The HCT116 human colon cancer cell line was obtained from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI 1640 medium (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum, 10 U/ml penicillin, and 10 μg/ml streptomycin in a humidified atmosphere containing 5% CO₂ at 37°C.

Dox was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). The primary polyclonal rabbit antibodies against human Smad4, Smad2, Smad3, phospho-Smad2 Ser465 (p-Smad2), phospho-Smad3 Ser425 (p-Smad3) and MDR p-gp (p-gp) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA) and primary polyclonal rabbit antibodies against human E-cadherin (sc-7870), Vimentin (sc-5565), N-cadherin (sc-7939), Snai1 (sc-28199) and Slug (sc-15391) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). These antibodies were used in western blot analysis and immunofluorescence staining at a dilution of 1:1,000. The TGFβ1 enzyme-linked immunosorbent assay (ELISA) kit was obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and other materials, including secondary antibodies were purchased from Beyotime Institute of Biotechnology (Shanghai, China).

The short hairpin RNA (shRNA)-encoding complementary single stranded oligonucleotides corresponding to Smad4 were designed as previously described (11). The RNAi sequence (5′-GTACTTCATACCATGCCGA-3′) was complementary to the human Smad4 cDNA sequence and was annealed and cloned into the pGCSIL-green fluorescent protein vector (GeneChem, Shanghai, China). The control sequence of RNAi (5′-TTCTCCGAACGTGTCACGT-3′) was used as a negative control. The recombinant virus was packaged using the Lentivector expression system (GeneChem). HCT116 cells were transfected with the recombinant lentivirus using Lipofectamine 2000 (Invitrogen Life Technologies) and transfected cells were screened under 800 μg/ml G418 (Calbiochem, Darmstadt, Germany) for 4 weeks to generate stable monoclonal cell lines (Smad4 stable downregulation cell lines, RNAi-Smad4 and control stable cell lines, RNAi-NC). The expression of Smad4 was confirmed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis.

Cells were divided into four groups, termed HCT116 cells, RNAi-NC cells, Smad4-RNAi cells and HCT116 cells untreated by Dox as a control (the control group). Cells were treated with 50 nmol/l Dox, or an equal volume of phosphate-buffered saline (PBS) for the controls, for 7 days (the medium was changed every alternate day, with the addition of the same dosage of Dox on occasion) for RT-qPCR, western blot analysis, immunofluorescence staining and ELISA.

Following drug treatment, cells were washed twice with PBS and lysed in ice-cold radioimmunoprecipitation assay buffer (20 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.5% Nonidet P-40; 1 mM EDTA; 50 μg/ml leupeptin; 30 μg/ml aprotinin; 1 mM Na₃VO₄; and 1 mM phenylmeth-ylsulfonyl fluoride). The cell lysate (20 μg) was separated on a sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and then transferred onto a nitrocellulose membrane (Pall Corporation, Port Washington, NY, USA) by use of the wet transfer system (Bio-Rad, Hercules, CA, USA). Nonspecific binding sites were then blocked for 2 h at room temperature in Tris-buffered saline (TBS, pH 7.4) containing 0.1% Tween-20 and 10% bovine serum albumin (BSA). The primary antibodies were diluted (1:1,000) in TBS containing 0.1% Tween-20 and 5% BSA and incubated overnight at 4°C. The appropriate peroxidase-conjugated secondary antibodies (immunopure rabbit anti-goat IgG; goat anti-mouse IgG; and goat anti-rabbit IgG; Pierce Biotechnology, Inc., Rockford, IL, USA) were used at a dilution of 1:1,500. Positive antibody reactions were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Inc.).

Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions and cDNA was synthesized from the mRNA obtained by RT-PCR using the SuperScript first-strand synthesis system (Fermentas, Vilnius, Lithuania). PCR was performed according to the standard protocol using a Roche LightCycler (Roche Diagnostics GmbH, Mannheim, Germany) with SYBR Green supermix (Bio-Rad). The PCR conditions were as follows: 31 Cycles of denaturation for 5 sec at 95°C, annealing for 30 sec at 60°C and primer extension for 60 sec at 72°C. cDNA was used as the template for the qPCR reaction with the following specific primers: Smad4, F 5′-TGTGACAGTGTCTGTGTGA-3′ and R 5′-CCTACCTGAACGTCCATTTC-3′; actin, F 5′-GTCCACCGCAAATGCTTCTA-3′ and R 5′-TGCTGTCACCTTCACCGTTC-3′. The human actin gene was amplified as an endogenous control. The gene expression of mRNA from each sample was calculated by normalizing against that of the reference gene, β-actin. Primer sequences are available upon request.

The cell lines (HCT116, RNAi-NC and RNAi-Smad4) were seeded onto 96-well plates (6.0×10³ cells/well) and allowed to attach overnight. Following cellular adhesion, freshly prepared Dox at the appropriate concentration (10, 30, 50, 80 or 100 nmol/l), was added for the 24 h treatment, and 50 μmol/l Dox was added for the 7 day treatment, respectively. The viability of the cells was evaluated using an MTT assay, according to the manufacturer’s instructions (Roche Applied Science, Indianapolis, IN, USA). Briefly, MTT was added at a concentration of 500 mg/l and the cells were incubated for 4 h at 37°C. The absorbance reading of each well was determined using a computer-controlled microtiter plate reader (iMARK; Bio-Rad) at a wavelength of 570 nm. The cell viability rates were defined as the relative absorbance of treated vs. untreated cells.

Cells were plated on 20 mm circular microscope coverslips. According to the manufacturer’s (Beyotime Institute of Biotechnology) instructions, cells were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 15 min. Non-specific binding sites were then blocked for 1 h at room temperature in PBS (pH 7.4) containing 0.1% Tween-20 and 5% BSA. Primary antibodies (Snail and Slug, 1:200) in PBS containing 0.1% Tween-20 and 1% BSA were added overnight at 4°C in a humidified chamber. Cells were then incubated with the appropriate fluorescein isothiocyanate-conjugated secondary antibodies (1:200) for 1 h at room temperature in a humidified chamber in darkness. The samples were subsequently treated with 4′,6-diamidino-2-phenylindole (10 μg/ml) for 30 sec to detect the cell nuclei. Images were obtained using fluorescence microscopy (BX53; Olympus, Tokyo, Japan) at magnification, ×400.

HCT116 cells, RNAi-NC cells and Smad4-RNAi cells were treated with 50 nmol/l Dox for 7 days and HCT116 cells, which were not subject to treatment with Dox were used as a control group. The concentration of TGFβ1 in the supernatant of the cells was measured using ELISA kits (Anogen-Yes Biotech Laboratories Ltd., Mississauga, ON, Canada) according to the manufacturer’s instructions.

Statistical comparisons between the two groups were performed using an unpaired t-test. All groups were compared using a one-way analysis of variance, followed by Tukey’s post hoc test where appropriate. SPSS software, version 17.0 (SPSS, Inc., Chicago, IL, USA) was used for all analyses. P<0.05 was considered to indicate a statistically significant difference. All data are expressed as the mean ± standard deviation from at least three independent experiments.

Dox has been observed to induce expression of circulating TGFβ in xenograft and transgenic animal models (12,13). In order to identify whether and how the TGFβ/Smad4 signaling pathway was affected by Dox administration, the effects of Dox treatment on TGFβ1 concentration and p-Smad2/Smad3 expression level in HCT116 cells were examined using ELISA and western blotting. Following treatment with 50 nmol/l Dox for 7 days, it was shown that the expression of TGFβ1 in HCT116 cells was significantly higher (P<0.05) than that in the control (HCT116 cells treated with PBS; Fig. 1A). Similarly, the levels of p-Smad2 and Smad3 in the HCT116 cells increased in comparison with the control group (Fig. 1B). Thus, the present results indicated that long-term administration of Dox may activate the TGFβ/Smad4 pathway in HCT116 cells.

To further examine the effects of Dox on HCT116 cells, the cell viability and p-gp levels were assessed using an MTT assay and western blotting. HCT116 cells were treated with 10, 30, 50, 80 and 100 nmol/l Dox for 24 h, and with 50 nmol/l Dox for 1, 2, 3, 4, 5, 6 and 7 days, respectively. Cells treated with PBS alone were used as a control. The results from the MTT assay indicated that the cell viability of HCT116 cells decreased in a dose-dependent manner (Fig. 1C). A concentration of 50 nmol/l was determined to be appropriate for long-term Dox administration for the MTT assay. It was observed that the viability of HCT116 cells decreased between 1 and 3 days in a time-dependent manner. However, it did not change markedly between 3 and 7 days, following treatment (Fig. 1C). Following administration of 50 nmol/l Dox, the p-gp expression of HCT116 cells increased in a time-dependent manner (Fig. 1D). These data demonstrated that HCT116 cells acquired resistance to Dox following long-term treatment at a low concentration.

In order to assess the knockdown efficiency of the lentiviral vector, the mRNA and protein expression of Smad4 in HCT116 cells, RNAi-NC cells and Smad4-RNAi cells was assessed. The results of the RT-qPCR and western blotting experiments revealed that mRNA and protein levels of Smad4 in Smad4-RNAi cells were significantly lower than that in HCT116 cells or RNAi-NC cells (P<0.01), while no significant difference was observed between HCT116 cells and RNAi-NC cells (P>0.05; Fig. 2).

To determine whether the acquisition of Dox resistance was associated with specific molecular changes that are consistent with EMT, the expression of epithelial and mesenchymal phenotypic markers, following 50 nmol/l Dox administration, were examined using RT-qPCR and western blotting. From the PCR data, a significant reduction in the expression of E-cadherin, and an upregulation of Vimentin and N-cadherin, in HCT116 cells was observed compared with that in the control group. The expression of the transcription factors, Snail and Slug, were significantly increased in resistant cells and the western blotting results were in accordance with the PCR data (Fig. 3A and B). Furthermore, immunofluorescence staining revealed that Snail and Slug were more localized in the cytoplasm of the HCT116 cells than it was in the control group (Fig. 4). These results suggested that EMT occurred in HCT116 cells following treatment with Dox.

Following successful silencing of the Smad4 gene using a lentiviral vector, Smad4-specific RNAi was used to investigate the effect of inhibiting the TGFβ/Smad4 signaling pathway on the process of EMT, which was induced following Dox treatment. RNAi-NC cells and Smad4-RNAi cells were also treated with 50 nmol/l Dox for 7 days, and the expression of EMT markers and transcription factors were examined using RT-qPCR and western blotting. The PCR data demonstrated that the expression of E-cadherin in Smad4-RNAi cells was higher than that observed in the RNAi-NC cells (P<0.05), and that the expression levels of Vimentin, N-cadherin, Snail and Slug in Smad4-RNAi cells was significantly lower than that detected in the RNAi-NC cells (P<0.05). The western blotting results were in accordance with the PCR data (Fig. 3A and B). Furthermore, immunofluorescence staining revealed that in the RNAi-NC cells, Snail and Slug were more localized to the cytoplasm than they were in the Smad4-RNAi cells (Fig. 4). These results suggested that downregulation of Smad4 reverses EMT, which is induced following Dox treatment.

In order to examine the effect of the downregulation of Smad4 on HCT116 cell sensitivity to Dox, the cell viability and MDR p-gp levels were also investigated using an MTT assay and western blot analysis, in RNAi-NC cells and Smad4-RNAi cells. Following administration of the same treatment to HCT116 cells, the MTT assay results indicated that the cell viability ratio of RNAi-NC and Smad4-RNAi cells decreased in a dose-dependent manner (Fig. 1C). Concerning the MTT data from the long-term administration of 50 nmol/l Dox, it was shown that the cell viability ratio of RNAi-NC cells decreased between 1 and 3 days following treatment, in a time-dependent manner, whilst it did not alter markedly between 3 and 7 days following treatment. This result was similar to that obtained in the HCT116 cells. However, the cell viability ratio of Smad4-RNAi cells decreased in a time-dependent manner for the duration of the experiment (Fig. 1C). Following 7 days of 50 nmol/l Dox administration, the p-gp expression of RNAi-NC cells was higher than that in the control and Smad4-RNAi cells (Fig. 1E). These data suggested that downregulation of Smad4 increases HCT116 cell sensitivity to Dox following long-term treatment at a low concentration.