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Published on: August 12, 2015
Potent subunit-specific effects on cell growth and drug sensitivity from optimised siRNA-mediated silencing of
Glen Reid1, Natacha Coppieters 't Wallant, Rachna Patel
1Genesis Research and Development Corporation Limited, PO Box 50, Auckland 1140, New Zealand.
Abstract:
Ribonucleotide reductase (RR) has an essential role in DNA synthesis and repair and is a therapeutic target in a number of different cancers. Previous studies have shown that RNAi-mediated knockdown of either the RRM1 or RRM2 subunit sensitizes cells to the cytotoxic effects of the nucleoside analogs and more recently it has been shown that RRM2 knockdown itself has a growth inhibitory effect. Here we compare the effects of siRNA-mediated knockdown of both RRM1 and RRM2 subunits of RR in A549 and HCT-116 cells using an optimized transfection protocol. Growth of A549 cells was strongly inhibited by efficient siRNA-mediated silencing of either RRM1 or RRM2, and knockdown of each subunit led to long-term growth inhibition and cell-cycle arrest. Knockdown with sub growth inhibitory siRNA concentrations sensitized A549 and HCT-116 cells to gemcitabine when RRM1 was targeted, whereas RRM2 knockdown led to hydroxyurea sensitization. These results suggest that the inhibition of cell growth, rather than drug sensitization, is the major effect of RRM1 and RRM2 knockdown. In an A549 xenograft model, cells transfected with RRM1-specific siRNA failed to form tumors in 6 out of 8 CD1 nude mice, whereas those transfected with RRM2-specific siRNA grew but at a reduced rate. Taken together, these data demonstrate that siRNA-mediated knockdown of the RRM1 subunit is more effective than knockdown of RRM2 in inhibiting the growth of cancer cell lines and suggest that RRM1 is a potential target for nucleic acid-based cancer therapies, either alone or in combination with gemcitabine.
Insights
Silencing Ribonucleotide reductase (RR) subunits RRM1 or RRM2 inhibits cancer cell growth. RRM1 subunit silencing showed greater efficacy in preclinical models, suggesting RRM1 as a promising target for nucleic acid-based cancer therapies.
Area of Science:
- Molecular Biology
- Cancer Therapeutics
- Genetics
Background:
- Ribonucleotide reductase (RR) is crucial for DNA synthesis and repair, making it a key target in cancer therapy.
- Previous research indicated RNAi-mediated knockdown of RR subunits (RRM1, RRM2) sensitizes cancer cells to cytotoxic drugs and RRM2 knockdown inhibits growth.
Purpose of the Study:
- To compare the effects of siRNA-mediated knockdown of RRM1 and RRM2 subunits of RR in A549 and HCT-116 cancer cell lines.
- To evaluate the potential of RRM1 and RRM2 as targets for novel cancer therapies.
Main Methods:
- Utilized an optimized transfection protocol for siRNA delivery in A549 and HCT-116 cells.
- Assessed cell growth inhibition, cell-cycle arrest, and drug sensitization (gemcitabine, hydroxyurea) following RRM1/RRM2 knockdown.
- Evaluated RRM1 and RRM2 siRNA efficacy in an A549 xenograft mouse model.
Main Results:
- Efficient siRNA-mediated silencing of RRM1 or RRM2 strongly inhibited A549 cell growth, inducing long-term inhibition and cell-cycle arrest.
- RRM1 knockdown sensitized cells to gemcitabine, while RRM2 knockdown sensitized cells to hydroxyurea, at sub-growth inhibitory concentrations.
- RRM1-specific siRNA transfection prevented tumor formation in 6/8 mice, whereas RRM2-specific siRNA resulted in reduced tumor growth in a xenograft model.
Conclusions:
- siRNA-mediated knockdown of RRM1 is more effective than RRM2 in inhibiting cancer cell line growth.
- RRM1 inhibition demonstrates significant potential as a therapeutic strategy, either alone or in combination with gemcitabine, for nucleic acid-based cancer treatments.
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