Tissue specimens were acquired from 128 patients (mean age, 66 years; 68.0% male) with ESCC who underwent cancer resection at Shanghai Jiao Tong University Affiliated with Sixth People’s Hospital between July, 2005 and June, 2011. Patients had received no treatment for ESCC prior to surgery. Tumors were staged using the tumor-node-metastasis classification system according to the International Union Against Cancer criteria. Ethical approval for the use of patient tissues was provided by the Ethics Committee of Shanghai Sixth People’s Hospital (Shanghai, China). Informed consent was obtained from all patients. Data regarding patient age, sex, pathological grading, lymph node metastasis (LNM), tumor size and depth were collected followed the criteria proposed by International Union Against Cancer criteria (19). The follow-up period ranged between 5.3 and 48.8 months, and mean follow-up time was 32.1 months.

The three human ESCC cell lines ECA109, TE1 and KYSE30 were purchased from the China Academy of Science cell library (Beijing, China). Cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin (100 μg/ml) at 5% CO₂ and 37°C. In cisplatin-related experiments, TE-1 cells were seeded onto 6-well cell culture cluster plates (Corning inc, Corning, NY, USA) at a concentration of 1×10⁶ cells/well in 2 ml culture medium. After being grown overnight, the cells were treated with cisplatin (cat. no., CAS 15663-27-1, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at various concentrations (0-50 μM) for 48 h.

IHC was performed following a previously described protocol (20). The primary antibodies used were anti-CCT8 (1:800; cat. no. sc-13891, Santa Cruz Biotechnology, Inc.) and anti-E-cadherin (1:200; cat. no. sc-71009, Santa Cruz Biotechnology, Inc.). IHC analyses (mouse anti-goat IgG-HRP: cat. no. sc-2354, 1:2,000; m-IgGκ BP-HRP: cat. no. sc-516102, 1:2,000, Santa Cruz Biotechnology, Inc.) were performed as previously described (21). A staining index (SI) score of ≥6 indicated high expression and an SI score of ≤4 indicated low expression (21).

Western blot analysis was performed following a previously described protocol (22). Protein was obtained using a lysis buffer (1% SDS, 10 mM Tris-HCl, pH 7.6, 20 μg/ml aprotinin, 20 μg/ml leupeptin and 1 mM AEBSF). Protein were separated on a 10% SDS-PAGE gel. Polyvinylidene fluoride (PVDF) membranes (Millipore, Inc., Billerica, MA, USA) were blotted with anti-CCT8 (cat. no. sc-13891, 1:500), anti-caspase-3 (cat. no. sc-166589, 1:1,000), anti-E-cadherin (cat. no. sc-71009, 1:1,000), anti-N-cadherin (cat. no. sc-53488, 1:1,000), anti-vimentin (cat. no. sc-66001, 1:1,000), α-actin (cat. no. sc-32251, 1:1,000), β-tubulin (cat. no. sc-5274, 1:1,000) and anti-GAPDH (cat. no. sc-137179, 1:500; all Santa Cruz Biotechnology, Inc.). Secondary antibodies used as follows: mouse anti-goat IgG-HRP: cat. no. sc-2354, 1:1,000; m-IgGκ BP-HRP: cat. no. sc-516102, 1:1,000 (Santa Cruz Biotechnology, Inc.). The band density was measured with a computer-assisted image-analysis system (Adobe Systems, San Jose, CA, USA) and normalized against GAPDH level.

Primer pairs for the CCT8 (NM_006585.2) siRNA expression vector had the following target sequences: 5′-CCUCCAUAAUGAGUAAACA-3′, 5′-GGCUGUGUAUAGAAACAUA-3′ and 5′-CUGGAGUUA AGGCCAAUGA-3′. These sequences are pertaining to pires2-egfp. The CCT8 siRNA vector (1 μg/1×10⁵ cells) and the non-specific vector (2 μl/1×10⁵ cells) were transfected for 48 h using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Cell proliferation and migration were measured as previously described (23,24). Migration assays were performed using the microliter scale radial monolayer migration assay. Tumor cells (5×10⁵) were seeded in 6-well plates and grown to confluence overnight. Twenty-four hours after incubation with 0.1% bovine serum albumin (BSA, Gibco; Thermo Fisher Scientific, Inc.) or 10 μg/ml laminin (cat. no. sc-29012, Santa Cruz Biotechnology, Inc.) conditions a line was scratched within the confluent cell layer using the fine end of a 10-ml pipette tip (time 0). Cells were washed with phosphate-buffered saline (PBS, Procell Inc., Wuhan, China) and photographed under a 20X objective lens every 6 h using an inverted Leica phase-contrast microscope (Leica DFC 300 FX, Leica Microsystems, Inc., Wetzlar, Germany). The invasiveness of cells was determined using a Transwell assay on a 24-well Transwell plate (8-μm pore size; Corning Inc., Corning, NY, USA). A total of 1×10⁵ cells were plated in the top chamber. BD Matrigel™ Basement Membrane Matrix was added to the upper membrane and allowed to gel for 1 h. Subsequently, 500 μl medium containing chemotactic factor was placed in the lower chamber. Cells were plated in the upper chamber of quadruplicate wells at a density of 1×10⁵/ml and incubated for 24 h. Cells were then fixed with paraformaldehyde, stained with crystal violet (cat. no. sc-207460, Santa Cruz Biotechnology, Inc.), and counted immediately after staining.

The Predictive Analytics Software 18 software package (SPSS, Inc., Chicago, IL, USA) was used for statistical analyses. Data are presented as the means ± standard deviations (SD) of 3 independent experiments. The association between CCT8 expression and patient clinicopathological features was calculated using the χ² test. Kaplan-Meier methods were used to construct survival curves. Multivariate analysis was performed using the Cox proportional hazards model. One-way analysis of variance followed by Tukey’s post hoc test was used to determine whether differences between groups were significant and P<0.05 was considered to indicate a statistically significant difference.

CCT8 and E-cadherin expression in the 128 patients with ESCC was assessed using IHC. CCT8 expression (Fig. 1) and its association with the clinicopathological variables of ESCC is presented in Tables I and II. Following a previously described protocol (22), patients were divided into two groups: One group contained patients exhibiting high CCT8 expression (score ≥6) and one group contained patients exhibiting low CCT8 expression (score ≤4). The results demonstrated that CCT8 expression was associated with pathological grading (P<0.001), tumor depth (P<0.011) and LNM (P<0.001); however, it was not associated with other prognostic factors, including tumor size (Table I). These results indicate that there is an association between CCT8 expression and the degree of ESCC malignancy. A total of 38 out of 57 (66.7%) patients in the high CCT8 expression group succumbed compared with only 22 of 71 (31.0%) patients in the low expression group (P<0.001; Table II). Furhthermore, patients with poor pathological grading (P<0.001), LNM (P=0.041), greater tumor depth (P=0.035) and high E-cadherin expression (P=0.004) exhibited significantly lower rates of survival (Table II). Kaplan-Meier survival curves were then constructed to determine the survival rates of patients in the high CCT8 expression group compared with those in the low CCT8 expression group. The results indicated that the survival time of patients in the high CCT8 expression group was significantly lower than that of patients in the low CCT8 expression group (P<0.05; Fig. 2). A multivariate Cox proportional hazards model was then used to predict patient survival. The results demonstrated that tumor pathological grade, LNM, tumor depth, CCT8 and E-cadherin expression were significant predictors of patient survival (P<0.05; Table III).

As CCT8 expression is associated with LNM in ESCC, it was hypothesized that it may also be associated with the migration of ESCC cells. Therefore, the expression of CCT8 in ESCC tissues was evaluated (Fig. 3A). CCT8 expression was high in tissues taken from patients with LNM and low in tissues from patients without LNM. In addition, CCT8 expression was measured in the TE-1, ECA109 and KYSE30 ESCC cell lines (Fig. 3B). Quantification of the results indicated that TE-1 cells exhibited the highest level of CCT8 expression among the ESCC cell lines (Fig. 3C); therefore, TE-1 cells were used in subsequent experiments.

It was hypothesized that CCT8 expression is associated with tumor migration. A monolayer wound healing assay was used to assess the migration rate of cells plated onto the nonpermissive substrate BSA and the migration-stimulating substrate, laminin (24,25). It was demonstrated that laminin significantly increased the migration of TE-1 compared with those cells treated with BSA (P<0.05; Fig. 4A). To determine whether the migratory phenotype affected cell proliferation, a Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamato, Japan) was used and the cell growth rate was determined. The results demonstrated that laminin stimulated TE-1 cell migration in vitro but did not affect cell proliferation (Fig. 4B). When TE-1 cells were plated on laminin, CCT8 expression increased markedly, as did the expression of N-cadherin and vimentin; by contrast, the expression of E-cadherin markedly decreased (Fig. 4C). These results suggest that CCT8 may serve a role in the migration of ESCC cells.

To assess the function of CCT8 in ESCC cells, CCT8 expression was knocked down and western blotting was used to verify this loss of expression (Fig. 5A). Three target sites were selected to knockdown CCT8 expression. The most significant decrease in CCT8 expression was following CCT8 knockdown with si#1 and si#2 (P<0.01 and P<0.05, respectively; Fig. 5B); therefore, these were used in subsequent studies. CCT8 knockdown also significantly inhibited the wound healing process (P<0.05; Fig. 5C and D) and significantly decreased the invasiveness of TE-1 cells (Fig. 5E and F). CCT8 knockdown also markedly increased the expression of E-cadherin and decreased the expression of N-cadherin and vimentin (Fig. 5G). Furthermore, expression of the apoptosis marker, cleaved caspase-3, was increased. It has been reported that CCT8 interacts with α-actin and β-tubulin, which can stimulate cell motility (26). The results of western blotting demonstrated that α-actin and β-tubulin were markedly downregulated following CCT8 knockdown (Fig. 5G). These observations suggest that CCT8 facilitates cell motility and is associated with the regulation of α-actin and β-tubulin.

Cisplatin is a chemotherapy drug widely used to treat different types of cancer, including ESCC (27); therefore, it was determined whether cisplatin downregulates CCT8 expression in ESCC. TE-1 cells treated with cisplatin for 48 h exhibited a dose-dependent decrease in CCT8 expression and a dose-dependent increase in cleaved caspase-3 expression (Fig. 6A). α-actin and β-tubulin expression also decreased in a dose-dependent manner (Fig. 6A). Cisplatin induced the upregulation of E-cadherin and decreased the expression of N-cadherin, vimentin, α-actin and β-tubulin. These effects were enhanced following CCT8 knockdown (Fig. 6B). These data demonstrate that CCT8 knockdown significantly increases the apoptosis of TE-1 cells and enhanced the effects of cisplatin on such cells.