DOWN-REGULATION OF BETA-CATENIN IN CANCER CELLS BY SIRNA COMPLEXED WITH CARBON NANOTUBE
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Date
2023-03-14
Authors
Ahmad Ghareeb
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Abstract
Globally, according to the World Health Organization, cancer is the second leading cause of death worldwide. In order to manage cancer, many approaches have been used, such as surgery, radiation, chemotherapy, and gene therapy.
Medicine research has taken advantage of biotechnology-based therapy in the past several years, and one of the newest strategies in cancer therapy; is the utilization of small interference RNA (siRNA) to control gene expression. However, effective delivery of siRNA into cells is a challenge, particularly in vivo. Lipofectamine is currently used in vitro for this purpose, but unfortunately, the compound has demonstrated unacceptable toxicity in vivo. To deliver these types of molecules without causing significant harm, researchers are developing new technological delivery systems. The development of drug delivery systems based on carbon nanotubes (CNTs) has received a lot of attention in this respect.
Our project aims to develop a new method for delivering siRNA into colon cancer cells in order to target the expression of the β-catenin protein, which is known to play a role in a variety of developmental processes; such as cell growth and proliferation, embryonic patterning, cell differentiation, and other cellular functions. The new approach is based on the synthesis of a new nano-system of multi-walled carbon nanotubes (MWCNTs) functionalized with a tetra-amine linker and mannose sugar as a targeting agent to increase the nano-system`s uptake and selectivity.
We successfully functionalized MWCNTs with tetra-amine groups (f-MWCNTs (8)), which were then functionalized with mannose molecules (f-MWCNTs (12)), confirmed by atomic force microscopy. In addition, the amount of amine in f-MWCNTs (8) was 12.7 x 103 nmol/mg and 40 x 103 nmol/mg in f-MWCNTs (12), while the amount of loaded mannose in f-MWCNTs (12) was 20.02 μg/mg as confirmed by the anthrone method. The N/P ratio required to trap β-catenin siRNA starts at 5:1 for f -MWCNTs (8) and 15:1 for f -MWCNTs (12), as determined by agarose gel. Western blot analysis revealed that our compounds significantly reduced β-catenin protein expression (p > 0.05). The knockdown percentage was 20% in f-MWCNTs (8) and 50% of f-MWCNTs (12). We hypothesized that the possible up-regulation of mannose receptors on the surface of caco-2 cells may increase the transfection efficacy of f-MWCNTs (12) compared with f-MWCNTs (8).
The MTS results showed that the down-regulation of β-catenin protein can restrict cancer cell growth and prevent cell proliferation which was shown significantly in both transfection approaches with an IC50 equal to 66.48 for f-MWCNTs (8)-siRNA slightly lower than control (IC50 = 70.01μg/ml) while 19.32 μg/ml for f-MWCNT (12)-siRNA. Also, this effect can increase the efficacy of 5-FU in colon cancer significantly compared with control. Therefore, we propose that our approach could be implemented in the setting of colon cancer therapy.
Keyword: Beta-catenin; Colorectal Cancer; Multi-walled Carbon Nanotubes.