HCT116 and SW480 CRC cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Paisley, Scotland, UK) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA), and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) in a humidified 5% CO₂ atmosphere at 37°C. Sodium selenite was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against cleaved caspase-9, cleaved PARP, LC3 or Beclin-1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies to β-actin were purchased from Sigma-Aldrich. The p62 antibody was purchased from Abcam (Cambridge, MA, USA).

Cells were lysed in RIPA buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na₃VO₄, 1 µg/ml leupeptin and 1 mM PMSF). The total cell lysates were sonicated and collected by centrifugation prior to concentration determination using the Bradford method. The proteins were resolved on 8–15% SDS-PAGE, and then electro-transferred to nitrocellulose membranes. Subsequently, the blots were incubated with the indicated primary antibodies and the corresponding HRP-conjugated secondary antibodies. The immunoreactive bands were visualized by chemiluminescent reagents from Thermal Scientific.

Cells were grown on glass slides for 24 h before treatment with 10 µM selenite for 24 h. The cells were incubated with LC3 primary antibodies overnight at 4°C, and were then incubated with FITC fluorescence-labeled secondary antibodies for 1 h at room temperature, followed by staining with DAPI solution to visualize the cell nuclei. The punctate of LC3 protein before and after treatment with selenite in the CRC cells was detected by an Olympus laser scanning confocal FV1000 microscope (Olympus, Tokyo, Japan) and analyzed by Olympus FluoView software.

GFP-LC3 plasmids were transfected into HCT116 and SW480 CRC cells using Lipofectamine 2000 according to the manufacturer's instructions. After another 24 h, the cells were treated with selenite or phosphate-buffered saline (PBS) as a solution control. The transfection efficiency was confirmed by western blotting.

The apoptotic rates of cells were determined using an Annexin V/PI double staining kit (Merck, Germany) according to the manufacturer's instructions. Then the cells were subjected to analysis by a C6 Accuri flow cytometer.

TEM was used to observe autophagy and ultrastructural changes in the HCT116 and SW480 cells 24 h after selenite treatment. Fixed cells were post-fixed in 2% OsO₄, dehydrated in graded alcohol and flat-embedded in Epon 812 (Electron Microscopy Sciences, Fort Washington, PA, USA). Ultra-thin sections (100 nm) were prepared, stained with uranyl acetate and lead citrate, and examined under an electron microscope (H-600; Hitachi, Japan).

The present study was approved by the Ethics Committee of the Institute of Basic Medical Science. Principles of laboratory animal care were followed and complied with standards equivalent to the guidelines for the welfare of animals in experimental neoplasia.

BALB/c nude mice (4 weeks old) were purchased from the Institute of Laboratory Animal sciences. Twenty-eight nu/nu mice were randomly assigned to four groups and subcutaneously injected with HCT116 or SW480 CRC cells which were suspended in serum-free DMEM at a concentration of 2×10⁷ cells/ml in the left shoulder of the nude mice. After the tumors were palpable, half of the mice were injected i.p. with sodium selenite (2 mg/kg/day). The control group was injected with 0.9% sodium chloride, at a volume of ~200 µl/20 g/day. At the end of the experiment, the mice were sacrificed by cervical dislocation, and the tumors and livers were rapidly removed and weighed.

Tumor tissues from the control and selenite-treated groups were sectioned and deparaffinized in xylene and dehydrated with graded ethanols in accordance with the routine method. The slides were incubated with primary antibodies against cleaved caspase-9, Beclin-1, p62 or LC3 overnight at 4°C. After being washed in PBS, the slides were incubated with HRP-conjugated secondary antibody at room temperature for 2 h treated with diaminobenzidine working solution, and then counterstained with Mayer's hematoxylin for 1 min. Finally, the slides were dehydrated with increasing concentrations of ethanol and clarified with xylene.

All of the above experiments were repeated at least three times. The results are expressed as the mean ± SD (n≥3). In addition, Student's t-test was applied to assess the statistically significant difference (P<0.05).

We previously showed that supranutrional selenite treatment induced apoptosis in HCT116 and SW480 CRC cells (21). As shown in Fig. 1A, the expression of the autophagy markers and Beclin-1, was increased. p62 is a specific substrate of autophagy, which was decreased in response to selenite treatment. We detected the conversion of microtubule-associated protein light chain 3 (LC3) (from LC3-I to LC3-II) in response to selenite treatment. Increased punctate of GFP-LC3 was noted in the cells treated with selenite (Fig. 1B). Consistently, from the electron micros-copy results (Fig. 1C), we observed more autophagosomes in the selenite-treated CRC cells. All the results collectively showed that sodium selenite treatment increased autophagy in the HCT116 and SW480 CRC cells.

3-Methyladenine, an inhibitor of autophagy initiation (22), was used to inhibit selenite-induced autophagy. Bafilomycin A1 was used to inhibit the degradation of autophagosomes by lysosomes (23). Using western blotting, we showed that the conversion of LC3 was reduced in the 3-MA-treated samples, and in contrast, however, LC3-II was accumulated in the bafilomycin A1-treated cells. From the western blot results, cleaved PARP and cleaved caspase-9 were greatly increased when autophagy was inhibited compared with selenite treatment alone (Fig. 2A). Additionally, from the Annexin V/PI double staining assay (Fig. 2B), we also concluded that when autophagy was inhibited, the apoptotic rate increased. Rapamycin is widely used to activate autophagy through its inhibitory effect on mTOR. From the western blot results, we discovered that LC3 conversion was increased in the rapamycin-treated cells compared with the control or selenite treatment group. By detecting cleaved PARP and caspase-9, we found that the levels of cleaved caspase-9 and PARP were decreased when autophagy was activated (Fig. 2C). In accordance with the western blot results using Annexin V/PI double staining assays, we found that the apoptotic rate was decreased from 30.1 to 15.2% and 31.1 to 10.3% in the HCT116 and SW480 cells, respectively (Fig. 2D). When the cells were treated with pancaspase inhibitor, Z-VAD-fmk, the punctate of LC3 disappeared compared with the control (Fig. 2E). This demonstrated that selenite-induced autophagy was elicited by selenite when apoptosis was induced in CRC cells. These results indicate that autophagy may facilitate the survival of cells by antagonizing the proapoptotic effect of selenite.

To further investigate the effect of sodium selenite on the growth of tumors in xenograft models, we subcutaneously inoculated HCT116 and SW480 cells in nude mice and developed tumors to a palpable size. Sodium selenite diet was given daily. After 21 days, the tumors were extracted and photographed. The results are shown in Fig. 3A and B; 2 mg/kg/day selenite treatment inhibited both HCT116 and SW480 tumor volume. The tumor weight was analyzed and the results are show in Fig. 3A and B (upper panel). Tumor weight in the 2 mg/kg/day selenite treatment group was significantly decreased compared with the control. Moreover, sodium selenite treatment had no obvious effect on the body weight of the mice (Fig. 3C and D). By H&E staining of the tumor and liver, compared with the control group, the selenite-treated group showed more pathological changes including some necrotic and apoptotic cells. Both HCT116 and SW480 tumors from the control group showed uniform large polymorphic, hyperchromatic spindle-shaped cells and irregularly dispersed chromatin with a high nuclear/cytoplasmic ratio (Fig. 3E). These results collectively showed that selenite treatment inhibited tumor growth in both the HCT116 and SW480 colon xenograft models.

To further analyze the effect of selenite on apoptosis and autophagy in the xenograft models, we exploited western blotting and immunohistochemical assays to analyze changes in levels of apoptosis and autophagy markers in the tissues. More cleaved PARP and caspase-9 were observed in the selenite-treated samples (Fig. 3G). Autophagy markers, Beclin-1 and LC3 were increased in the context of selenite treatment. Consistently, p62 was downregulated in the selenite-treated tumors, and these results were consistence with those in the cell culture experiments. Additionally, in the immunohistochemical experiments (Fig. 3F) we also verified the conclusion that selenite treatment induced apoptosis and autophagy in the xenograft tumors.

To explore the effect of ROS on selenite-induced apoptosis and autophagy, we modulated the ROS level in cells using MnTMPyP and H₂O₂. When ROS in CRC cells were scavenged with MnTMPyP, the punctate of LC3 disappeared (Fig. 4A) and cleaved PARP was decreased significantly even in the presence of selenite treatment (Fig. 4C). While the cells were pretreated with H₂O₂ to augment ROS level, we observed increased punctate of LC3 from confocal (Fig. 4A) and increased PARP cleavage from western blot results (Fig. 4C). Accordingly, from Annexin V/PI double staining assay, we found that the apoptotic rate was decreased when MnTMPyP was added. The opposite results were shown when using H₂O₂ compared with the MnTMPyP-treated cells (Fig. 4B). Finally, we detected the change in the autophagy marker LC3 in the samples treated with MnTMPyP and found a decrease in the conversion of LC3. We also observed an opposite trend of change in the H₂O₂-treated cells (Fig. 4C). We concluded that selenite-induced apoptosis and autophagy may be caused by ROS through some unknown mechanism.