Overexpression of Bcl-2 as a proxy redox stimulus to enhance activity of non-viral redox-responsive delivery vectors

Abstract

Redox-sensitive non-viral delivery systems exploit intracellular reducing environment to improve the efficacy of the delivery of nucleic acids by selectively releasing the cargo in the subcellular space. Bcl-2 overexpression is frequently observed in human cancers and is closely associated with increased resistance to chemotherapy and radiotherapy. One of the biochemical alterations accompanying Bcl-2 overexpression is the increase in cellular glutathione (GSH) levels. In this study, we hypothesize that such increase of GSH concentrations will selectively enhance the transfection activity of redox-sensitive delivery systems in cells overexpressing Bcl-2. Transfection studies were conducted in MCF-7 mammary carcinoma cells and MCF-7 clones overexpressing Bcl-2. It was confirmed that Bcl-2 overexpression resulted in the expected increase in GSH concentrations. Redoxsensitive complexes containing plasmid DNA, mRNA, antisense oligodeoxynucleotides, and siRNA exhibited selectively increased activity in cells overexpressing Bcl-2 compared to non-redox complexes. The effect of Bcl-2 overexpression on the selective enhancement of transfection was highly dependent on the type of the delivered nucleic acid, and was most pronounced for mRNA. This study shows that Bcl-2 overexpression can serve as a proxy redox stimulus to enhance the activity of all major classes of potential nucleic acid therapeutics, when delivered using redoxsensitive vectors.

Opposing influence of intracellular and membrane thiols on the toxicity of reducible polycations

“…The total intracellular GSH content (i.e., both oxidized and reduced forms) was determined using a previously published enzymatic recycling method [19]. Briefly, 3×10 5 Panc-1 and Panc-28 cells were seeded in a 6-well plate.…”

Delivery of Nucleic Acids via Disulfide‐Based Carrier Systems

“…One hypothesis could be that the disulfide reduction is likely to have a smaller effect on low-molecular-weight nucleic acids due to a smaller influence of the polycation size on the unpacking rates. [78] REVIEW www.advmat.de…”

Effect of innate glutathione levels on activity of redox-responsive gene delivery vectors

“…Cellular GSH levels [21, 24], subcellular distribution of GSH [21], concentration of cell surface protein thiols [25] and population doubling time [21] were measured for all the cell lines. Intracellular GSH concentration was measured on reverse-phase ion-exchange Waters HPLC system and normalized to cell volume determined using Packed-Cell-Volume tubes using the protocol described by the manufacturer (MidSci).…”

“…We previously tested the effect of increased GSH levels associated with overexpression of Bcl-2 (an anti-apoptotic protein overexpressed in multiple human cancers). The results demonstrated that Bcl-2 overexpression can be utilized as a proxy redox stimulus to selectively enhance the activity of all four major classes of potential nucleic acid therapeutics, when delivered by redox-sensitive vectors [21]. …”