Activation of p53 was required for AIF/EndoG-dependent apoptosis and autophagic cell death induced by FK-16.

(A) HCT116 cells were incubated with FK-16 for 24 h or 48 h. Cytosolic and nuclear p53 and total expression of Bcl-2 members (PUMA, Bcl-2, Bax and Bak) were determined by Western blot. GAPDH and Lamin A/C were used as internal controls for cytosolic and nuclear proteins, respectively. (B) Knockdown of p53 reversed the upregulation of pro-autophagic factors (Atg5 and Atg7) and pro-apoptotic factors (Bax, Bak, nuclear AIF and nuclear EndoG) as well as downregulation of Bcl-2 by FK-16. (C) FK-16 failed to induced phosphotidylserine externalization in p53-depleted HCT116 cells. After transfection with control- or p53-siRNA for 48 h, cells were treated with or without FK-16 (40 µM) for another 24 h followed by propidium iodide/annexin V-double staining. (D) Knockdown of 53 markedly reduced the number of LC3⁺ autophagic vacuoles in FK-16-treated cells (40 µM; 48 h) as determined by confocal immunofluorescence (400×). Nuclei (blue) were stained with DAPI. (E) Knockdown of p53 partially reversed the inhibitory effect of FK-16 on cell viability in HCT116 as determined by MTT assay. Data are presented as means ± S.D. of three separate experiments. *, p<0.05; **, p<0.01 significantly different from the respective control group. ^†, p<0.05; ^††, p<0.01 significantly different from control siRNA-transfected cells treated with FK-16.