FIGURE 2.

RIS exposure results in drug resistance by modulation of proapoptotic molecules. A, HCT-116, p53 knockout, and MIC-1-deficient cells were pre-exposed to increasing doses (100, 300, and 500 μg/ml) of DON or 25, 50, and 100 ng/ml of ANS for 24 h and treated with 375 μm 5-FU for 48 h. Detached cells were collected, stained with trypan blue, and counted with a hemocytometer for evaluation of cytotoxicity to 5-FU. Statistically significant results by unpaired t test are presented by repetitive experiments (***, p < 0.001). Con, control. B, HCT-116 cells pre-exposed to corresponding doses of DON or ANS for 24 h were treated with 100 μm 5-FU for 48 h. Whole cell lysates were analyzed by Western blotting with components of apoptosis-signaling molecules. hnRNP, heterogeneous nuclear ribonucleoprotein. C and E, DON-pre-exposed (500 ng/ml DON for 24 h), p53 knockout, or MIC-1-deficient HCT-116 cells were treated with 100 μm 5-FU or 100 nm paclitaxel, respectively, for 48 h and then analyzed by Western blotting with PARP1/2, MIC-1, and p53 apoptotic signaling molecules. Heterogeneous nuclear ribonucleoproteins were used as a loading control. D and F, HCT-116 cells were pre-exposed to 500 ng/ml DON for 24 h. p53 knockout cells and MIC-1-deficient cells were treated with 100 μm 5-FU (D) and 100 nm paclitaxel (F), respectively, for 24 h. Cells were fixed, and PI-positive apoptotic cells were verified by flow cytometry. G, HCT-116 cells pre-exposed to 50 ng/ml ANS for 24 h were treated with 100 nm paclitaxel for 24 h. Whole cell lysates were analyzed with MIC-1, p53, and PARP 1/2 preapoptotic components by Western blotting. H, PI-positive dead cells were verified by flow cytometry. HCT-116 cells pre-exposed to 50 ng/ml ANS for 24 h were evaluated for PI-positive apoptotic cells in response to paclitaxel.