Fetal bovine serum (10%; FBS; BeNa Culture Collection) was added to Dulbecco's Modified Eagle's Medium-high glucose (DMEM-H; BeNa Culture Collection), F-12K medium (BeNa Culture Collection), L-15 medium (BeNa Culture Collection) and Roswell Park Memorial Institute (RPMI)-1640 medium (BeNa Culture Collection) at 37˚C with 5% CO₂ for the incubation of CRC cell lines (Caco2, LoVo, SW480 and HCT116) procured from BeNa Culture Collection, separately. Human intestinal epithelial cell line (HIEC-6) procured from Zhejiang Meisen Cell Technology Co., Ltd., was also cultured in DMEM-H with 10% FBS. In addition, HCT116 cells were stimulated by 80 µM WZB117 (Shanghai Topscience Co., Ltd.) for 24 h (22) or 5 µM SC79 (Shanghai Topscience Co., Ltd.) for 2 h (23).

Using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), the specific small interfering (si)RNAs for ENO1 (siRNA-ENO1-1/2) and the corresponding negative control (siRNA-NC) from Guangzhou Geneseed Biotech Co., Ltd., were transfected into HCT116 cells. Briefly, 2x10⁵ cells were seeded in a 6-well plate. When the cell fusion efficiency was 30-50%, 2 ml of a pre-prepared transfection complex containing 50 nM siRNA was added to each well. The cells were cultured at 37˚C with 5% CO₂ for 6 h and then the medium was changed to continue the culture. The cells were collected 48 h post-transfection for further experiments. The siRNA sequences used were as follows: siRNA-ENO1-1: 5'-GTGTCCCTTGCCGTCTGCAAAGC-3', siRNA-ENO1-2: 5'-ATCAATGGCGGTTCTCATGCTGG-3', siRNA-NC: 5'-TCGTTGGCACTTCGGGTCTGCTAG-3'.

Transfected HCT116 cells in 96-well plates were exposed to 80 µM WZB117 or 5 µM SC79 after the adjustment of cell density to 5x10³ cells/well. Each well was cultivated for extra 2 h at 37˚C following addition of 10 µl CCK-8 solution (Selleck Chemicals). Cell activity was assessed by estimating OD450 nm value by a microplate reader (Huaan Magnech Bio-Tech Co., Ltd.).

Cell proliferation was measured via the employment of iClick EdU Andy Fluor 488 Imaging Kit (Guangzhou FulenGen Co., Ltd.). In brief, the transfected HCT116 cells (1x10⁴ cells/well) subjected to 96-well plates were exposed to WZB117 or SC79, prior to the addition of EdU (20 µM per well) for 2 h at 37˚C as per the manufacturer's instructions. Afterwards, cells were probed with iClick EdU reaction buffer and stained with Hoechst 33342 for 10 min at room temperature following 15 min of fixation with 3.7% paraformaldehyde and 20 min of permeabilization with 0.5% Triton X-100 at room temperature. Images were prepared for observation under a fluorescence microscope (Motic China Group Co., Ltd.).

Following centrifugation at 3,000 x g for 10 min at 4˚C, total iron level in the cell supernatants was examined with Iron test kit (cat. no. ST1020; Leagene; Beijing Regan Biotechnology Co., Ltd.). OD562 nm value was determined using a spectrophotometer (Shimadzu Corporation).

Following the centrifugation of HCT116 cells at 10,000 x g for 10 min at 4˚C, glutathione (GSH) and malondialdehyde (MDA) levels were examined with GSH Assay Kit (cat. no. TO1036; Leagene; Beijing Regan Biotechnology Co., Ltd.) and MDA Assay Kit (cat. no. TO1011; Leagene; Beijing Regan Biotechnology Co., Ltd.). The absorbance value at 412 and 535 nm was read with a spectrophotometer (Shimadzu Corporation).

Intracellular ROS was estimated using a Reactive Oxygen Species Assay Kit (cat. no. BES-BK2782B; Shanghai Bolson Biotechnology Co., Ltd.). After indicated transfection and treatment, HCT116 cells plated into 24-well plate (3x10³ cells/well) were treated with 10 µΜ dichlorodihydrofluorescein diacetate (DCFH-DA) at 37˚C for 20 min in the dark. The cells were then centrifuged at 1,000 x g for 5 min at 4˚C. Subsequently, the supernatant was removed and the cell pellet was resuspended in PBS. ROS production was determined under a fluorescence microscope at 480 nm excitation/590 nm emission.

The Seahorse XF Glycolysis Stress Test Kit (Agilent Technologies, Inc.) was used for the ECAR assay. Briefly, 5x10⁴ HCT116 cells were plated on 96-well cell culture XF microplates (Agilent Technologies, Inc.) for 24 h at 37˚C, which was then replaced by the medium without glucose and pyruvate. 10 mM glucose, 1 µM oligomycin and 100 mM 2-deoxy-glucose (2-DG) were sequentially added to the cell medium, with two measurements after each treatment. The results were subjected to analysis with Seahorse Bioscience XF96 Extracellular Flux Analyzer (Agilent Technologies, Inc.).

HCT116 cells inoculated in 96-well plates at a density of 2x10⁶ cells/well at 37˚C received the indicated transfection and treatment. Using a L-Lactate Assay Kit (cat. no. ab65330; Abcam) and Glucose Colorimetric Assay Kit (cat. no. ab282922; Abcam), lactate production and glucose consumption were respectively determined and were normalized to total protein concentration quantified by the BCA method (Shanghai Rongsheng Biotech Co., Ltd.).

Following the homogenization of HCT116 cells in RIPA buffer (Fude Biological Technology, Co. Ltd.), the BCA method (Shanghai Rongsheng Biotech Co., Ltd.) was used to measure protein content. Subsequently, 12 µg of protein was fractionated on 12% SDS-PAGE and transferring onto the PVDF membranes. The membranes were then blocked with 5% BSA for 1 h at room temperature (Beijing Solarbio Science & Technology Co., Ltd.) and immunoblotted with primary antibodies including ENO1 (cat. no. ab227978; 1:1,000), glucose transporter type 1 (GLUT1; cat. no. ab115730; 1:100,000), hexokinase 2 (HK2; cat. no. ab209847; 1:1,000), pyruvate kinase M2 isoform (PKM2; cat. no. ab85555; 1:1,000), glutathione peroxidase 4 (GPX4; cat. no. ab125066; 1:1,000), ferritin heavy chain 1 (FTH1; cat. no. ab75972; 1:1,000), acyl-CoA synthetase long-chain family member 4 (ACSL4; cat. no. ab155282; 1:10,000), AKT (cat. no. ab179463; 1:10,000), phosphorylated (p-)AKT (cat. no. ab38449; 1:1,000), STAT3 (cat. no. ab109085; 1:10,000), p-STAT3 (cat. no. ab267373; 1:1,000) and β-actin (cat. no. ab213262; 1:1,000) from Abcam at 4˚C overnight, prior to being incubated with HRP-conjugated secondary antibody (ab6721; 1:2,000; Abcam) for 1.5 h at 37˚C. The visualization of the blots was performed by the Super ECL Plus from Biorigin (Beijing) Inc. and the densitometry was performed using ImageJ software (v. 1.4; National Institutes of Health).

Following analysis with GraphPad Prism 8 software (GraphPad Software; Dotmatics), all data were reported as mean ± standard error of mean. The unpaired student's t-test was used to compare the significance between two groups, and the one-way ANOVA and Tukey's post hoc test were used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

To identify the role of ENO1 in CRC, ENO1 expression in CRC cells was initially tested. Using western blotting, it was found that ENO1 displayed higher expression in CRC cell lines (Caco2, LoVo, SW480 and HCT116) in contrast to a human intestinal epithelial cell line (HIEC-6; Fig. 1A). Accordingly, HCT116 cells were used in the following experiments. To determine the effects of ENO1 on the biological processes of CRC, ENO1 expression was distinctly weakened after transfection of siRNA-ENO1-1/2 plasmids. SiRNA-ENO1-2 was chosen for the follow-up assays since ENO1 showed lower expression in SiRNA-ENO1-2 group compared with the SiRNA-ENO1-1 group (Fig. 1B).

As depicted in Fig. 2A, following ENO1 knockdown, the ECAR was markedly decreased in HCT116 cells. In addition, inhibition of ENO1 resulted in the downregulation of glucose consumption and lactate production (Fig. 2B and C). Western blot analysis revealed that the expression of glycolysis-related GLUT1, HK2 and PKM2 were all lowered when ENO1 was depleted (Fig. 2D). All these findings suggested that ENO1 downregulation produced protective properties on the glycolysis of CRC cells.

As reported, glycolysis can serve as a driver of tumor cell proliferation. To investigate whether ENO1 participated in CRC cell proliferation via mediating glycolysis, WZB117, a specific inhibitor of GLUT1, was used. The experimental data from CCK-8 assay showed that the cell viability was reduced in the WZB117 group compared with the control group, and further attenuated after the simultaneous use of WZB117 and siRNA-ENO1 (Fig. 3A). As expected, EdU staining results showed that the fluorescence intensity of WZB117 group was slightly decreased compared with the control group, while after the use of SiRNA-ENO1, the fluorescence intensity of WZB117+iRNA-ENO1 group was significantly decreased compared with the other three groups (Fig. 3B). In conclusion, the suppressive role of GLUT1 inhibitor in CRC cell proliferation was strengthened by knockdown of ENO1.

Ferroptosis remains a pivotal event during the process of CRC (24). It was noted that ENO1 reduction evidently raised total iron and MDA levels but lessened GSH level (Fig. 4A and B). The results of DCFH-DA staining reflected that intracellular ROS level was noticeably elevated when ENO1 was downregulated (Fig. 4C). Western blotting also revealed that GPX4, FTH1 expressions were depleted and ACSL4 expression was augmented in ENO1-silenced HCT116 cells (Fig. 4D). Overall, ENO1 insufficiency might contribute to the ferroptosis of CRC cells.

At the same time, the expressions of proteins involved in AKT/STAT3 signaling were examined and it proved that inhibition of ENO1 significantly depleted p-AKT/AKT and p-STAT3/STAT3 expressions, which were then both partially recovered by treatment with SC79, an activator of AKT (Fig. 5A), suggesting that ENO1 deletion blocked AKT/STAT3 signaling in CRC cells. Moreover, SC79 was further applied to show that ENO1 participated in the glycolysis and ferroptosis of CRC cells through mediating AKT/STAT3 signaling. SC79 was discovered to increase the ECAR which was on a downward trend in HCT116 cells transfected with SiRNA-ENO1 (Fig. 5B). In addition, the suppressed lactate production and glucose consumption caused by ENO1 interference were partially reversed by SC79 (Fig. 5C and D). GLUT1, HK2 and PKM2 expressions all fell in ENO1-silencing HCT116 cells, but were restored by SC79 (Fig. 5E). CCK-8 and EdU staining assays revealed that the attenuated proliferative capacity of HCT116 cells attributed to ENO1 insufficiency was improved by activation of AKT (Fig. 6A and B). Furthermore, the increasing total iron and MDA levels and the falling GSH level following knockdown of ENO1 in HCT116 cells were all reversed by SC79 (Fig. 7A and B). Similarly, absence of ENO1 accelerated the generation of intracellular ROS, which was halted by SC79 (Fig. 7C). ENO1 deletion also reduced GPX4 and FTH1 expressions while increasing ACSL4 expression, which were all nullified by SC79 (Fig. 7D). Activation of AKT/STAT3 signaling counteracted the effects of ENO1 deficiency on the glycolysis, proliferation and ferroptosis of CRC cells.