 cells took up fluorescently-labeled siRNA after 24 h. All siRNA sequences caused growth inhibition of cells, with variable efficiency in a dose-dependent manner. Significant decreases in Bcl-2 and Bcl-xL proteins were caused by two siRNA sequences. Both caused significant growth inhibition in concentrations as low as 100 nM. These two siRNAs caused the greatest increase in caspase-7 activity and DNA fragmentation level. Addition of CaCl 2 as a transfection enhancer resulted in marked increase of growth inhibition and Bcl-2 gene suppression by siRNA. Our lipoplexes containing siRNA showed equal or superior efficacy in comparison with commercial siRNA transfection kits. Efficiency of cell growth inhibition per RNA molecule using siRNA was found to be twenty fold higher than by a well-established Bcl-2 antisense oligonucleotide (ODN) molecule after optimization of ODN delivery to cells in culture. This study indicates the potential for efficient delivery of siRNA for treatment of various malignancies.

Multi-Target Inhibition of Cancer Cell Growth by SiRNA Cocktails and 5-Fluorouracil Using Effective Piperidine-Terminated Phosphorus Dendrimers

“…For instance, BLAST (Basic Local Alignment Search Tool provided by the U.S. National Center for Biotechnology Information, NCBI) search revealed partial complementarity (13 nucleotides) of Scrambled siRNA sequences to several transcripts of genes coding metabolically active proteins, such as 5-methyltetrahydrofolate-homocysteine methyltransferase (involved in the metabolism of essential amino acids), kinesin family member 3B (takes part in chromosome movement during cell division) and DNA topoisomerase I (regulates transcription). On the other hand, Scrambled siRNA sequence did not induce any negative effect on viability of MCF-7 cells (breast cancer) in the original study [25]. Thus, we can also assume cell type specific susceptibility towards investigated Scrambled siRNA.…”

“…Three pro-apoptotic siRNAs designed for silencing of anti-apoptotic genes from BCL-2 family were synthesized: siBCL-2 (sense 5 -GCUGCACCUGACGCCCUUCtt-3 , antisense 5 -GAAGGGCGUCAGGUGCAGCtt-3 ) [25], siBCL-xL (sense 5 -CAGGGACAGCAU AUCAGAGtt-3 , antisense 5 -CUCUGAUAUGCUGUCCCUGtt-3 ) [25], siMCL-1 (sense 5 -GGAC UUUUAUACCUGUUAU tt-3 , antisense 5 -AUAACAGGUAUAAAAGUCCtt-3 ) [29]. The following sequence of Scrambled siRNA was used: 1st strand 5 -ACUCUAGCGGCACCAUCGUGCCtt-3 , 2nd strand 5 -GGCACGAUGGUGCCG-CUAGAGUtt-3 [25].…”

“…The following sequence of Scrambled siRNA was used: 1st strand 5 -ACUCUAGCGGCACCAUCGUGCCtt-3 , 2nd strand 5 -GGCACGAUGGUGCCG-CUAGAGUtt-3 [25]. RNA oligonucleotides bear two deoxyribonucleotides at 3 -termini to increase their stability towards exonuclease digestion.…”

“…Being overexpressed in many types of cancer, these genes allow malignant cells to survive and grow by impairing essential signaling processes that lead to programmed cell death [23]. RNAi-based downregulation of the aforementioned genes triggers cell death in ovarian cancers [24], breast cancers [25,26], pancreatic cancers [27], acute myeloid leukemia [28] and skin cancers [29]. Silencing of BCL-2 family genes is also known to increase the sensitization of various cancer cells types to drugs commonly used in chemotherapy such as cisplatin [24], doxorubicin [26], etoposide [28] and 5-fluorouracil [30].…”

Anticancer siRNA cocktails as a novel tool to treat cancer cells. Part (B). Efficiency of pharmacological action

“…Scramble siRNA was used as follows (Scr1 5 0 -ACUCUAGCGGCACCAUCGUGCCtt 3 0 , Scr2 5 0 -GGCACGAUGGUGCCGCUAGAGUtt3 0 ) (Chetoui et al, 2008;Saad et al, 2008;Vestin et al, 2008).…”