Retrovirally mediated RNA interference targeting the M2 subunit of ribonucleotide reductase: A novel therapeutic strategy in pancreatic cancer
Other Scholarly Work
Duxbury, MS, Ito, H, Benoit, E et al. (2004). Retrovirally mediated RNA interference targeting the M2 subunit of ribonucleotide reductase: A novel therapeutic strategy in pancreatic cancer
. SURGERY, 136(2), 261-269. 10.1016/j.surg.2004.04.029
Duxbury, MS, Ito, H, Benoit, E et al. (2004). Retrovirally mediated RNA interference targeting the M2 subunit of ribonucleotide reductase: A novel therapeutic strategy in pancreatic cancer
. SURGERY, 136(2), 261-269. 10.1016/j.surg.2004.04.029
Background Ribonucleotide reductase M2 subunit (RRM2) overexpression enhances tumor chemoresistance and cellular invasiveness. We hypothesized that the RNA interference (RNAi) induced by retrovirally delivered small interfering RNA (siRNA) would sensitize pancreatic adenocarcinoma cells to gemcitabine and attenuate their invasive potential. Methods Stable suppression of RRM2 expression in PANC1, MIAPaCa2, BxPC3, and Capan2 cells was induced by exposure to a novel replication-deficient retrovirus, engineered to express RRM2-specific siRNA (psiRRM2), and confirmed by Western blot analysis. Single-base mismatch vector (psiControl) served as control. Ribonucleotide reductase activity was quantified, and gemcitabine 50% inhibitory concentrations were calculated. TUNEL staining and caspase profiling were performed after gemcitabine exposure. Cellular invasiveness was quantified in a Matrigel Boyden chamber. NF-κB activity and matrix metalloproteinase-9 (MMP-9) expression and activity were measured. Results RRM2 expression was stably and specifically suppressed in psiRRM2, but not psiControl transfectants. psiRRM2 transfectants exhibited lower 50% inhibitory concentrations, increased gemcitabine-induced apoptosis, and greater caspase-3 activation, relative to psiControl transfectants. Invasiveness was attenuated in psiRRM2 transfectants, as was NF-κB activity, MMP-9 expression, and MMP-9 activity, relative to psiControl transfectants. Conclusions RRM2 gene silencing attenuates pancreatic adenocarcinoma cellular invasiveness and gemcitabine chemoresistance. Retroviral siRNA delivery can efficiently induce stable RNAi, allowing dissection of gene function and potentially representing a new therapeutic modality.
