A Systems Biology Approach Identifies SART1 as a Novel Determinant of Both 5-Fluorouracil and SN38 Drug Resistance in Colorectal Cancer

Results from the initial siRNA screen in the HCT116 cells. The graphs show the relative toxicity, as assessed from MTT and toxiLight assays, for all positive genes from the clinical analysis (A), the 5-FU in vitro analysis (B), and the SN38 in vitro analysis (C). Displayed are those genes that show an interaction between gene silencing and either 5-FU or SN38 treatment as measured by 2-way ANOVA. Synergistic interactions are highlighted as *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Results from the cell line panel siRNA screen. The graphs show the relative toxicity, as assessed from MTT and toxiLight assays, for SART1, PTRF, and RAPGEF2 for the LoVo (A), RKO (B) LS174T (C), HT29 (D), and SW620 (E) cell lines. Displayed is the effect of gene silencing alone and in combination with either 5-FU or SN38 treatment. Statistical significance between gene silencing alone (siRNA) and gene silencing in combination with chemotherapy was assessed by an unpaired 2-way t test. Statistical significance are highlighted as *, P < 0.05; **, P < 0.01; ***, P < 0.001. Two siRNA sequences were included for each gene, indicated by (1) and (2). ns, not significant.

A, Western blot analysis of SART1 expression and PARP cleavage in HCT116 cells transfected with control (−) or SART1 (+) siRNAs and cotreated with 5-FU (IC_30(48h)) or SN38 (IC_30(48h)) for 24 and 48 hours. B–D, flow cytometric analyses of apoptosis following SART1 silencing alone or in combination with either 5-FU (IC_30(48h)) or SN38 (IC_30(48h)) in HCT116 (B), RKO (C), and LS147T (D). All cells were reversed transfected with SART1 siRNA for 24 hours before a 48 hours treatment with chemotherapy.

Cell viability assays were conducted in HCT116 (A and B), LS174T (C and D), and RKO cell lines (E and F) in response to SART1 siRNA (0.5, 1 and 5 nmol/L) and either SN38 (1, 5, and 10 nmol/L) or 5-FU (1, 5 and 10 μmol/L) for 72 hours. To evaluate the interaction between chemotherapy and SART1 silencing, we used the method of Chou and Talalay (26). CI values <1, = 1, and >1 indicate synergism, additivity, and antagonism, respectively. For synergistic interactions, CI values between 0.8 and 0.9 indicate slight synergy, 0.6 and 0.8 indicate moderate synergy, 0.4 and 0.6 indicate synergy and those less than 0.4 indicate strong synergy.