Multiple siRNA delivery against cell cycle and anti-apoptosis proteins in breast cancer and normal cells

Abstract

Event Abstract Back to Event Multiple siRNA delivery against cell cycle and anti-apoptosis proteins in breast cancer and normal cells Manoj Parmar1*, Bárbara E. Arteaga Ballesteros2*, Timothy Fu2*, Remant Bahadur Kc2*, Hamidreza Montazeri Aliabadi3*, Judith C. Hugh4* and Hasan Uludag1, 2, 5* 1 University of Alberta, Faculty of Pharmacy and Pharmaceutical Sciences, Canada 2 University of Alberta, Department of Chemical and Materials Engineering, Canada 3 Chapman University, School of Pharmacy, United States 4 University of Alberta, Department of Laboratory Medicine and Pathology, Canada 5 University of Alberta, Department of Biomedical Engineering, Canada Introduction: Current therapies for breast cancer induce severe side effects that warrant a search for alternative therapies. Short-interfering RNA (siRNA)-based therapeutics directly target and silence specific genes critical for cancer cells, thus holding promise as an effective therapy with less side effects. Given the unregulated cell cycle progression in cancer cells, proteins involved in cell cycle regulation such as spindle assembly checkpoint kinase TTK and cell-division cycle protein 20 (CDC20) are promising targets to stop uncontrolled cell growth. Other targets that might act in conjunction with cell cycle proteins are anti-apoptosis proteins such as survivin [1], which are often up-regulated in transformed cells. We hypothesize that the dual silencing of cell cycle and anti-apoptosis proteins might be exceptionally effective and specific for treatment of breast cancer. Here, we explored the combinational siRNA therapy in breast cancer as well as effects on normal cells in vitro. Methods & Results: Since siRNA is highly labile and anionic, we synthesized a library of cationic and hydrophobic (lipid-modified) polymers from 0.6, 1.2 and 2.0 kDa polyethylenimines (PEI). We screened this library for delivery of CDC20 siRNA in MDA-MB-231 cells, where 1.2 kDa PEI substituted with linoleic acid (PEI-LA) was found to be the most effective [2,][3]. We additionally determined efficacy of 6 siRNAs targeting at different locations of TTK gene; all TTK siRNAs inhibited growth of MDA-MB-231 and MCF7 breast cancer cells (as assessed by MTT Assay), which was further confirmed with the down-regulation of TTK transcripts by qPCR. We then explored the feasibility of combinational siRNA therapy by mixing specific siRNAs with polymers at different weight ratios to form complexes. TTK/CDC20 and CDC20/Survivin siRNA combinations decreased the growth of MDA-MB-231 cells significantly, while only TTK/CDC20 combination inhibited MCF7 cell growth significantly at a relatively low (15 nM each) siRNA concentration (Figure 1). The transcripts of each of these targets were significantly down-regulated in specific siRNA treated cells compared to scrambled siRNA treated cells. The combinational siRNA delivery was also performed in normal breast epithelial MCF10A cells, human umbilical vein endothelial cells (HUVEC) and human bone marrow stromal cells (HBMSC) to determine the side effects of siRNA treatments using 15 nM each siRNA at different siRNA:polymer ratios. The combinational siRNA therapy at lower ratio (1:2) showed higher side effects of siRNA treatment in these cells compared to higher ratios (1:4, 1:8; Figure 2). Based on relative effect of combinational siRNAs in breast cancer and normal cells, TTK/CDC20 siRNA combination inhibited MDA-MB-231 (33%) and MCF7 (24%) cell growth at 1:4 siRNA:polymer ratio without showing much side effects in MCF10A and HUVEC cells (Figure 2D). However, this combination showed significant side effects in HBMSC cells (33%). Conclusions: This study has indicated improved efficacy of combinational siRNA therapy against cell cycle and anti-apoptosis proteins in breast cancer therapy. The lipid-substituted polymers could serve as a viable platform for delivery of multiple siRNAs against critical targets. However, the siRNA therapy has also showed side effects on non-malignant (normal) cells in vitro and more selective therapies might be needed to target cancer cells solely. Manoj Parmar is a recipient of Women and Children's Health Research Institute (WCHRI) Graduate Studentship Grant and Alberta Innovates: Health Solutions (AIHS) Graduate Studentship.; This study was supported by operating grants from Canadian Breast Cancer Foundation (CBCF) and Natural Sciences and Engineering Council of Canada (NSERC).