Figure 2.
siRNA-mediated silencing of STAT3 results in a significant sensitization to chemoradiotherapy. (a) Total STAT3 and pSTAT3Tyr705 protein levels decreased 96 hr after transfection (and stimulation with 100 ng/ml IL-6 for 30 min) with two different siRNAs targeting STAT3 compared to a nonspecific negative-control (siNEG) in SW480 (left) and SW837 (right). STAT3 mRNA silencing was established 72 hr after transfection using RT-PCR (P < 0.05). (b) STAT3 transcriptional activity was determined 96 hr after transfection (and stimulation with 100 ng/ml IL-6 for 30 min) using a dual luciferase reporter assay in SW480 (left) and SW837 (right). A negative reporter plasmid served as control. Silencing of STAT3 led to a significant reduction of STAT3 transcription factor activity (P < 0.05). (c) Seventy-two hours after transfection, SW480 (left) and SW837 cells (right) were preincubated with 3 μM of 5-FU for 16 hr, and subsequently irradiated at 1, 2, 4, 6 and 8 Gy of X-rays (without stimulation with IL-6). Silencing of STAT3 significantly increased the sensitivity of both SW480 (P = 0.018; ANOVA model) and SW837 (P = 0.006) to 5-FU-based chemoradiotherapy. Each experiment was repeated three times. Data are displayed as mean values, n = 3, error bars ± SEM.