A total of 629 colorectal adenocarcinoma cases were downloaded from TCGA database (http://www.cbioportal.org/). The mutations of Smad4, and the expression of Smad4 in CRC and matched normal tissues were analyzed.

Normal human colon epithelial cells (CCD 841 CoN cells) and four CRC cell lines (SW480, LoVo, SW620 and LS174T) were obtained from Invitrogen; Thermo Fisher Scientific, Inc. The cells were cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 0.03% glutamine and 100 µg/ml streptomycin at 37°C in an incubator with 5% CO₂. The cells were digested and subcultured with 0.25% trypsin when confluence reached 80–90%.

The Smad4 silencing plasmid (siSmad4) (Forward: 5′-CGAAUACACCAACAAGUAATT-3′, Reverse: 5′-UUACUUGUUGGUGUAUAUUCGTA-3′), Smad4 overexpression plasmid and empty control plasmid (negative control, NC) were purchased from Invitrogen; Thermo Fisher Scientific, Inc. SW480 cells were seeded in a 6-well plate (1.0×10⁵ cells /well) for 24 h before transfection and divided into three groups: i) the control (0.1% PBS) subgroup, siNC subgroup, siSmad4 subgroup, NC subgroup and Smad4 overexpression plasmid subgroup; ii) the control (0.1% PBS) subgroup, cetuximab (20 µg/ml; Merck KGaA) subgroup, NC + cetuximab (20 µg/ml) subgroup, siSmad4 + cetuximab (20 µg/ml) subgroup and siSmad4 subgroup; iii) the control (0.1% PBS) subgroup, NC + cetuximab (20 µg/ml) subgroup and Smad4 overexpression plasmid + cetuximab (20 µg/ml) subgroup. Transient transfection was effected by Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 20 µM NC, siSmad4 or Smad4 overexpression plasmid, and 5 µl Lipofectamine^® 2000 were added to Opti-MEM and incubated at 25°C for 10 min. Lipofectamine^® 2000 was then mixed into each well and the cells were cultured in Opti-MEM. After 6 h of culturing, the medium was replaced with RPMI-1640 containing 10% FBS. After 24 h, the cells were used to perform the subsequent experiments.

In the present study, the CCK-8 assay was conducted in triplicate. Cells were plated into 96-well plates at a seeding density of 1×10⁴ cells/well for 24 h. Initially, fresh medium containing 0.1, 1, 10, 30, 50 or 100 µg/ml Cetuximab was used to treat the cells for 24 or 48 h at 37°C, after which the CCK-8 assay was performed. In addition, the CCK-8 assay was performed to explore the effects of silencing or overexpressing Smad4 on the viability of cetuximab-treated cells. Briefly, 10 µl CCK-8 solution was added to the cells for 2 h at 37°C. Optical density (OD) was measured at 450 nm (Thermo Fisher Scientific, Inc.).

Cells were seeded in 6-well plates (6×10⁴ cells/well) and incubated at 37°C for 24 h. A sterile 100-µl pipette tip was used to generate a wound in the center of the plate. PBS was used to gently wash the cells three times, and serum-free medium was added to the cells. Cell migration was observed using an inverted light microscope at 24 h. The wound area was measured using ImageJ software version 1.49 (National Institutes of Health).

Cell invasion was detected using a Matrigel-coated Transwell assay. Briefly, 100 µl serum-free medium containing 10⁵ cells was added into the upper chamber (Corning, Inc.) and 600 µl culture medium containing 10% fetal bovine serum was added to the lower chamber. The cells were incubated for 24–48 h at 37°C in order to detect invasion into the lower membrane. Subsequently, the cells were fixed with 95% ethanol for 10 min at 25°C and stained with 0.5% crystal violet solution for 5 min at room temperature after the Matrigel on the filter membrane was removed.

Cell apoptosis was assessed by flow cytometry. Cells were harvested, collected and washed with cold PBS twice. The suspension (5×10⁵ cells/well) in binding buffer was cultured with Annexin V-APC/7-AAD apoptosis kit [cat. no. AP105, Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd.] in the dark at 25°C for 20 min. Binding buffer was then added to each well and the samples were analyzed by flow cytometry within 1 h using BD CellQuest Pro Software version 1.2; BD Biosciences).

Smad4, apoptosis-related genes (Bax and Bcl-2), E-cadherin and vimentin were detected by RT-qPCR in the different groups. Total RNA was extracted from cultured cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was reverse transcribed with the PrimeScript™ RT reagent kit (Takara Bio, Inc.) at 37°C for 45 min and then at 85°C for 5 min. cDNA was amplified using SYBR Fast qPCR Mix (Invitrogen; Thermo Fisher Scientific, Inc.). For quantitative real-time PCR, the PCR reaction was set at 95°C for 5 min, followed by 33 cycles at 94°C for 30 sec, 55°C for 30 sec, 72°C for 45 sec and 72°C extension for 10 min. The primer sequences used are listed in Table I. mRNA expression was quantified using the 2^−ΔΔCq method (30). GAPDH served as an internal control.

Total protein was extracted from cultured cells using lysis buffer; RIPA buffer (Thermo Fisher Scientific, Inc.) was added to each well and cells were centrifuged at 14,000 × g for 15 min at 4°C. Bicinchoninic acid protein quantification (Thermo Fisher Scientific, Inc.) was used to detect protein concentration. Proteins (20 µg/lane) were separated by 10% SDS-PAGE and were then transferred onto polyvinylidene difluoride membranes, which were blocked in TBS-0.1% Tween-20 (TBST) containing 1% milk at room temperature for 2 h. The membranes were then incubated with the following primary antibodies: Rabbit anti-Smad4 (cat. no. ab40759, 1:5,000; Abcam), anti-Bcl-2 (cat. no. ab182858, 1:2,000; Abcam), anti-Bax (cat. no. ab32503, 1:1,500; Abcam), mouse anti- E-cadherin (cat. no. ab1416, 1:50; Abcam), anti-vimentin (cat. no. ab8978, 1:1,000; Abcam) and anti-GAPDH (cat. no. ab8245, 1:1,000; Abcam). The membranes were washed with TBST, and were then incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:2,000; SA00001-2; ProteinTech Group, Inc.) and with horseradish peroxidase -conjugated goat anti-mouse IgG (1:2,000; sc-516102; Santa Cruz Biotechnology, Inc.) as secondary antibodies. The blots were visualized by enhanced chemiluminescence (ECL; Thermo Fisher Scientific, Inc.). An ECL system (Amersham; GE Healthcare) was used to detect the bands. The density of the blots was measured using Quantity One software version 2.4 (Bio-Rad Laboratories, Inc.).

Statistical analysis was performed using Prism GraphPad version 6.0 software (GraphPad Software, Inc.). All data are presented as the means ± standard deviation. Differences were analyzed using one-way analysis of variance following Tukey's multiple comparison post hoc test. P<0.05 was considered to indicate a statistically significant difference.

TCGA data (Fig. 1A and B) demonstrated that Smad4 was mutated in 131 (21%) out of 629 sequenced cases/patients with colorectal adenocarcinoma. The majority of the mutations were deletions. In addition, mRNA and protein expression levels of Smad4 in cancer tissues were lower than those in normal tissues in the cases downloaded from TCGA (Fig. 1C). The expression levels of Smad4 in normal colonic epithelial cells and four CRC cell lines were determined by RT-qPCR and western blot analysis. The results demonstrated that expression of Smad4 in CRC cell lines (SW480, LoVo, SW620 and LS174T) was significantly decreased compared with in CCD 841 CoN cells at the mRNA and protein levels (P<0.01; Fig. 1D and E). The SW480 cell line was selected as the subject of subsequent experiments as it had the lowest expression of Smad4 at the mRNA and protein levels.

SW480 cells were treated with or without different concentrations (0.1, 1, 10, 30, 50 and 100 µg/ml) of cetuximab. There was a difference in OD 450 nm with 0 µg/ml Cetuximab since the cells proliferated after 24 and 48 h. Cell viability was increased in response to treatment with different concentrations of cetuximab for 24 and 48 h compared to 0 h (P<0.05; Fig. 2A). The results indicated that certain cells already had drug resistance, and therefore, the inhibitory effect of cetuximab was not significant at 24 h. However, at 48 h, cell viability was downregulated in a dose-dependent manner until the cells were treated with 20 µg/ml cetuximab. Therefore, 20 µg/ml cetuximab (48 h) was selected as an effective concentration in subsequent experiments. The transfection efficiency of siSmad4 was demonstrated in Fig. 2B and C; the expression levels of Smad4 were significantly decreased post-transfection with siSmad4. In addition, treatment with 20 µg/ml cetuximab (P<0.05) or siSmad4 (P<0.01) significantly inhibited Smad4 protein (Fig. 2D) and mRNA (Fig. 2E) expression. When cetuximab was used in combination with siSmad4, the inhibitory effect on Smad4 expression was at its most significant (P<0.01). Furthermore, cetuximab treatment alone and NC + cetuximab markedly downregulated SW480 cell viability (P<0.01; Fig. 2F). However, the effect of cetuximab treatment was clearly inhibited when cetuximab was used in combination with siSmad4, compared with single cetuximab or NC + cetuximab (P<0.01). In addition, compared with groups treated with cetuximab, siSmad4 alone significantly enhanced cell viability (P<0.01). Silencing Smad4 not only increased SW480 cell viability, but also partly reversed the cell viability decrease induced by cetuximab, indicating that silencing Smad4 may attenuate cell sensitivity to cetuximab in the SW480 CRC cell line.

Cell migration was detected using a scratch assay. The results revealed that cetuximab and NC + cetuximab treatment significantly increased the open wound area, compared with the control (P<0.01; Fig. 3A). Silencing Smand4 markedly reduced the open wound area (P<0.01; Fig. 3A). The open wound area was decreased by siSmad4 in combination with cetuximab compared with siSmad4 treatment alone; however, the decreased effect was not significant compared with cetuximab or cetuximab + NC. A Transwell assay was used to determine cell invasion and the result demonstrated that the invasion rate of SW480 cells was markedly decreased in groups treated with cetuximab (P<0.01; Fig. 3B). Conversely, invasion of cells transfected with siSmad4 was significantly increased (P<0.01; Fig. 3B). However, silencing Smad4 only partly increased the cell invasion attenuated by cetuximab. The effects of silencing Smad4 on the apoptosis of SW480 cells with or without cetuximab were detected by flow cytometric analysis. A noticeable increase in apoptosis (P<0.01; Fig. 3C) was identified in the cetuximab (17.47%), NC + cetuximab (18.4%) and siSmad4 + cetuximab (9.18%) groups, compared with in the control group (4.48%). Silencing Smad4 (4.17%) significantly downregulated apoptosis rate (P<0.01) compared with in the cetuximab (17.47%) or siSmad4 + cetuximab (9.18%) groups. In addition, silencing Smad4 partly reversed the increase in cell apoptosis induced by cetuximab (P<0.01).

The expression levels of the apoptosis-related genes Bax and Bcl-2 were detected by RT-qPCR and western blot analysis. The results also demonstrated that cetuximab downregulated Bcl-2 (P<0.01; Fig. 4A, B and F) and upregulated Bax protein (P<0.01; Fig. 4A and C) and mRNA expression (P<0.01; Fig. 4G) compared with control. Silencing Smad4 partly reversed the decreased expression of Bcl-2 and the increased expression of Bax (protein, P<0.01) by cetuximab. Silencing Smad4 treatment alone could attenuate Bax expression in comparison with siSmad4 + cetuximab (protein, P<0.05; mRNA, P<0.01). To further determine how silencing Smad4 could affect EMT in SW480 CRC cells with or without cetuximab treatment, the expression of EMT-associated genes (E-cadherin and vimentin) was detected using RT-qPCR and western blot analysis. The results demonstrated that both protein (Fig. 4A and D) and mRNA levels (Fig. 4H) of E-cadherin were increased in the cetuximab and NC + cetuximab groups compared with in the control group (P<0.01). Conversely, the mRNA and protein expression levels of E-cadherin were significantly downregulated in the siSmad4 and siSmad4 + cetuximab groups compared with in the cetuximab or NC + cetuximab groups (P<0.01). The effects of siSmad4 on vimentin expression demonstrated opposite results to those on E-cadherin at the mRNA and protein levels (P<0.01; Fig. 4A, E and I). These results indicated that silencing Smad4 partly reversed the effects of cetuximab on the expression of apoptosis- and EMT-related genes.

The transfection efficiency of the Smad4 overexpression plasmid was demonstrated in Fig. 2B and C; Smad4 expression was significantly increased post-transfection with the overexpression plasmid. As shown in Fig. 5A-C, cetuximab significantly inhibited Smad4 protein (P<0.05) and mRNA expression (P<0.01). Smad4 overexpression partly reversed the expression of Smad4 decreased by cetuximab (P<0.01). Cell viability was detected using the CCK-8 assay; the results demonstrated that NC + cetuximab significantly decreased cell viability (P<0.01; Fig. 5D). Cetuximab in combination with Smad4 overexpression further decreased cell viability compared with the control group (P<0.01). In addition, cell viability was attenuated in the Smad4 overexpression + cetuximab group compared with in the NC + cetuximab group (P<0.05), suggesting that overexpressing Smad4 may enhance the sensitivity of SW480 cells to cetuximab.

As shown in Fig. 6A, NC + cetuximab slightly increased the wound area compared with in the control group (P<0.05). Overexpression of Smad4 had no evident effect on the inhibition of migration induced by cetuximab (P>0.05). The Transwell assay demonstrated that NC + cetuximab and Smad4 overexpression + cetuximab significantly downregulated invasion rate (P<0.01; Fig. 6B). For cell apoptosis, the results demonstrated that cetuximab treatment alone (18.79%), and also Smad4 overexpression + cetuximab (25.25%) significantly upregulated the apoptosis rate compared with in the control group (4.64%, P<0.01; Fig. 6C). Additionally, the apoptotic effect of Smad4 overexpression + cetuximab was significantly different compared with treatment with cetuximab alone (P<0.01).

The results revealed that the anti-apoptosis gene Bcl-2 was markedly decreased in the Smad4 overexpression + cetuximab group compared with in the control and NC + cetuximab groups, at the mRNA and protein levels (P<0.01; Fig. 7A, B and F). At the protein level, the expression of Bax was significantly increased in the Smad4 overexpression + cetuximab and NC + cetuximab groups compared with in the control group (P<0.01; Fig. 7A and C). Notably, the increase in Bax expression was more noticeable in the Smad4 overexpression + cetuximab group compared with in the NC + cetuximab group. The effects of treatments on the mRNA expression levels of Bax were consistent with those on protein expression (P<0.01; Fig. 7G). Furthermore, the protein and mRNA expression levels of E-cadherin were significantly upregulated in the Smad4 overexpression + cetuximab group compared with in the control and NC + cetuximab groups (P<0.01; Fig. 7A, D and H). Conversely, the protein expression levels of vimentin were significantly decreased in the Smad4 overexpression + cetuximab group compared with in the control and NC + cetuximab groups (P<0.01; Fig. 7A and E). At the mRNA level, the Smad4 overexpression + cetuximab group exhibited no clear difference in vimentin expression compared with the NC + cetuximab group (P>0.05; Fig. 7I). However, the mRNA expression levels of vimentin were markedly downregulated compared with in the control group (P<0.01; Fig. 7I).