The human colon cancer cell line HCT116 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (both from Invitrogen-Gibco, Carlsbad, CA, USA), 100 U/ml penicillin, and 0.1 mg/ml gentamycin in a humidified incubator maintained at 37°C and 5% CO₂. Cells were treated for 24 h with DFO and then exposed for 6 h to 200 ng/ml TRAIL with or without the autophagy inhibitor chloroquine (10 μM) (both from Sigma-Aldrich, St. Louis, MO, USA).

Cell morphology was assessed microscopically (inverted microscope, Nikon), and cell viability was determined by crystal violet staining, as previously described (23). Briefly, cells were stained for 10 min at room temperature with crystal violet solution (0.5% crystal violet in 30% ethanol and 3% formaldehyde), washed five times with water, and then dried. After that, the cells were lysed with 1% sodium dodecyl sulphate (SDS), and the absorbance was measured at 550 nm. Cell viability was calculated from the relative dye intensity of the samples compared to the controls.

The number of viable cells was determined by trypan blue dye exclusion (Sigma-Aldrich) using a hemocytometer. The result was expressed as a percentage relative to the vehicle-treated controls.

HCT116 cells were lysed in lysis buffer [25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, 100 mM NaCl, 1 mM ethylene diamine tetraacetic acid (EDTA), 5 mM MgCl₂, 0.1 mM dithiothreitol (DTT) and a protease inhibitor mixture], and whole cell proteins were electrophoretically resolved on a 10–15% sodium dodecyl sulfate polyacrylamide gel and transferred to a nitrocellulose membrane. Immunoreactivity was detected through sequential incubation with primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and enhanced chemiluminescence reagents (Westsave Gold Detection kit; AbFrontier Inc.). The primary antibodies used for immunoblotting were anti-human HIF-1α (BD Biosciences), anti-LC3B (Cell Signaling Technology), anti-P62 (Millipore Corporation), anti-phospho-AKT (Epitomics, Burlingame, CA, USA) and anti-β-actin (Sigma-Aldrich). Images were examined using a Fusion FX7 imaging system (Vilber Lourmat, ZI Sud Torcy, France). The densitometry of the signal bands was analyzed using Bio-1D software (Vilber Lourmat, Marne la Vallée, France).

The unpaired t-test or Welch’s correction was used for comparison between the two groups. For multiple comparison, the one-way ANOVA followed by the Tukey-Kramer test was used. All statistical analysis was performed using GraphPad Prism software. Results were considered significant for values p<0.05, p<0.01 or p<0.001.

We examined whether DFO promotes or inhibits TRAIL-induced cell death and whether this effect is associated with induction of autophagy. We investigated the influence of DFO on TRAIL-mediated cytotoxicity in HCT116 colon cancer cells by images and crystal violet assay. HCT116 cells were exposed to DFO with or without TRAIL. The cell viability of the TRAIL-treated cells was decreased more than half according to images captured by light microscopy and crystal violet assay. The cell viability of the cells treated with DFO only was comparable to that of the untreated controls. These results revealed that DFO treatment inhibited TRAIL-induced cytotoxicity in HCT116 colon cancer cells (Fig. 1A–C). Trypan blue exclusion assay was implemented for cell viability (Fig. 1D).

If cell death is induced by cytotoxicity in cells, then cells cannot generate exocytosis. Thus, trypan blue particles can flow in cells easily. For this reason, trypan blue dyed cells represent dead cells. These comprehensive results exhibited that DFO treatment inhibited cell death induced by TRAIL treatment dose-dependently. These results indicate that DFO was effective in preventing TRAIL-induced cell death in HCT116 colon cancer cells.

We identified the proliferative and protective effects of DFO on cancer cells. We hypothesized that DFO would play a role of an HIF-1α inducer not an iron chelator, since HIF-1α is involved in critical aspects of cancer biology such as angiogenesis and cell survival (15). We investigated whether DFO induces HIF-1α stabilization by western blot analysis. As shown in Fig. 2A and B, the level of HIF-1α was increased in the DFO-treated group in a dose-dependent manner compared with the control group as detected by western blot analysis and densitometry. These results suggest that DFO leads to stabilization of HIF-1α. We investigated whether DFO affects survival and death signals. Akt affects cell survival and metastasis in many types of cells including colon cancer cells. Akt acts as a key signal that is associated with oncogenic receptors to many essential prosurvival cellular functions in human cancer (24). Caspase-3 plays a key role in regulating programmed cell death or apoptosis, a normal process required for maintenance of the regulation of physiological functions (25). Our data showed that DFO treatment recovered Akt activation from reduction by TRAIL treatment (Fig. 2A and C) and decreased the caspase-3 cleavage induced by TRAIL (Fig. 2A and D). Thus, these results suggest that DFO confers a protective effect from TRAIL in colon cancer cells.

LC3 protein is considered to be an autophagy marker and is localized and aggregated on autophagosomes. LC3 transforms from LC3-I to LC3-II during autophagosome formation (26). We examined whether DFO induces autophagy by assessing LC3 transformation. As shown in Fig. 3, the level of LC3-II was increased and P62 expression was decreased in the DFO-treated group in a dose-dependent manner compared with the control group as detected by western blot analysis and densitometry (Fig. 3A and B). P62 is considered as a crucial mediator for target protein to the autophagy system in removal of aggregated proteins. P62 is degraded by itself during autophagy (27). We used the autophagy inhibitor, chloroquine (CQ), that is widely used to inhibit the maturation of autophagosomes into degradative autolysosomes (18,28). We identified that DFO-induced upregulation of LC3-II was increased by CQ treatment since CQ inhibited the fusion of autophagosomes and autolysosomes (Fig. 3C). For this reason, P62 was increased by CQ treatment (Fig. 3C and D).

We analyzed the cell viability using CQ to investigate the effect of DFO on the activation of autophagy. We identified that the protective effect of DFO was reversed by CQ in regards to cell viability by crystal violet assay and graphical analysis (Fig. 4A and B). Photographed images and trypan blue exclusion assay were implemented for cell viability. Inhibition of cell death by DFO treatment was reversed by CQ pre-treatment (Fig. 4C and D). These results strongly indicate that DFO treatment inhibits TRAIL-mediated cytotoxicity through induction of autophagy.