Inhibition of pancreatic tumor growth by NOX inhibition.

<p>(A) Decrease of NOX activity by stable shRNA knockdown of p22phox (p22phox-shRNA) in Panc-1 cells, but not in cells with control shRNA (c-shRNA) knockdown. *** <i>p</i><0.001 (<i>n</i> = 3). Insert, Panc-1 cells were infected with p22phox-shRNA lentiviral particles, and the knockdown efficiency was detected by anti-p22phox antibody using Western blot. Non-targeting control shRNA lentiviral particles (c-shRNA) were used as negative control. (B) p22phox knockdown significantly decreased Panc-1 cell growth. 1×10⁵ cells were seed to six-well plates. Cell numbers were counted using Z2 coulter counter (Beckman Coulter) during 6 d of culture. (C) p22phox knockdown significantly suppressed colony formation of Panc-1 cells. 1×10⁴ cells were seed in 0.35% of the soft agar and assayed for colony formation after 2 wk. The numbers of colonies formed on soft agar were counted. Error bars, ±SD. *** <i>p</i><0.001 (<i>n</i> = 3). (D) p22phox knockdown decreased glucose uptake in Panc-1 cells. Cells (1×10⁶) were incubated in 5 ml glucose-free DMEM medium for 4 h, followed by incubation with 0.2 µCi/mL ³H-2-deoxyglucose for 1 h. Cellular uptake of ³H-2-deoxyglucose was determined by liquid scintillation counting after the cells were washed 2 times with PBS and normalized by cell number. Error bars, ±SD. * <i>p</i><0.05 (<i>n</i> = 3). (E) p22phox knockdown decreased lactate generation in Panc-1 cells. Lactate was measured 24 h after changing to fresh culture medium and normalized by cell number. Error bars, ±SD. ** <i>p</i><0.01 (<i>n</i> = 3). (F–I) Each side of athymic nude mice (<i>n</i> = 7) received subcutaneously injections of 5×10⁶ Panc-1 cells bearing p22phox-shRNA (left flank) or c-shRNA (right flank). The mice were monitored for tumor growth and body weight throughout the experiment. All the mice were sacrificed when tumor size reached about 10% of body weight as mandated by the IACUC protocol. Tumor volume was calculated using the following equation: tumor volume (mm³) = L * W *(L+W)/2 * 0.526. (F) The tumor sizes were measured throughout the experiment to evaluate p22phox knockdown effect. Data represent tumor volume: mm³±SEM. *** <i>p</i><0.001 (<i>n</i> = 7). (G) Photographs of athymic nude mice bearing p22phox-shRNA (left flank) or c-shRNA (right flank) xenografts. (H) Photograph and comparison of excised tumor size. (I) Tumor weight derived from p22phox-shRNA knockdown or c-shRNA knockdown was measured. Error bars, ±SEM. <i>p</i><0.05 (<i>n</i> = 7).