Testimonial:

mRNA Killing Lung Cancer A549 Cells with RJH Reagents and Cytotoxic siRNAs

Background

Lung cancer is a deadly disease. Cancers originating in lungs or metastasized to lungs from other sites are particularly difficult to treat due to several mechanisms that facilitate cancer cells to resist the actions of chemotherapy agents. Once the drug resistance sets in, it is difficult to control abnormal proliferation of cells ultimately leading to therapeutic failure. New types of therapeutic agents are urgently needed especially for advanced stage lung cancers. Short interfering RNA (siRNA) is a relatively new therapeutic agent that can silence targeted genes and reduce corresponding protein levels. It has been possible to slow cell proliferation or induce apoptosis of cancerous cells with the choice of right siRNAs and protein targets. However, to realize the full potential of siRNA technology, an effective delivery system is needed since siRNA on its own cannot enter the cells and hence cannot silence the targeted genes. It is possible to use viral delivery system or electroporation to import siRNA into cells under culture conditions; however, these approaches are difficult to implement in a clinical setting and cannot be used for therapeutic purposes. RJH Biosciences developed a range of transfection reagents that are effective on a wide range of cells, including lung cancer cells. The transfection reagents can be readily formulated with siRNAs and used in culture, as well as in preclinical animal models. Being able to use the same transfection reagent in both cell culture and animal models provides assurance on the delivery method of the siRNA. Here we summarize the delivery of commercially available cytotoxic siRNAs to induce cell death in lung cancer A549 cells.

Methodology

Lung cancer A549 cells (ATCC) were seeded in 96-well plates with 200 µL of tissue culture medium for 24 hours before transfection with the siRNA complexes. A broadly-cytotoxic siRNA from ThermoFisher (identity not revealed) was formulated with the RJH transfection reagents ALL-Fect, Prime-Fect, InVivo-Fect and an proprietary formulations of RJH-X at ratios of 1:23, 1:10, 1:23 and 1:10 (w/w), respectively. The siRNA complexes were prepared in Opti-MEMTM by incubating siRNAs with the transfection reagents for 30 min before addition to the cells.


As control treatments, (i) an equivalent concentration of the chosen transfection reagents and (ii) scrambled (non-specific) siRNA formulated with the chosen transfection reagents were used to treat the cells under similar conditions. Cells were transfected with a final concentration of 50 nM of siRNA (except the cells that received transfection reagent alone). After incubation for 96 hours in a humidified CO2/O2 (95/5%) atmosphere, the MTS viability assay (Abcom) was run to determine the total cell activity in wells. The obtained absorbance readings (490 nm) were taken as the total viable cells numbers. The cells treated with the transfection reagent alone were used as a reference (taken as 100% relative viability). The viabilities of the remaining cells were normalized with the viabilities of the cells treated with the transfection reagent alone.

Results and Discussion

The experimental results from the cell viability assay are shown below. The relative cell viabilities are summarized separately for each group of cells treated with different transfection reagents.


Cells treated with the transfection reagents alone typically yielded relative viabilities that were similar to the untreated cells in the case of transfection reagents Prime-Fect and ALL-Fect, indicating no effect of these transfection reagents alone on the cell viabilities. InVivo RNA-Fect and RJH-X was not tested in this regard. Cells treated with scrambled siRNA complexes of Prime-Fect and InVivo RNA-Fect gave relative viabilities equivalent to untreated cells, again indicating no toxicity of the non-specific complexes on the viabilities of A549 cells. Complexes from ALL-Fect and RJH-X displayed some loss of viabilities (~10%) indicating slight toxicity of the non-specific complexes compared to un-treated cells. In contrast, the cytotoxic siRNA complexes gave significant reduction of toxicity, with Prime-Fect complexes displaying ~40% relative cell viability and InVivo RNA-Fect complexes displaying ~16% relative cell viability, and ALL-Fect and RJH-X complexes displaying less than 10% relative cell viabilities. Therefore, multiple RJH reagents were found to be very effective in this particular application intended to induce death of lung cancer cells. The differential activity obtained between the scrambled siRNA and cytotoxic siRNA were very high, suggesting that effective therapeutic effects could be readily demonstrated. A relatively low concentration of siRNAs was used (50 nM) to obtain the desired therapeutic effect so that translation into animal models should be facilitated at such low doses.

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Figure 1. Relative cell viability

Relative cell viability obtained with 2 separate transfection reagents. The viabilities are summarized separately for each transfection reagent. The study groups were untreated cells, cells treated with the transfection reagent alone (in the absence of siRNA), cells treated with scrambled siRNA, and cells treated with cytotoxic siRNA. Note the very drastic drop of the cell viabilities with the cytotoxic siRNAs delivered with all transfection reagents.
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Figure 2. Relative cell viability

Relative cell viability obtained with 2 separate transfection reagents. The viabilities are summarized separately for each transfection reagent. The study groups were untreated cells, cells treated with the scrambled siRNA plus transfection reagent and cells treated with the cytotoxic siRNA. Note the very drastic drop of the cell viabilities with the cytotoxic siRNAs delivered with all transfection reagents.

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