Three colorectal cancer cell lines, HCT-116, HT-29 and Caco-2, were purchased from the Cell Bank of the Chinese Academy of Sciences (CCAS; Shanghai, China) and maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS) (both from Invitrogen, Shanghai, China). PT (10 µM) was added to the complete medium used in the culture of HCT-116/PT, HT-29/PT and Caco-2/PT. The viral packaging cell line 293TN was purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA) and maintained in Dulbeccos minimum essential medium, (DMEM; Invitrogen) supplemented with 10% FBS. All cells were incubated at 37°C in a humidified 95% air and 5% CO₂ incubator. All adherent cells were passaged using 0.25% trypsin (Invitrogen) digestion.

HCT-116, HT-29 and Caco-2 cells were maintained according to routine protocols, except for the addition of PT (starting at 0.5 µM) (Sigma-Aldrich, Carlsbad, CA, USA) to the medium; the concentration of PT was doubled every 3 passages, i.e., 1, 2, 4, 8, 16 and 32 µM. After being cultured in medium containing 32 µM PT for 3 passages, resistant cells were named as HCT-116/PT, HT-29/PT and Caco-2/PT. We then examined the IC₅₀ values for PT in the resistant cell lines and their parental cells. The resistant cells as well as their parental cell lines were seeded into 96-well plates (Corning, Corning, NY, USA) at a concentration of 1×10⁵ cells/well and cultured for 48 h in medium containing PT at final concentrations of 1, 2, 4, 8, 16 or 32 µM. The Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assay was employed to assess the sensitivity of the cells to PT, and the IC₅₀ values were calculated. Moreover, PT-resistant cell lines and their parental cell lines (1×10⁶ cells) were harvested for total RNA and protein extraction, followed by real-time PCR and western blotting for Smad4 assessment.

The coding sequence (CDS) of human Smad4 (NM_005359.5) was amplified using the primers 5′-GGAATTCGCCACCATGGACAATGTGTCTATTACG-3′ and 5′-CGGGATCCTCAGTCTAAAGGTTGTGGGTCTG-3′, which contained an EcoRI site, a Kozak sequence and a BamHI site. cDNA was prepared by reverse transcription using RNA isolated from 293TN cells. The PCR product was digested and cloned into a pcDH1-CMV lentiviral expression vector (System Biosciences, Mountain View, CA, USA); the recombinant vector was named pcDH1-Smad4. The construct integrity was confirmed by DNA sequencing, and endotoxin-free DNA was prepared in all cases. The products of the vectors were confirmed by DNA sequencing, and endotoxin-free DNA was prepared.

One day prior to transfection, 293TN cells were seeded into 10-cm dishes and co-transfected with 2 µg of the pcDH1-Smad4 vector and 10 µg of pPACK Packaging Plasmid mix (System Biosciences) using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer's protocol. The medium was then replaced with DMEM containing 1% FBS. After 48 h, the supernatant was harvested and then cleared by centrifugation for 5 min at 5,000 × g and 4°C before being passed through a 0.45-µm polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The viral titer was determined by gradient dilution, and the packaged lentivirus was named Lv-Smad4. A control virus carrying no insert gene, Lv-control, was constructed as well.

HCT-116/PT, HT-29/PT and Caco-2/PT cells in logarithmic growth phase were seeded into 6-well plates at 5×10⁵ cells/well, and cultured for 24 h in complete medium at 37°C and 5%CO₂. One day later, a viral solution (Lv-control or Lv-Smad4) was added at a multiplicity of infection (MOI) of 10. The infection efficiency was observed using a fluorescent marker at 72 h after infection. Total RNA and protein were isolated from the cells and subjected to real-time PCR and western blotting for evaluation of Smad4 mRNA and protein levels, respectively.

The 3 cell lines and their derivatives were seeded into 96-well plates at 5×10⁴ cells/well and cultured in medium containing PT at final concentrations of 1, 2, 4, 8, 16 or 32 µM for 48 h, followed by a CCK-8 assay to assess cell viability. Briefly, 10 µl of CCK-8 solution was added, and the cells were then cultured under normal conditions for an additional 4 h before measuring the absorbance at 450 nm. The inhibition ratio was calculated, and the fitted equation between the inhibition ratio and PT concentration was obtained for computation of the IC₅₀ values for PT at 48 h.

The resistant cells were seeded into 6-well plates at 1×10⁵ cells/well in medium containing 10 µM PT and cultured for 48 h. The cells were collected, and apoptosis was assessed using flow cytometry (FACSCalibur) after treatment with Annexin V/FITC apoptosis detection kit II (cat. 556570) (both from BD Biosciences, San Jose, CA, USA). Each cell line was grouped as control cells, cells infected with Lv-control and cells infected with Lv-Smad4, and then cultured in the presence or absence of PT. For lentiviral infection, cells were employed in the experiment 72 h after infection. The experiments were carried out in 3 resistant cell lines.

Cell migration was assessed using a QCM™ 24-well Fluorimetric Cell Migration Assay kit (Chemicon, Temecula, CA, USA) in accordance with the manufacturer's instructions. Cells were seeded into the upper chamber at 1×10⁶ cells/well, and 500 µl of complete medium containing 10% FBS was added to each well in the lower chamber. Cells that had migrated to the underside of the membrane were fixed in 4% paraformaldehyde after 48 h. The invading cells were stained with DAPI, and fluorescence was quantified. Data are reported as relative fluorescence units (RFUs). The grouping was the same as that in the apoptosis assay.

HCT-116/PT, HT-29/PT and Caco-2/PT and those infected with Lv-Smad4 for 72 h were seeded into 6-well plates at 1×10⁵ cells/well and treated with PT (10 µM) or without PT. After being cultured at 37°C under 5% CO₂ for 48 h, the cells were harvested, and total protein was extracted for western blotting to assess the levels of Smad4, MDR1, Bcl-2, Bax and caspase-3, as well as the phosphorylation of NF-κB p65.

Six-week-old male BALB/c nude mice, provided by the Animal Experiment Center of the Second Military Medical University, were randomly divided into 3 groups: the model group (16 mice, inoculated with Caco-2/PT), the Lv-control group (8 mice, inoculated with Caco-2/PT infected with Lv-control) and the Smad4 overexpression group (8 mice, inoculated with Caco-2/PT infected with Lv-Smad4). Suspensions of Caco-2/PT, Caco-2/PT + Lv-control or Caco-2/PT + Lv-Smad4 (1×10⁷ cells) cells were prepared in 50 µl of dPBS and subcutaneously injected into the left fore-flank of each mouse; tumors formed within 15 days. The model group was divided into treated and untreated groups. The treated, Lv-control and Smad4 overexpression groups were administered daily with 10 mg/kg PT [200 ng/µl, prepared in normal saline from a 20 µg/µl stock solution prepared with dimethyl sulfoxide (DMSO)] via caudal vein injection, and the untreated group was injected with normal saline. The treatment period was 5 weeks. The tumor diameter was assessed every week, and the tumor volumes were calculated. All mouse experiments were approved by the Medical Ethics Review Committee of Harbin Medical University.

Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instruction and reverse-transcribed into cDNA using M-MLV reverse transcriptase and an oligo(dT)18 primer (both from Takara, Shiga, Japan). The following specific primers were used for quantitative PCR of human Smad4 and β-actin to generate amplified products of 222 and 211 bp, respectively: Smad4, 5′-ACCACCAAAACGGCCATCTTCAG-3′ and 5′-GGTCCACGTATCCATCAACAGTA-3′; β-actin, 5′-CCTGTACGCCAACACAGTGC-3′ and 5′-ATACTCCTGCTTGCATCC-3′. Real-time PCR was performed using a SYBR^® Premix Ex Taq™ kit and a TP800 system (both from Takara). cDNA from 200 ng of total RNA was used as the template. The PCR reactions were carried out under the following conditions: 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 20 sec and extension at 72°C for 20 sec. The mRNA levels of Smad4 were normalized to those of an endogenous housekeeping gene, β-actin, using the ΔΔCt method.

Total protein was extracted from cells using M-PER mammalian protein extraction reagent (Pierce, Rockford, IL, USA). Equal amounts of protein (25 µg/lane), as estimated using the bicinchoninic acid (BCA) protein assay (Pierce), were separated by 11% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. The blots were probed with a monoclonal antibody against human Smad4 (1:1,000), anti-MDR1 (1:500), anti-p-p65/p65 (1:500), anti-Bcl-2 (1:200), anti-Bax (1:500), anti-caspase-3 (1:300) and anti-β-actin (1:800), followed by a secondary horseradish peroxidase (HRP)-conjugated anti-mouse/rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After being washed, the bands were detected using chemiluminescence and exposed to X-ray films. β-actin was used as an endogenous reference for normalization.

All data are expressed as the mean ± standard deviation (SD). Factorial analysis was employed for inter- and intra-group comparisons. Statistical significance was determined by Student's t-test for comparison of two groups, and means were compared with a Chi-square test. P-values <0.05 were considered to be statistically significant. All statistical analyses were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA).

The real-time PCR and western blotting results for Smad4 in 3 PT-resistant cell lines revealed decreases at both the transcriptional level (Fig. 1A-a, B-a and C-a) and protein level (Fig. 1A-b, B-b and C-b). There was a significant difference in PT-resistant cells and parental cells (P<0.01). Lentiviral-mediated overexpression of Smad4 in these 3 PT-resistant cell lines resulted in a gene delivery efficiency close to 100% at 72 h after infection, as demonstrated by green fluorescent protein (GFP) expression (Fig. 1A-c, B-c and C-c). Furthermore, protein assessments revealed that the protein levels of Smad4 (Fig. 1A-d, B-d and C-d) were significantly increased in the groups infected with Lv-Smad4 compared with the control cells or the Lv-control group (P<0.01), and there was no significant difference in the control cells or the Lv-control group (P>0.05), indicating that the lentiviral system effectively delivered exogenous Smad4 gene in these cells.

We analyzed the IC₅₀ values to ascertain the successful establishment of PT-resistant cell lines. As expected, the IC₅₀ values of the HCT-116/PT, HT-29/PT and Caco-2/PT cell lines increased from 4.09±0.76, 3.97±0.58 and 7.13±1.21 µM to 20.31±3.21, 17.65±1.89 and 18.64±3.02 µM after 48 h, respectively (P<0.01, vs. the parental cells; Fig. 2A). Overexpression of Smad4 in HCT-116/PT, HT-29/PT and Caco-2/PT cells significantly decreased the IC₅₀ values to 6.12±2.11, 5.75±1.08 and 7.86±1.95 µM (P<0.01, vs. the control cells or the Lv-control group; Fig. 2B), respectively.

We employed a Transwell experiment to explore changes in tumor cell migration, which plays an important role in tumor metastasis. In the absence of PT, there was no statistical difference in the number of cells penetrating the basal membrane between the Lv-Smad4 and the control cells or the Lv-control group (P>0.05); in the presence of 10 µM PT, overexpression of Smad4 decreased the number of cells that penetrated the basement membrane (P<0.01, vs. the control cells or the Lv-control group; Fig. 3A), and there was no statistical difference between the control cell group and Lv-control group (P>0.05). The data also revealed that treatment with 10 µM PT had no significant impact on HT-29 and HT-29 infected with Lv-control (P>0.05), however it significantly inhibited the migration in HT-29 cells infected with Lv-Smad4 (P<0.05, PT group vs. non-PT group;Fig. 3A). The results indicate that overexpression of Smad4 has no direct impact on the migration of HT-29/PT, but may increase its sensitivity to PT, thus an increased response to PT was observed. Consistent with the data of the migration assay, overexpression of Smad4 did not induce apoptosis in HT-29/PT cells, but it restored its sensitivity to PT, thus PT exerts its apoptosis-inducing activity (Fig. 3B). We also performed these assays in HCT-116/PT and Caco-2/PT cell lines, and observed similar results (data not shown).

The results of the in vivo tumor inhibition experiment revealed that administration of PT for 5 weeks only slightly inhibited tumor formation by Caco-2/PT cells, but significantly inhibited tumor formation by Caco-2/PT cells overexpressing Smad4 (Fig. 4). At the fifth week after administration, the tumor volumes were 535.29±80.29 mm³ in the model group and 431.40±60.00 mm³ in the PT administration group, corresponding to an inhibition rate of 19.42% and a non-significant difference. However, the tumor volume in the Smad4-overexpressing group that received PT averaged 189.75±42.18 mm³, corresponding to an inhibition rate of 64.55% and a significant difference compared to the control group (P<0.01). The data from the in vivo tumor inhibition experiment were in line with the overexpression of Smad4 resensitizing the 3 PT-resistant cell lines.

To elucidate how Smad4 overexpression increases the sensitivity of resistant colorectal cancer cells to PT, we examined a number of proteins that are related to Smad4 expression and are associated with drug resistance. In the absence of PT, the level of Smad4 in the Lv-Smad4 group was significantly higher than that in the control cells (P<0.01; Fig. 5). The opposite was observed with MDR1. No observable difference was found in the phosphorylation of NF-κB p65, and in the expression of Bcl-2, caspase-3 and Bax (P>0.05). When the cells were treated with 10 µM PT for 48 h, caspase-3 and Bax in the cells infected with Lv-Smad4 were higher than in the control group (P<0.01), and the expression of MDR1 and Bcl-2 and the phosphorylation of NF-κB p65 in the Lv-Smad4 group were lower than in the control group (P<0.05). The data of HCT-116/PT and HT-29/PT cell lines were in agreement with those of Caco-2/PT (data not shown).