Figure 7.

Dual absence of p53 and p21 causes resistance to the MC3-mediated cytotoxicity. (A) HCT116 WT and p53^−/− cells were transfected with either NC- or anti-p21 siRNA and treated with a concentration of 5 μM of MC3 for 24 h, after which cell viability was assessed using MTT assay. Error bars represent the SD from two independent experiments, each was done in triplicates. (B) Confirmation of p21 knock-down determined by immunoblotting. Numbers denote values obtained from densitometric analyses of the presented blots normalized to the respective loading controls (β-actin). (C) The ROS scavenger, NAC attenuates the MC3-triggered p21 induction in the three CRC cells, as well as pp38 MAPK (T180/Y182) levels and PARP cleavage in the HCT116 clones, detected by immunoblotting. Cells were treated with increasing concentration of MC3 as indicated. NAC (10 mM) was added 1 h prior to the addition of MC3 at the concentrations indicated. (D) Densitometric analyses show a significant reduction in MC3-induced pp38 and p21 protein levels in the presence of anti-oxidant treatment. Error bars represent the SEM of four biological replicates, one of which is depicted in (C). Vinculin served as the loading control. 0.1% DMF was used as mock treatment. Multiple comparisons were performed using two-way ANOVA and a post-hoc Tukey test. (E) Schematic overview of the small molecule's anti-proliferative and pro-apoptotic actions in CRC cells with different p53 backgrounds. *, **, and **** are representative of p-values ≤ 0.05, 0.01, and 0.0001, respectively.