Figure 1.
Induction of caspase-independent but AIF/EndoG-dependent apoptosis by the anticancer peptide FK-16 in colon cancer cells.
(A) Effects of LL-37, FK-16, and scrambled FK-16 (48 h) on viability of colon cancer cells (HCT116, LoVo) were determined by MTT assay. The cytotoxic effect of FK-16 was also assayed in human normal colon mucosal epithelial NCM460 cells. (B) Phosphotidylserine externalization in HCT116 cells treated with or without FK-16 was determined by flow cytometry of PI/Annexin V-stained cells. (C) Effects of FK-16 on PARP cleavage and caspase activation in HCT116 cells were determined by Western blot. Cisplatin was used as a positive control for caspase activation. (D) Pre-treating HCT116 cells with the pan-caspase inhibitor z-VAD-fmk for 1 h did not prevent FK-16-induced loss of cell viability as determined by MTT assay (24 h). (E) Nuclear expression of AIF and EndoG in HCT116 cells were analyzed by Western blot analysis of fractionated proteins. GAPDH and Lamin A/C were used as internal controls for cytosolic (Cy) and nuclear (Nu) proteins, respectively. (F) Effects of FK-16 (6 h) on subcellular localization of AIF (green) and EndoG (red) in HCT116 were determined by confocal immunofluorescence (400×). (G) Knockdown of AIF and EndoG partially reversed phosphotidylserine externalization induced by FK-16 (40 µM; 24 h) in HCT116. Cells were challenged with FK-16 at 48 h post-transfection of AIF- and EndoG-siRNAs. Data were presented as mean ± SD from three separate experiments. *, p<0.05; **, p<0.01, significantly different from the respective control group.
Figure 2.
Induction of autophagic cell death by FK-16 in colon cancer cells.
(A) HCT116 cells exhibited increased protein expression of LC3-I/II after treatment with FK-16 for 24 h and 48 h. LC3 expression induced by FK-16 was abolished by the autophagy inhibitor 3-MA (5 mM, 1 h pre-treatment). (B) FK-16 also induced Atg5 and Atg7 protein expression. (C) Treating HCT116 with FK-16 for 24 h or 48 h prominently enhanced the formation of autophagic vacuoles as determined by immunofluorescent staining for LC3 (400×). The formation of LC3+ autophagosomes induced by FK-16 was blocked by 3-MA. (D) Ultrastructural analysis by electron microscopy revealed the formation of autophagosome or secondary lysosomes with the residual digested material in FK-16-treated HCT116 cells (40 µM; 48 h). (E) The accumulation of acidic vesicular organelles, which emitted bright red fluorescence, induced by FK-16 (40 µM) was visualized by acridine orange staining (400×). (F) Increased autophagic flux was confirmed by co-treating HCT116 cells with FK-16 and bafilomycin A1. Treatment with bafilomycin A1 (10 nmol/L) did not prevent the upregulation of LC3-I/II in cells incubated with FK-16 (40 µM). (G) Knockdown of Atg5 or Atg7 attenuated the cytotoxic effect of 48-h treatment of FK-16 (40 µM) in HCT116. 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.
Figure 3.
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.
Figure 4.
Altered expression of Bcl-2 and Bax was required for AIF/EndoG-dependent apoptosis and autophagic cell death induced by FK-16.
(A) Restoration of Bcl-2 expression by transfection with Bcl-2-encoding plasmid reversed the upregulation of pro-autophagic (Atg5, Atg7 and LC3-I/II) and pro-apoptotic (nuclear AIF and nuclear EndoG) factors induced by FK-16 (40 µM; 48 h) in HCT116. (B) Genetic ablation of Bax (Bax KO) in HCT116 reversed FK-16-induced apoptotic and autophagic signals. Bax+/− HCT116 treated with or without FK-16 was used as control. (C) Restoration of Bcl-2 expression or genetic ablation of Bax in HCT116 abolished the formation of LC3+ autophagic vacuoles induced by FK-16 (40 µM; 48 h) as determined by confocal immunofluorescence (400×). (D) Restoration of Bcl-2 expression or genetic ablation of Bax 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<0.01 significantly different from empty vector-transfected or Bax+/− cells treated with FK-16.
Figure 5.
Reciprocal regulation of AIF/EndoG-dependent apoptosis and autophagic cell death induced by FK-16.
(A) The knockdown efficacies of AIF-, EndoG-, Atg5- and Atg7-siRNAs were confirmed by downregulation of protein levels of respective targets in HCT116. (B) Knockdown of Atg5 or Atg7 enhanced basal and FK-16-induced (40 µM; 6 h) nuclear expression of AIF and EndoG. (C) Knockdown of Atg5 or Atg7 increased FK-16-induced (40 µM; 48 h) phosphotidylserine externalization as determined by annexin V staining and flow cytometry. (D) Knockdown of AIF or EndoG enhanced basal and FK-16-induced (40 µM; 48 h) Atg5 and Atg7 protein expression. (E) Knockdown of Atg5 or Atg7 abolished whereas Knockdown of AIF or EndoG enhanced FK-16-induced (40 µM; 48 h) LC3-I/II expression in HCT116. Data are representative of three independent experiments.
Figure 6.
Schematic diagram showing the common regulation of caspase-independent apoptosis and autophagic cell death by the p53-Bcl-2/Bax cascade and their reciprocal regulation induced by FK-16 in colon cancer cells.