Phycoerythrin (PE)-conjugated mouse anti-human CD29 (cat. no. 557332), allophyco-cyanin-conjugated CD44 (cat. no. 559942), fluorescein isothiocyanate (FITC)-conjugated CD47 (cat. no. 556045), CD54-PE (cat. no. 555511), VIM-PE (cat. no. 562337), CD49d-FITC (cat. no. 560840), CD49e-PE (cat. no. 555617), CD51/61-PE (cat. no. 550037), Alexa Fluor 647-conjugated FN (cat. no. 563098), CD133-PE (cat. no. 566593), CD166-PE (cat. no. 559263), CD324-PE (cat. no. 562870), CD325-Alexa Fluor 488 (cat. no. 562119) and CD326-PE (cat. no. 347198) and rat anti-human CD49f-PE (cat. no. 555736) antibodies were purchased from BD Pharmingen (BD Biosciences, Franklin Lakes, NJ, USA). Rabbit anti-human p21 (cat. no. 2947), p27 (cat. no. 3686), CDK2 (cat. no. 2546), CDK4 (cat. no. 12790), Cyclin D1 (cat. no. 2978), phosphorylated (p)-cell-division control protein 2 (CDC2; also known as CDK1; cat. no. 4539), β-actin (cat. no. 4970), mouse anti-human CDK6 (cat. no. 3136), Cyclin D3 (cat. no. 2936) and β-actin (cat. no. 3700) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Mouse anti-HK1 (cat. no. BM1910) and mouse anti-human HK2 (cat. no. BM1911) antibodies were purchased from Boster Biological Technology (Pleasanton, CA, USA). Rabbit anti-human HIF-1α (cat. no. WL01607) and glucose transporter type (GLUT)-1 (cat. no. WL01163) antibodies and an Annexin V-Light 650/propidium iodide (PI) Reagent kit (cat. no. WLA002a) were from Wanleibio Co., Ltd (Shanghai, China). Horseradish peroxidase (HRP)-conjugated goat anti-mouse (cat. no. 70-GAM007) and goat anti-rabbit (cat. no. 70-GAR007) secondary antibodies were purchased from Hangzhou MultiSciences (Lianke) Biotech Co., Ltd. (Hangzhou, China). Oligomycin A (Oligo A) was purchased from Selleck Chemicals (Houston, TX, USA). An ATP assay kit was purchased from PerkinElmer, Inc. (Waltham, MA, USA). FBS, L-15 medium and TRIzol^® reagent were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). PI, McCoy's 5A media, 2-Deoxy-D-glucose (2-DG), dimethyl sulfoxide, Triton X-100, RNase, 4% paraformaldehyde, 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] and a Bicinchoninic Acid (BCA) Protein assay kit were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Trypsin-EDTA (0.25%), penicillin/streptomycin (100X), tetramethylethylenediamine, ammonium persulfate, tris(hydroxymethyl)aminomethane, glycine, Tween-20, sodium dodecyl sulfate (SDS), an Efficient Chemiluminescence kit, radioimmunoprecipitation assay (RIPA) lysis buffer and bovine serum albumin were obtained from Gen-view Scientific, Inc. (El Monte, FL, USA). A PrimeScript RT Reagent kit and SYBR Premix Ex Taq PCR kit were purchased from Takara Bio, Inc. (Otsu, Japan). Polyvinylidene difluoride (PVDF) membranes, 30% polyacrylamide and dithiothreitol were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).

The human colon cancer cell lines SW480 and SW620 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in L-15 medium containing 10% FBS and 1% penicillin/streptomycin. All cells were maintained at 37°C in a humidified atmosphere of 5% CO₂, and harvested with 0.25% trypsin before use.

Intracellular ATP concentrations were determined by luminescence assay. SW480 and SW620 cells (1×10⁴ cells/well) were sorted and collected in 96-well plates using a BD FACSAria III flow cytometer (BD Biosciences). To determine the effects of Oligo A on the intracellular ATP levels over time, cells were cultured in L-15 containing 1 µM Oligo A, 1% FBS and 3 mg/ml glucose at 37°C for 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48 and 60 h. To evaluate the effects of Oligo A or 2-DG on the intracellular ATP levels over time, SW620 cells (1×10⁴ cells/well) were sorted and collected in 96-well plates as aforementioned. Cells were cultured in L-15 containing 1 µM Oligo A, 1% FBS and 3 mg/ml glucose or L-15 containing 1% FBS and 3 mg/ml 2-DG at 37°C for 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48 and 60 h.

To detect the ATP synthesis ability of the two cell lines in different microenvironment, cells were cultured for 24 h at 37°C in different media conditions, as follows: i) NOR, in which cells were cultured in L-15 containing 1% FBS and 3 mg/ml glucose, and cells metabolized through OXPHOS and glycolysis; ii) OXPHOS, in which cells were cultured in L-15 containing 1% FBS without glucose, and cells metabolized through OXPHOS; iii) GLY, in which cells were cultured in L-15 containing 1% FBS, 3 mg/ml glucose and 1 µM Oligo A, and cells metabolized through glycolysis; and iv) UN-(O+G), L-15 containing 1% FBS and 1 µM Oligo A, in which cells did not metabolize through either OXPHOS or glycolysis.

ATP production was determined with the ATPlite 1-step Luminescence ATP Detection Assay system (PerkinElmer, Inc.), according to the manufacturer's protocol. Briefly, following culture in different media in 96-well plate, ATP assay solution (100 µl) was added to each well and shaken at 400 rpm, 37°C for 5 min. Subsequently, cells were incubated in the dark for 10 min at 37°C and luminescence was measured with a TECAN M200 PRO microplate luminometer at the wavelength of 540-600 nm (Tecan Group, Ltd., Männedorf, Switzerland).

Expression of the cell surface proteins CD29, CD44, CD47, CD54, CD49d, CD49e, CD49f, CD51/61, CD133, CD166, CD324, CD325 and CD326, and the intracellular proteins VIM and FN1 were detected by BD FACSAria III flow cytometry. Briefly, SW480 and SW620 cells (5x10⁵ cells/well) were seeded in 6-well plate and cultured with L-15 media containing 10% FBS and 1% penicillin/streptomycin at 37°C for 24 h. Subsequently, cells were collected and washed with phosphate-buffered saline (PBS) three times. The cells were centrifuged and resuspended with 200 µl staining buffer (PBS + 1% FBS). Anti-cell surface protein antibodies were added (all 1:10) and incubated at 4°C in the dark for 20 min. For intracellular proteins, cells were fixed with Fixation Buffer (BD Biosciences) for 20 min at 4°C. The cells were washed with PBS and permeabilized with Perm Buffer III (BD Biosciences) for 30 min at room temperature. Subsequently, cells were washed with Perm/Wash Buffer I (BD Biosciences) and stained with mouse anti-human VIM-PE and mouse anti-human FN-Alexa Fluor 647 antibodies. Background staining was determined by staining with the respective isotype control antibodies. Following staining, cells were washed with staining buffer and resuspended in 500 µl of the staining buffer. Flow cytometric analysis was performed on the BD FACSAria III. Data were acquired with BD FACSDiva software 6.0, and the obtained data were analyzed with FlowJo software 7.6 (FlowJo LLC, Ashland, OR, USA).

Apoptosis was examined using the Annexin V-Light 650/PI Reagent kit, according to the manufacturer's protocol. SW480 and SW620 cells (5×10⁵ cells/well) were seeded in 6-well plates and cultured at 37°C for 24 h in the media conditions aforementioned: i) NOR; ii) OXPHOS; iii) GLY; iv) UN-(O+G). Following incubation, cells were collected and washed twice with PBS. Subsequently, cells were stained with 5 µl Annexin-V-Light 650 and 5 µl PI at room temperature for 15 min in the dark. Cells were analyzed with a BD FACSAria III flow cytometer and the obtained data were analyzed with FlowJo software 7.6.

∆Ψm was detected by flow cytometry using DiOC6(3), as described in our previous study (23). Briefly, SW480 and SW620 cells were seeded (5×10⁵ cells/well) in 6-well plates and cultivated in the media conditions aforementioned: i) UN-(O+G) and ii) GLY. Cells were cultured for 0, 1, 4, 6, 8 h and 24 h at 37°C. Cells were harvested and incubated with 3 µM DiOC6(3) at room temperature for 20 min in the dark and subsequently analyzed by the BD FACSAria III. Data were acquired with BD FACSDiva software 6.0, and the obtained data were analyzed with FlowJo software 7.6.

To evaluate the expression levels of glycolysis-regulated proteins HIF-1α, HK1, HK2 and GLUT1 in different microenvironments, western blotting was performed as previously described (24). SW480 and SW620 cells (5×10⁵ cells/well) were seeded in 6-well plates and cultivated in three different media conditions, as aforementioned, at 37°C for 24 h: i) OXPHOS; ii) NOR: and iii) GLY. To investigate the expression of associated proteins in condition of glycolysis, ‘OXPHOS’ group served as the control group. To investigate the expression of glycolysis regulatory proteins (HK1 and HK2) and cell cycle regulators (CDK2, CDK4, CDK6, p21 and p27) over time when OXPHOS was suppressed, SW480 and SW620 cells (5×10⁵ cells/well) were seeded in 6-well plates and cultured in L-15 supplemented with 1 µM Oligo A, 1% FBS and 3 mg/ml glucose at 37°C for 0, 1, 4, 8, 24 and 48 h. To determine the expression of cell cycle regulators (CDK6, Cyclin D3, Cyclin D1, pCDC2 and CDK4) in different microenvironment, cells were cultured in three different media named above as OXPHOS, GLY and NOR.

Following incubations, cells (1×10⁶ cells/well) were rinsed with PBS and lysed with RIPA lysis buffer on the ice for 5 min. The lysates were centrifuged at 18,000 × g at 4°C for 15 min. Protein concentrations were determined with the BCA protein assay kit. The samples were denatured with SDS running buffer [50 mM Tris-Cl (pH 6.8), 2% (w/v) SDS, 0.1% (w/v) bromophenol blue, 20% (v/v) glycerol and 200 mM dithiothreitol] at 100°C for 5 min. Proteins (1 mg/ml) were separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked in 5% skim milk for 1 h at room temperature. The membranes were incubated with the following primary antibodies overnight at 4°C: Anti-HK1, anti-HK2 and anti-HIF-1α (all 1:200 dilution); anti-GLUT1, anti-CDK2, anti-CDK4, anti-CDK6, anti-pCDC2, anti-Cyclin D1, anti-Cyclin D3, p21, p27 and the loading control mouse anti-human and rabbit anti-human β-actin (all 1:1,000). Following washing with TBS + Tween-20, the membranes were incubated with an HRP-conjugated goat anti-rabbit (1:10,000 dilution) or goat anti-mouse (1:10,000) secondary antibody for 1 h at room temperature. Visualization was performed on ChemiDoc XPS system (Bio-Rad Laboratories, Inc.) using the Efficient Chemiluminescence kit. Densitometric analysis was performed with Image Lab 5.2.1 (Bio-Rad Laboratories, Inc.); relative protein expression levels were normalized to β-actin.

SW480 and SW620 (5×105 cells/ well) were seeded in 6-well plates and cultured at 37°C for 24 h in three different media conditions, as aforementioned: i) OXPHOS; ii) NOR; and iii) GLY. RNA was extracted using TRIzol reagent, according to the manufacturer's protocol, and resuspended in 20 µl RNase-free water. Following determination of the RNA concentration with Quawell Q5000 Micro-volume Spectrophotometer (Quawell Technology, Inc., San Jose, CA, USA), RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent kit. cDNA was used for each of three replicates of qPCR using the SYBR Premix Ex Taq PCR kit and a CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories, Inc.) with the following thermocycling conditions: Initial denaturation at 95°C for 30 sec; followed by 40 cycles of 95°C (5 sec) and 60°C (30 sec). Target mRNA expression levels were normalized to β-actin and calculated by the 2^−ΔΔCq method (25). Results are presented as the mean ± standard deviation (SD) of three independent experiments. mRNA expression levels were analyzed for HK1, HK2, HIF-1α, CDH1, CDH2, ZEB1, SNAI1, SNAI2, FN1, VIM, CD133, TWIST and β-actin; all primer sequences for PCR analysis are presented in Table I.

In addition, SW480 and SW620 cells (5×10⁵ cells/well) were seeded in 6-well plates and cultivated in L-15 supplemented with 1 µM Oligo A, 1% FBS and 3 mg/ml glucose at 37°C for 0, 1, 4, 8, 24 or 48 h, and the mRNA expression levels of the aforementioned genes were detected by RT-qPCR.

The tolerance and sensitivity of SW480 and SW620 cells to FBS deprivation was determined by starvation culture. Cells (5x10⁵ cells/well) were seeded in 6-well plates cultivated in FBS-free L-15 medium. The FBS-free medium was replaced every 2 days. Cell morphology and viability were observed under Zeiss Observer A1 microscope (Zeiss AG, Oberkochen, Germany).

A wound-healing assay was performed to assess the migratory ability of SW480 and SW620 cells. Cells (2.5×10⁴ cells/well) were seeded onto a 24-well plate and cultured for 24 h. Subsequently, a scratch was made across the cell monolayer with a pipette tip. Cells were washed three times with PBS and L-15 medium containing 0.5% FBS was added to each well. Cells were cultured in incubator at 37°C and cell migration within the scrape line was recorded at 0, 8, 24 and 48 h using a light microscope (Zeiss AG); migration distance was used to evaluate migratory ability.

BALB/c nude mice (n=12 females; age, 6-8 weeks; weight, 20 g) were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Mice were housed in clean, pathogen-free rooms in an environment with controlled temperature (26°C), filtered atmosphere and humidity (55%), with a 12-h light/dark cycle, and provided free access to pellet food and water in microinsulator cages. All animals used in investigations were handled in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The Institutional Animal Care and Use Committee of Fuzhou University (Fuzhou, China) reviewed and approved all animal use procedures based on the above regulations, including the use of appropriate species, quality and number of animals, avoidance or minimization of discomfort, distress and pain of animals with a good scientific basis, and use of appropriate anesthesia and euthanasia. BALB/c nude mice were randomly allocated to two groups (n=6/group), which received an intravenous injection of PBS (200 µl; pH 7.4) containing either SW480 or SW620 cells (1.0x10⁶ cells) into the tail vein. At 30 days post-injection, mice were sacrificed and the lungs were excised and stained with Bouin's solution at room temperature for 24 h for pulmonary nodule counting.

Statistical analyses were performed with the SPSS statistical software package 22 (IBM Corp., Armonk, NY, USA). Results are expressed as the mean ± SD. Statistical significance values were evaluated through one-way analysis of variance with a Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

As previously mentioned, the SW480 cell line was established from a primary tumor, and the SW620 cell line was derived from a lymph metastatic lesion from the same patient (22). Results from the wound-healing assay indicated that SW620 cells exhibited a stronger migratory ability compared with SW480 cells; SW620 cells migrated further compared with the SW480 cells over the same period time (Fig. 1A). In vivo metastatic ability assay revealed that mice injected with SW620 cells had significantly higher numbers and total volumes of tumor nodules in the lung compared with mice injected with SW480 cells (Fig. 1B-D); tumor metastatic lesions were not observed in other organs.

Metastatic cells that escape from the primary tumor to distant organs, must possess certain traits that allow them to survive in a new microenvironment, including its conditions of nutrient scarcity (5). Cell viability and survival depend on the efficiency of ATP production through OXPHOS or glycolysis. To determine the differences in metabolic performance between SW480 and SW620, cells were treated with Oligo A, an inhibitor of ATP synthase and the OXPHOS pathway (26), or with 2-DG, a glucose molecule that has the 2-hydroxyl group replaced by hydrogen, and is thus unable to undergo further glycolysis (27), to investigate the effect on intracellular ATP levels. When OXPHOS was inhibited, the ATP levels of SW480 cells decreased rapidly and remained stable between 24 and 60 h post-treatment (Fig. 1E); ATP levels were maintained at ~0.8 of the basal value (basal value was determined from the same cells cultured in L-15 medium containing FBS and glucose, without Oligo A or 2-DG; in these conditions cell metabolism is through both OXPHOS and glycolysis). By contrast, at 4 h following Oligo A treatment, the ATP levels of SW620 cells increased to 1.2 of the basal value and decreased to the basal value at 24 h following treatment and remained stable at ~0.9 of the basal value between 36 and 60 h post-treatment. Therefore, it was speculated that when treated with Oligo A in the first 24 h, SW620 cells may be able to initiate emergency cellular metabolism pathways to compensate for the depleted energy supply.

To evaluate whether SW620 cells upregulated OXPHOS when glycolysis was suppressed, SW620 cells were treated with 2-DG. It was determined that the ATP levels were below the basal value at all time points because of the action of 2-DG (Fig. 1F). A similar ATP suppression effect was not observed in the Oligo A group. These data suggested that glycolysis may serve a key role when cells are confronted with microenvironmental changes.

OXPHOS and glycolysis are the two principal metabolism processes to maintain cell viability and biosynthesis. In the present study, the potency of OXPHOS and glycolysis were compared in the two cell lines. Glycolysis and OXPHOS were inhibited by 2-DG or Oligo A, respectively. Cells were treated with 2-DG for 24 h; the ATP levels in SW480 cells were 0.856 of the NOR, whereas SW620 cells exhibited a higher level, at 0.967 of the NOR (Fig. 1G). Similarly, when cells were treated with Oligo A and metabolized through glycolysis, the ATP levels were 0.755 and 0.890 of the NOR in SW480 and SW620 cells, respectively. When both OXPHOS and glycolysis were suppressed at the same time, ATP levels in the two cell lines dropped to only 0.10 of the NOR. These results indicated that SW620 cells exhibited stronger OXPHOS and glycolysis metabolism potential compared with SW480 cells.

In summary, SW620 cells exhibited higher energy metabolism capability compared with SW480 cells; when the cells were confronted with an energy supply shortage, SW620 cells exhibited a stronger capability for OXPHOS and glycolysis.

To further investigate the association between ATP levels and glycolysis, SW480 and SW620 cells were exposed to three different environments: i) OXPHOS, ii) NOR and iii) GLY. The expression levels of regulatory proteins of glycolysis, including HK1, HK2, GLUT1 and HIF-1α, were measured by western blot analysis. In the presence of glycolysis metabolism (NOR and GLY), the expression levels of HK1, HK2 and GLUT1 proteins in SW620 cells were all higher compared with the OXPHOS group, whereas the expression levels in SW480 cells were all decreased compared with the OXPHOS group (Fig. 2A-F). In addition, the expression levels of HIF-1α, HK1, HK2 and GLUT1 proteins in NOR and GLY treatments in SW620 cells were all significantly higher compared with the respective expression levels in SW480; OXPHOS was used as the control. These results suggested, compared with SW480 cells, SW620 cells are more likely to metabolize through glycolysis when glucose is sufficient.

In SW480 and SW620 cells treated with Oligo A for 48 h, the protein expression levels of HK1 and HK2 in SW480 cells gradually decreased in the first 8 h of treatment and increased slightly at 24 and 48 h; however, expression remained below the basal value (0 h post-treatment; Fig. 2G-I). By contrast, the expression of HK1 in SW620 cells increased gradually in the first 8 h of treatment and subsequently reduced slightly, remaining above the basal value (the expression levels at 0 h) up to 48 h post-treatment (Fig. 2G and H). HK2 expression in SW620 did not exhibit any significant changes compared with 0 h (Fig. 2G-I). In conjunction with the data presented in Fig. 1E, these results indicated that when OXPHOS was suppressed, the intracellular ATP levels were positively associated with HK1 and HK2 expression.

The study of metabolic regulations of the cell cycle is a relatively new field that is gaining increasing attention, as metabolic signals are integrated into, and coupled with, cell cycle progression (10,28). In the present study, the effects of different microenvironments on the cell cycle were investigated.

Tumor-associated cell cycle defects are often mediated by alterations in CDK activity (29). CDK catalytic activities are often modulated by interactions with cyclins and CDK inhibitors (28). In the present study, the expression profiles of cell cycle regulatory proteins were compared in SW480 and SW620 cells following exposure to three different microenvironments. It was identified that the expression levels of cell cycle regulatory proteins in these two cell lines were significantly different (Fig. 3). When cells metabolized by OXPHOS and glycolysis (NOR), the expression of Cyclin D3, pcdc2, and CDK4 in SW620 cells was significantly higher compared with SW480 cells, OXPHOS was used as the control (Fig. 3). However, when cells were metabolized by glycolysis (GLY), the expression of Cyclin D3 and CDK4 in SW620 cells was significantly lower compared with SW480 cells. Moreover, CDK6 and Cyclin D1 did not express in SW480 cells but its expression decreased in SW620 cells. CDC2 is a master regulator of mitosis, as it controls the centrosome cycle as well as mitotic onset, and can be activated by phosphorylation (30). Cyclin D1 and Cyclin D3 control the G1-S progression of the cell cycle in complex with CDK4 and CDK6 (28). These data indicated that SW620 cells may manage their proliferation through upregulating the expression of cyclins when energy supply is sufficient. However, in the GLY environment, the expression of Cyclin D3 and CDK4 in SW620 cells was significantly lower compared with SW480 cells (Fig. 3C and E, respectively). These results suggested that SW620 cells may inhibit the expression of positive cell cycle regulatory proteins to sustain cell survival when energy supplies are low.

Oligo A was used to inhibit OXPHOS, and the changes in cell cycle regulation were compared in SW480 and SW620 cells over a 48-h period. The expression levels of CDK2 and CDK4 in SW480 and SW620 cells decreased over time (Fig. 4), which indicated that the cell cycle was arrested when the expression of positive cell regulatory proteins was inhibited. Similarly, the expression of CDK2 and CDK4 exhibited the same trend in SW620 cells when treated with Oligo A. CDK6 expression was not detected in SW480 cells, whereas its expression decreased in SW620 cells over time. Furthermore, protein expression levels of the CDK inhibitors p21 and p27 were upregulated in SW620 cells as the treatment time increased, whereas their expression levels in SW480 cells decreased over time (Fig. 4A, D and E). Cell cycle upstream regulatory proteins p21 and p27 may inhibit the expression of CDKs and promote cell cycle arrest (31,32). These results indicated that cell cycle progression may be closely associated with intracellular metabolism.

The bioenergetics adaptability of SW480 and SW620 cells in different microenvironment was evaluated by apoptosis and ∆Ψm analysis. Cells in the NOR group metabolized through both OXPHOS and glycolysis; both SW480 and SW620 maintain a considerable proportion of viable cells (Fig. 5A). In the OXPH group, cells metabolized through OXPHOS and the apoptotic rates increased. In the UN-(O+G) group, cells did not metabolized through either OXPHOS or glycolysis; almost all cells are apoptotic or necrotic. In the GLY group, cells metabolized through glycolysis, and the proportion of apoptotic cells was significantly lower in SW620 cells compared with that in SW480 cells. These results indicated that when the cells metabolized by glycolysis, SW620 cells exhibited a stronger anti-apoptotic ability compared with SW480 cells.

ΔΨm is vital for sustaining the physiological function of the cellular respiration/electron transport chain to yield ATP; decreases in ΔΨm will lead to mitochondrial dysfunction and result in apoptosis (33). Changes in ΔΨm of SW480 and SW620 cells were evaluated when both glycolysis and OXPHOS were inhibited [UN-(O+G) group]. The ΔΨm of SW480 cells exhibited a sudden drop at 1 h and gradually decreased between 1 and 8 h, whereas the ΔΨm of SW620 cells declined gradually during the entire experimental period when both OXPHOS and glycolysis were restrained. In addition, as UN-(O+G) SW480 and SW620 cells exhibited increased rates of apoptosis and necrosis at 24-h culturing, ΔΨm could not be detected in cells at 24 h post-treatment (Fig. 5B and C). When only OXPHOS was suppressed (GLY group), the ΔΨm of SW620 cells was significantly increased at 8 and 24 h following Oligo A treatment compared with 0 h (Fig. 5D and E). The elevation of ΔΨm indicates the alleviation of mitochondrion impairment, and cells were protected from apoptosis (34). These data suggested that SW620 cells exhibit stronger adaptability under unfavorable conditions (Oligo A treatment) to prevent apoptosis by increasing ΔΨm.

SW620 cells are a type of metastatic cell derived from a lymph node lesion, which may possess stronger mesenchymal phenotypic properties compared with primary tumor-derived SW480 cells. To investigate the association between metabolism and EMT, the mRNA transcription levels of glycolysis regulatory proteins (HK1, HK2 and HIF-1) and EMT regulatory factors (CDH1, FN1, VIM, CD133, SNAI1, SNAI2, ZEB1, CDH2 and TWIST) were compared in different metabolic microenvironment. When cells were cultured in medium without glucose and metabolized through OXPHOS, the mRNA transcriptional levels of the mesenchymal phenotype-associated proteins FN1 and VIM, and the key glycolysis enzyme HK1 were lower in SW620 cells compared with expression levels in SW480 cells; the other mRNA transcriptional levels were significantly higher in SW620 cells (Fig. 6A). When cells metabolized through both OXPHOS and glycolysis (NOR), the transcriptional levels of FN1 and SNAI2 were lower compared with transcriptional levels in SW480 cells; for all other genes, the transcriptional levels were significantly higher in SW620 cells (Fig. 6B). However, when OXPHOS was inhibited by treating with glucose and Oligo A, in which cells metabolized through glycolysis, all the mRNA transcriptional levels were upregulated in SW620 cells compared with SW480 cells (Fig. 6C). These data suggested that SW620 cells may regulate the expression of HK1 according to alterations in the culture environment. The HK2 mRNA expression level was higher in SW620 cells compared with SW480 cells in each type of culture condition (Fig. 6A-C), which indicated that HK2 may be a key enzyme that is upregulated in highly metastatic tumor cells. Furthermore, compared with SW480 cells, SW620 cells exhibited higher mRNA transcriptional levels of the EMT promoting factors SNAI1, TWIST and ZEB1 (Fig. 6D), which suggested that SW620 cells exhibited stronger mesenchymal cell properties and metastatic potential.

When OXPHOS was inhibited by Oligo A for 48 h, the mRNA transcriptional levels of the key glycolysis enzymes HIF-1α, HK1 and HK2 were significantly upregulated in SW620 cells compared with SW480 cells (Fig. 7). These results were consistent with the data presented in Figs. 1E-H and 2G-I, which further indicated that SW620 cells may regulate the expression of HK1 and HK2 to enhance glycolysis to supplement the energy supply under conditions of energy shortage.

In addition, in SW620 cells, mRNA expression levels of the EMT promoting factors SNAI1, SNAI2, ZEB1 and TWIST, the mesenchymal phenotype-associated proteins FN1 and VIM and other EMT-related proteins CDH1, CDH2 and CD133 were upregulated over time (Fig. 7). This may result in an enhancement of migration and metastasis ability. By contrast, SW480 cells exhibited the opposite trends.

In the present study, the expression of metastasis-associated proteins CD29, CD44, CD47, CD54, VIM, CD49d, CD49e, CD49f, CD51/61, FN, CD133, CD166, CD324, CD325 and CD326 were compared in SW480 and SW620 cells by flow cytometry. The expression levels of CD44, CD54, CD133, CD166 and CD324 between SW480 and SW620 cells were markedly different; the most notable differences were that CD133 and CD166 were highly expressed in SW620 cells, but their expressions were not be detected in SW480 cells (Fig. 8). Also, the expression of CD324 in SW620 is higher than that in SW480. Conversely, the expression of CD44 and CD54 was lower in SW620 cells compared with expression in SW480 cells.

Serum is commonly used as a cell culture supplement and provides macromolecules, carrier proteins, trace elements, attachment and spreading factors, low molecular weight nutrients, hormones and growth factors; FBS is the most widely used serum and is often used to maintain cell growth and proliferation (35). In the present study, the cell growth status of SW480 and SW620 cells in FBS-free medium was compared. SW480 cells exhibited low adherence, nuclear condensation and a small degree of apoptosis (white arrows) following 5 days of starvation culturing, and almost all cells had undergone apoptosis at day 15 (Fig. 9). By contrast, SW620 cells maintained high adherence, integrated morphology and a seemingly high viability following starvation culturing for 5 or 15 days. These results indicated that SW480 cells were sensitive to FBS and that SW620 cells were strongly tolerant of FBS-free medium. By comparison with SW480 cells, SW620 cells appear to be better adapted to survive in nutrient-scarce microenvironments.