Human CRC cell lines HT-29 and SW620 were obtained from American Type Culture Collection (Manassas, VA, USA). Cells were respectively maintained in McCoy’s 5A medium or L-15 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum at 37°C in humidified tissue culture incubator containing 5% CO₂.

Radix Astragali [Astragalus membranaceus (Fisch.) Bunge var. mongolicus] was obtained from Shaanxi University of Chinese Medicine (Shanxi, China). The authenticity and quality of the crude herb were confirmed in the Quality Assurance Laboratory of the School of Chinese Medicine, Hong Kong Baptist University (Hong Kong, China) by microscopic and chromatographic analyses, as well as DNA finger-printing. To ensure consistency between batches, voucher specimens were kept at the herbarium center (Shanghai, China) for future reference. AST was extracted as described previously (13). In brief, 500 g of crude herb was refluxed in methanol for 1 h at room temperature. Following this, n-butanol was added to the reconstituted residue for phase separation to obtain the total AST. Butanol was removed in the rotary evaporator. The resulting residue was reconstituted with distilled water and lyophilized into dry powder. The dried and lyophilized powder was reconstituted in ultrapure water to form a 10 mg/ml stock and stored at −20°C.

HT-29 and SW620 cells were seeded in 96-well plates at a density of 1×10⁴ cells/well and treated with AST at the indicated concentrations (0, 0.02, 0.04, 0.08, 0.16 and 0.20 mg/ml) and time intervals (0, 12, 24, 48 and 72 h). Control cells received McCoy’s 5A or L-15 treatment only. A sterile MTT solution (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to each well, and the cells were incubated for an additional 4 h at 37°C. The medium was removed and 150 µl dimethyl sulfoxide was added to dissolve the formazan crystals formed in the viable cells. Plates were examined at 570 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). At least three independent experiments were performed.

HT-29 and SW620 cells were seeded in 6-well plates at a density of 500 cells/well and treated with 50 µg/ml AST. Cells treated with 25 µg/ml 5-fluorouracil (5-Fu, Sigma-Aldrich; Merck KGaA) were used as a positive control group. The medium (McCoy’s 5A or L-15) was refreshed every 2 days. Following two weeks of incubation at 37°C, colonies were fixed with methanol in 15 min at room temperature and then stained with Giemsa solution (Sigma-Aldrich; Merck KGaA) for 10-15 min at room temperature. Plates were washed with PBS, and colony formation was photographed and analyzed using ImageJ 1.48 (National Institutes of Health, Bethesda, MD, USA). Cloning efficiency is the number of colonies divided by the number of cells plated.

To investigate the effect of AST treatment on cell apoptosis, cells treated with 50 µg/ml AST served as the experimental group. Untreated cells were used as a control group. Cell apoptosis was analyzed using an Annexin V/propidium iodide apoptosis detection kit (BD Biosciences, San Jose, CA, USA) according to the instructions of the manufacturer. Following 48 h of AST treatment, the cells were harvested, washed with cold PBS (pH 7.4), centrifuged at 400 × g at 4°C for 5 min, and double-stained with Annexin V-Fluorescein isothiocyanate and propidium iodide in binding buffer [10 mM HEPES (pH 7.4), 140 mM NaCl and 2.5 mM CaCl₂] for 15 min in the dark. The samples were analyzed with Attune NxT Software version 2.5 and a flow cytometer (Invitrogen Attune NxT; Thermo Fisher Scientific, Inc.).

Total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. Subsequenetly, cDNA was synthesized with an iScript Advanced cDNA Synthesis kit (Bio-Rad Laboratories, Inc.). PCR was performed in duplicate with a CFX96 PCR system and iTaq Universal SYBR-Green Supermix according to the manufacturer’s instructions (Bio-Rad Laboratories, Inc.). PCR amplification conditions were as follows: 1 cycle of 95°C for 3 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 22 sec. Primers used were as follows: Human (h)β-actin forward, 5′-CCTGGGCATGGAGTCCTGTG-3′ and reverse, 5′-TCTTCATTGTGCTGGGTGCC-3′; (h)c-Myc forward, 5′-GGAGGAACAAGAAGATGAGGAAG-3′ and reverse, 5′-AGGACCAGTGGGCTGTGAGG-3′; (h) lactate dehydrogenase A (LDH-A), forward, 5′-CCCCAGAATAAGATTACAGTTATTG-3′ and reverse, 5′-GAGCAAGTTCATCTGCCAAGT-3′; (h) hexokinase 2 (HK2) forward, 5′-GATTGTCCGTAACATTCTCATCGA-3′ and reverse, 5′-TGTCTTGAGCCGCTCTGAGAT-3′; (h)Glut-1 forward, 5′-TCTGGGCATGTGCTTCCAGTA-3′ and reverse, 5′-ATCGAAGGTCCGGCCTTTAGTC-3′; mouse (m) β-actin forward, 5′-ATGCCATCCTGCGTCTGGACCTGGC-3′ and reverse, 5′-AGCATTTGCGGTGCACGATGGAGGG-3′; (m)c-Myc forward, 5′-TCTCCATCCTATGTTGCGGTC-3′ and reverse, 5′-TCCAAGTAACTCGGTCATCATCT-3′; (m)LDH-A forward, 5′-GCTCCCCAGAACAAGATTACAG-3′ and reverse, 5′-TCGCCCTTGAGTTTGTCTTC-3′; and (m)phosphofructokinase (PFK)U forward, 5′-CCGAGGAGCGTACAAAGT-3′ and reverse, 5′-CTGAGCGGTGGTGGTGAT-3′.

HT-29 and SW620 cells were lysed in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Following this, total protein concentration was determined using the BCA assay Kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein (20 µg per lane) were separated using SDS-PAGE (on 10% gels), transferred to nitrocellulose membranes (Amersham; GE Healthcare, Chicago, IL, USA). Following blocking in 5% non-fatty milk at room temperature for 60 min, membranes were incubated overnight at 4°C with primary antibodies directed against β-actin (1:500; BM5422, Wuhan Boster Biological Technology, Ltd., Wuhan, China), B-cell lymphoma 2 (Bcl-2; 1:1,000; ab692), Bcl-2 associated X protein (Bax; 1:1,000; ab32503; both Abcam), Bcl-2 homologous antagonist/killer (Bak; 1:1,000; 12105, CST Biological Reagents Co., Ltd., Shanghai, China), Bcl-2-associated agonist of cell death (Bad; 1:1,000; 9268, CST Biological Reagents Co., Ltd.), pro-caspase3 and poly (ADP-ribose) polymerase (PARP; 1:1,000; 9532, CST Biological Reagents Co., Ltd.), respectively. The membranes were then washed three times with phosphate buffered saline with 0.05% Tween-20 buffer and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; 14709, CST Biological Reagents Co., Ltd.) for 1 h at room temperature. The blots were detected using chemiluminescence (Pierce; Thermo Fisher Scientific, Inc.) and an Odyssey Imaging System (Li-Cor Biosciences, Lincoln, NE, USA).

Basal glucose uptake was measured as previously reported for human cells in vitro (14). Briefly, HT-29 and SW620 cells were plated at 1×10⁴ cells/well in 96-well cell culture plates and treated with 50 µg/ml AST at 37°C for 0, 12, 24 and 48 h. At every point in time, cells were treated with 100 µl of glucose-free DMEM culture medium containing 100 µg/ml 2-NBDG for 20 min. The medium was subsequently replaced with 200 µl Hank’s Balanced Salt Solution and the plate was centrifuged for 5 min at 30 × g (at room temperature). The fluorescence intensity was measured at 530 nm using a plate reader from Tecan Group Ltd. (Männedorf, Switzerland).

HK and LDH activities were measured according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute (HK cat. no. A077-1 and LDH cat. no. A020-2; Nanjing, China). Cell lactate production was determined as previously reported in human cells (15). In brief, HT-29 and SW620 cells were collected, lysed via centrifugation at 6,000 × g for 10 min (at 4°C). The supernatant was collected and total protein was examined using a BCA kit (Biyuntian, Shanghai, China).

A total of 32 male 9-week-old C57BL/6J mice (mean body weight of 21 g; range, 18-25 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China) and housed in individually ventilated cages (IVC; Tianhuan, Shanghai, China) supplied with filtered air and access to food and water. The mice were maintained under the following conditions: Temperature, 20-22°C; relative humidity, 50-60%; and a 12-h light/dark cycle. All mice were acclimatized for 1 week prior to starting dextran sulfate sodium (DSS; molecular weight, 36-50 kDa; MP Biomedicals, Solon, OH, USA) administration. Mice were randomly divided into the following three groups: Mice were exposed to 2% DSS (n=8, positive control), 0.1 mg/g AST (n=8, negative control) and 0.1 mg/g AST treatment combined with 2% DSS in drinking water (n=8), respectively. The body weight of all mice was monitored daily. All mice were sacrificed 8 days following the administration of DSS. Colitis was induced by administration of 2% DSS dissolved in distilled water for 8 days. Once the colon length was measured, the distal colon was extracted and fixed in 4% paraformaldehyde for 24 h at room temperature. Paraffin-embedded colon tissues (5 µm thick) were prepared for hematoxylin and eosin staining. The resulting tissue sections were stained with hematoxylin for 5 min and eosin for 3 min at room temperature. The sections were observed using a confocal microscope (LSM 5 PASCAL; Carl Zeiss AG, Oberkochen, Germany). The remaining colon tissues were embedded in paraffin or subjected to RNA and protein extraction. The procedures employed for the handing and care of animals were approved by the Renji Hospital Ethics Committee, Shanghai Jiao Tong University School of Medicine (Shanghai, China), and conformed to the national guidelines for animal use in research.

All analyses were performed using SPSS 17.0 statistical package (SPSS, Inc., Chicago, IL, USA). Data were expression as the mean ± standard error of the mean. Data were compared using one-way analysis of variance followed by an LSD post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

The MTT assay was performed to determine the growth inhibitory effect of AST on CRC cells. It was revealed that AST could inhibit the growth of CRC cells in a dose- and time-dependent manner (Fig. 1A and B). The IC₅₀ values at 24 h in HT-29 and SW620 cells were ~35 and 46 µg/ml, respectively. Thus, a final concentration of 50 µg/ml AST was used in all subsequent experiments unless otherwise specified. Following this, the inhibitory impact of AST on HT-29 and SW620 cell proliferation over an extended time interval was determined. To assess colony formation, cells were seeded in 6-well plates and treated with 50 µg/ml AST (cells treated with 25 µg/ml 5-Fu served as a positive control) for 2 weeks. Statistical analysis indicated that AST significantly reduced the number of colonies formed by CRC cells compared with the vehicle group (Fig. 1C and D). The results illustrated that AST has a directly inhibitory effect on CRC cell growth and viability over short- and long-term time points.

To determine whether the cytotoxic effects of AST are associated with the induction of apoptosis, flow cytometry was performed when AST was applied to cells. AST treatment significantly induced apoptosis in CRC cells (Fig. 2A and B). Furthermore, ~34% of HT-29 cells and ~31% of SW620 cells were apoptotic in response to AST. Notably, the expression levels of apoptosis-associated proteins confirmed the induction of apoptosis by AST, including the upregulation of Bax, Bad and Bak, and the downregulation of Bcl-2, pro-caspase3, PARP (Fig. 2C).

To explore the impact of AST on glycolysis metabolism in CRC cells, glucose uptake and lactate levels were measured. As indicated in Fig. 3A, the glucose uptake of CRC cells decreased with AST treatment. Furthermore, lactate production was also reduced (Fig. 3B). In addition, the enzymes activities of HK2 and LDH-A in CRC cells were assessed. AST significantly reduced the enzymes activities of HK2 and LDH-A (Fig. 3C and D). The results illustrated that AST could inhibit aerobic glycolysis and the activities of glycolytic enzymes.

To verify whether the expression levels of glycolytic enzymes were also decreased by AST, western blot analysis and RT-qPCR were performed. The relative mRNA expression levels of LDH-A, Glut-1 and HK2 were significantly reduced by AST in SW620 cells (Fig. 4A, B and C). Interestingly, the expression of c-Myc, a primary oncogene of carcinogenesis that is overexpressed in various cancer types (16,17), was significantly decreased in CRC cells treated with AST (Fig. 4D). In addition, western blot analysis revealed similar trends to RT-qPCR data (Fig. 4E).

To uncover the possible impact of AST on inflammation and glucose metabolism in vivo, a DSS-induced colitis mouse model was established. DSS is a well-known agent for inducing inflammation in order to identify the impact of inflammation on the pathogenesis and the clinical course of inflammation (18-20). In the DSS-induced colitis mouse model of the present study, compared with the combined treatment of AST and DSS, mice administered DSS alone were used as a positive control, and those with AST treatment alone served as a negative control. Body weight loss was observed over 8 days post-administration of DSS. In addition, colon length and histological analysis were observed 8 days post-administration of DSS. Following DSS treatment, the body weight and colon length of mice treated with DSS only were significantly reduced. Notably, there was only a slight decrease in the combined treatment group compared with control group and AST groups (Fig. 5A and B). In addition, the inflammatory response of the colonic mucosa was assessed using hematoxylin and eosin staining. As indicated in Fig. 5C, a large number of lobular necro-inflammatory cells were observed in the DSS group; however, this was reduced by AST treatment. The result suggests that AST treatment could attenuate the inflammatory response induced by DSS. Considering the decrease of body weight and the acute inflammatory reactions associated with DSS, it was proposed that glucose metabolism may be altered following inflammation enhancement. mRNA expression levels of c-Myc and glycolytic enzymes were increased in colon epithelial cells following 8-day DSS administration. However, AST treatment significantly inhibited the tumor-like aerobic glycolysis (the high level of glycose uptake and lactate production) in mice with DSS-induced colitis (Fig. 5D), revealing almost the same trends that were indicated in the in vitro cell experiments.