MK-2206 was purchased form Abmole Bioscience, Inc. (Houston, TX, USA). Bafilomycin A1 (BafiA1) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). DMSO and DAPI were purchased from Beyotime Institute of Biotechnology (Haimen, China). Anti-RI (cat. no. ab245627), anti-autophagy-related protein (ATG)5 (cat. no. ab228668), anti-ATG7 (cat. no. ab223365), anti-ATG13 (cat. no. ab201467), anti-beclin-1 (BECN1; cat. no. ab62557), anti-LC3/II (cat. no. ab51520), anti-β-Actin (cat. no. ab119716) and goat anti-rabbit immunoglobulin G (IgG; Alexa Fluor^® 488-conjugated; cat. no. ab150077) antibodies were purchased from Abcam (Cambridge, UK). Anti-Akt (cat. no. 9272), anti-phosphorylated (p)-Akt (cat. no. 9271), anti-mTOR (cat. no. 2972), anti-p-mTOR (cat. no. 2971), anti-ULK1 (cat. no. 6888), anti-p-ULK1 (cat. no. 5869), anti-AMPK (cat. no. 2532S), anti-p-AMPK (cat. no. 2535), anti-P62/sequestosome 1 (SQSTM1; cat. no. 38749), goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated (cat. no. 7074) and goat anti-mouse IgG HRP-conjugated (cat. no. 7076) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

The human CRC cell line HT29 was obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and was cultured in RPMI1640 Medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and 100 U/ml penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. Cells were treated with vehicle (0.1% DMSO) or Akt inhibitor MK-2206 (5 µM) at 37°C for 24 h, and the levels of LC3-II were examined by western blotting. Cells were seeded at a density of 2×10⁵ cells/well into six-well plates and were treated with DMSO (0.1%) or bafilomycin A1 (1 µM) at 37°C for 72 h.

HT29 cells were seeded into six-well plates, and the pcDNA3.1-RI plasmid (4 µg/µl), maintained in the laboratory (12), was transfected using Lipofectamine™ 2000 (Thermo Fisher Scientific, Inc.). Approximately 48 h after transfection, 800 µg/ml of geneticin (G418) (Sigma-Aldrich; Merck KGaA) was added to the medium and incubated for 2 weeks to select the positive clones that would be employed in generating a stable transfected cell line, HT29/RI, that overexpresses RI. Mock cells were transfected with an empty vector and used as a control (HT29/vector).

All of the plasmids required for shRNA-mediated knockdown assays were reconstructed and provided by Genepharm, Inc. (Sunnyvale, CA, USA). The pGPU6/GFP/Neo-shRNA vectors used to knock down RI, ATG13, BECN1, mTOR and ULK1 were shRI (4 µg/µl), shATG13 (5 µg/µl), shBECN1 (4 µg/µl), shmTOR (4 µg/µl) and shULK1 (4 µg/µl), respectively. The transfection reagent Lipofectamine^® 2000 was used to transfer plasmid DNA into the HT29 cells. Non-targeting shRNA was used as negative control (shCTL; 4 µg/µl). Cells were harvested 48 h following transfection.

The cells were harvested, and total cell lysates were prepared using a protein extraction kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufacturer's protocol. Protein concentrations were quantified using a bicinchoninic acid kit (Beyotime Institute of Biotechnology). Equal amounts (20 µg per lane) of protein samples were separated using 8–12% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked in TBS with 3% nonfat milk for 2 h at 37°C, followed by incubation with primary antibodies at 4°C overnight. The primary antibodies used for western blotting were the following: Anti-RI (1:2,000), anti-ATG5 (1:500), anti-ATG7 (1:1,000), anti-ATG13(1:1,000), anti-(BECN1) (1:600), anti-LC3/II (1:3,000), anti-β-Actin (1:1,000) anti-Akt (1:500), anti-p-Akt (1:1,000), anti-mTOR(1:1,000), anti-p-mTOR (1:1,000), anti-ULK1 (1:500), anti-p-ULK1 (1:500), anti-AMPK (1:500), anti-p-AMPK (1:500), anti-P62/SQSTM1 (1:1,000). The following secondary antibodies were incubated for 1 h at 37°C: Goat anti-rabbit HRP-linked IgG (1:2,000) or goat anti-mouse HRP-conjugated IgG (1:2,000). Chemiluminescent signals were detected using the Enhanced Chemiluminescent Plus kit (GE Healthcare, Chicago, IL, USA) and the signal intensity was measured by densitometric analysis using the Versa-Doc™ Imaging system (version 4.0; Bio-Rad Laboratories, Inc., Hercules, CA, USA). β-actin was used as the internal control to normalize the samples.

Cells were grown in six-well plates and fixed with 3.5% formaldehyde in PBS for 10 min at room temperature, washed twice with PBS, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 0.5% bovine serum albumin (Beyotime Institute of Biotechnology) for 15 min at room temperature. Following two washes with PBS, the cells were incubated with an LC3-II primary-antibody (1:500) for 2 h at room temperature, followed by incubation with an Alexa Fluor^® 488-conjugated secondary antibody (1:200) for 1 h at room temperature. Fluorescent signals were detected using a fluorescence microscope (Olympus Corporation, Tokyo, Japan; magnification, ×400). Autophagy was quantified by counting the number of LC3-II dots per cell (a minimum of 50 cells per preparation in three independent experiments).

For the clonogenic assay, cells were transfected with control shRNA (shCTL), RI shRNA (shRI), RI plasmid (RI) or empty vector (Vector) for 48 h respectively. Cells in different groups were subsequently seeded with appropriate dilutions into 6 cm dishes, followed by incubation at 37°C for 2 weeks. Colonies were fixed with 4% paraformaldehyde at 4°C for 30 min, and subsequently stained with crystal violet (0.5% w/v) at room temperature for 5 min. The dishes were imaged using a light microscope (Olympus Corporation; magnification, ×400) and the numbers of distinct colonies (≥50 cells per colony) were counted under the microscope (small, <0.3 mm; medium, 0.3–0.6 mm; large, >0.6 mm). The results were averaged for each treatment group.

The cell viability was determined by Cell Counting Kit-8 (CCK-8) assay (Beyotime Institute of Technology). Briefly, cells in each group were plated at a density of 3,000 cells/well in 96-well plates, and incubated at 37°C with 5% CO₂ for 12, 24, 48 and 72 h. CCK-8 solution (10 µl) was added to each well, the optical absorbance was determined at 450 nm following a 3 h incubation using BioTek™ Epoch™ (BioTek Instruments, Inc., Winooski, VT, USA).

The data were analyzed using SPSS software 16.0 (SPSS, Inc., Chicago, IL, USA). The results are expressed as the mean ± standard error of the mean. Each experiment was repeated at least three times and analyzed using a Students' t-test or one-way analysis of variance followed by Tukey's test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Previous studies have demonstrated that Akt inhibition strongly activates autophagy (19), and that overexpression of RI suppresses the PI3K/Akt pathway in human CRC cells (12). To study a possible regulatory effect of RI on autophagy in HT29 cells, these cells were transfected with pcDNA3.1-RI recombinant vectors, and successfully transfected cells were selected for further analyses. Overexpression of RI markedly increased LC3-II levels in the HT29 cells (Fig. 1A), suggesting that increased RI expression induces autophagy. In addition, fluorescence microscopy was used to verify the generation of autophagosomes. An increase in the number of LC3-II puncta was observed in the HT29/RI cells (Fig. 1B), further substantiating the previous observations. As visible in Fig. 1B, the majority of LC3-II puncta were concentrated in the nucleus. The number of LC3-II puncta/cell significantly increased by >3-fold compared with the HT29 and HT29/Vector cells controls (Fig. 1C). LC3-II levels were also elevated during autophagosome-lysosome fusion or autophagic vesicle degradation. To verify that this LC3-II elevation was induced by the upregulation of RI, the autophagic flux in the HT29/RI cells was analyzed in the absence (Vehicle) or presence of BafiA1, an inhibitor of autophagosome-lysosome fusion and LC3-II degradation. As indicated in Fig. 1D, BafiA1 increased the levels of LC3-II and of the autophagy-specific substrate P62/SQSTM1 compared with the control and empty vector groups, suggesting that LC3-II accumulation in HT29/RI cells was attributable to the promotion of autophagy but not to autophagic degradation. Taken together, these results confirmed that the overexpression of RI triggered autophagy in human CRC cells.

A previous study demonstrated that the upregulation of RI affects the morphology and proliferation of bladder cancer cells (20). To investigate the effects of RI on HT29 cell survival, RI expression was upregulated by introducing an exogenous RI gene using the overexpression vector pcDNA3.1/RI, or silenced using a specific shRNA (shRI)-mediated knockdown. As presented in Fig. 2A and B, RI expression levels significantly increased or decreased in the HT29/RI or HT29/shRI cells, respectively, compared with the controls, suggesting that the transfection and RI expression manipulation were successful.

A colony formation assay was conducted to elucidate the association between RI expression and HT29 cell metastasis. The overexpression of RI significantly inhibited CRC cell colony formation, whereas knocking down RI in HT29 cells increased it, indicating the inhibitory effect of RI on HT29 cell tumorigenic ability (Fig. 2C and D). Cell viability was subsequently assessed using the CCK-8 assay. The results further provided the evidence that RI expression is negatively associated with viability in CRC cells (Fig. 2E). These results therefore supported the observations made with the colony formation assay, demonstrating that RI overexpression may negatively affect the viability and tumorigenic abilities of CRC cells.

To determine whether autophagy induced by RI overexpression contributes to the regulation of specific proteins of the ATG family, the expression levels of ATG5, ATG7, ATG13 and BECN1 were assessed by western blotting. Fig. 3A indicates that overexpression of RI significantly increased the protein levels of BECN1 and ATG13, but not ATG5 and ATG7. To further validate these observations, specific shRNA sequences of ATG13 (shATG13) and BECN1 (shBECN1) were transfected into HT29/RI cells. The results demonstrated that LC3-II levels significantly decreased due to the knockdown of BECN1 and ATG13 in the HT29/RI cells (Fig. 3B and C). Furthermore, the formation of LC3-II autophagic vacuoles were observed under fluorescence microscopy. As exhibited in Fig. 3D, the knockdown of ATG13 or BECN1 significantly attenuated the accumulation of LC3-II in RI-overexpressing cells. Moreover, the number of LC3-II dots/cell significantly decreased following silencing of ATG13 or BECN1 in HT29/RI cells. Taken together, these results indicated that ATG13 and BECN1 may have been responsible for autophagy induced by RI overexpression in human CRC cells.

To evaluate which pathways may have mediated the effect of RI on autophagy and cell viability, signal transduction molecules, including Akt, mTOR, ULK1 and AMPK, were examined in CRC cells. A previous study demonstrated that the function of RI is associated with the PI3K/Akt pathway (12). Consistent with these reports, the present study revealed that Akt and mTOR were significantly inactivated in HT29/RI cells, whereas ULK1 was activated by phosphorylation, but there was no effect on the expression levels of phosphorylated AMPK (p-AMPK), total Akt, total mTOR, total ULK1 and total AMPK (Fig. 4A). These results suggested that RI overexpression may have induced autophagy by inhibiting the Akt/mTOR/ULK1 pathway, but not via the activation of AMPK. To further validate whether Akt, mTOR and its downstream target ULK1 were involved in RI induced autophagy, the effects of the Akt inhibitor, MK-2206 (21), were evaluated by assessing its effects on LC3-II levels. Fig. 4B demonstrates that the inhibition of activated Akt (p-Akt) resulted in an increase in the amount of LC3-II. In addition, the downregulation of mTOR (shmTOR) increased ULK1 phosphorylation and significantly promoted LC3-II accumulation (Fig. 4C), whereas silencing of ULK1 markedly reduced LC3-II levels, indicating that mTOR and ULK1, as the downstream elements of Akt signaling, may also be involved in the process of RI-induced autophagy in CRC cells (Fig. 4D). Based on these results, activation of the Akt/mTOR/ULK1 signaling pathway may have been responsible for RI-induced autophagy in CRC cells.