Paraffin-embedded tissue samples were sectioned for IHC from primary tumors and adjacent non-cancerous tissues. The tissues were obtained from 129 randomly selected stage II CRC patients who underwent surgery at the Sun Yat-sen University Cancer Center (Guangzhou, China) from January 1998 to December 2002. The CRC patients were histopathologically and clinically diagnosed with CRC (T3/4N0 M0, stage II) according to the American Joint Committee on Cancer (AJCC) TNM staging system. The clinical information was collected from unprocessed medical files and pathological reports. The study was carried out with the approval of the Ethics Committee of the Sun Yat-sen University Cancer Institutional Board, and prior written informed consent was obtained from all of the patients involved.

The corresponding tissue blocks were cut into 5 µm thick sections. Hematoxylin and eosin (H&E) sections were used for analyzing the tumor location. IHC was performed with a rabbit-RKIP antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to standard procedures (20). Human benign prostate tissues were used as a positive control. For the negative control, tissue slides were incubated with PBST instead of the rabbit-RKIP antibody. In the present study, a semi-quantitative estimation was made by both the extent and intensity of immunoreactivity: the percentage of staining was defined as follows: ≤5%=0, >5 to ≤25%=1, >25 to ≤50%=2, and >50%=3. The scores for intensity were evaluated as 0 for negative staining; 1 for weak staining; 2 for moderate staining; and 3 for strong staining. Finally, the indexed sum was acquired by the addition of the intensity grade and the percentage of the staining area. Slides were scored by two independent pathologists who were blinded to the patient data. Discrepancies were resolved by consensus after reevaluation of the slides. A score of 4.5 was suggested as a cut-off for dichotomizing RKIP levels for the prognosis of TTP and overall survival (OS) according to ROC curves. If the final score was <4.5, the tumor was considered to have low expression; otherwise, the tumor was considered to have high expression.

Human colon cancer cell lines (HT-29, HCT116, SW480 and LoVo) were obtained from the American Type Culture Collection and were maintained in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) in a humidified chamber with 5% CO₂ at 37°C. For stable overexpression of endogenous RKIP, the coding sequence of RKIP was amplified and subcloned into the LV5 (pGCMV/GFP+Puro) vector, a lentivirus purchased from GenePharma Biotech (Shanghai, China), according to the manufacturer's instructions. HCT116 cells were then transfected with the overexpressing RKIP lentivirus or negative control lentiviral vectors and named HCT116/RKIP and HCT116/vector cells, respectively. To generate stable RKIP-knockdown cells, an annealed short interfering RNA (siRNA) for RKIP was inserted into the pGPU6/GFP/Neo vector (GenePharma Biotech) according to the manufacturer's instructions to obtain pGPU6/GFP/Neo containing RKIP targeting short hairpin RNA (pGPU6/GFP/Neo-shRKIP). The target sequence for effective knockdown of RKIP expression was 5′-CCC ACC CAG GTT AAG AAT A-3′. pGPU6/GFP/Neo-shRKIP or empty pGPU6/GFP/Neo vectors, which served as negative controls, were transfected into SW480 cells named SW480/sh-RKIP and SW480/vector, respectively. All stable clonal cells generated were selected and cultured according to the manufacturer's instructions for further studies. Western blot assays were used to detect the expression of RKIP in all stable cell lines as described below.

Total RNA was isolated from the tissues or cell lines using TRIzol (Invitrogen). Then, 2 µg of total RNA was reverse-transcribed into cDNA in a 10 µl reaction solution for real-time PCR (RT-PCR) using GoTaq^® qPCR Master Mix (Promega, Madison, WI, USA) as directed by the manufacturer. The programmed parameters for RT-PCR were as follows: heating at 95°C for 10 min to activate AmpliTaq Gold polymerase, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing and extension at 60°C for 60 sec. The expression levels of β-actin were used as an endogenous control to ensure equal loading of the samples. Invitrogen synthesized both oligonucleotide primers for RKIP and β-actin (Shanghai, China). The primer sequences used were as follows: RKIP forward, 5′-CAA TGA CAT CAG TGG CAC AGT C-3′ and RKIP reverse, 5′-CAC AAG TCA TCC CAC TCG GCC TG-3′; β-actin forward, 5′-TGG ATC AGC AAG CAG GAG TA-3′ and β-actin reverse, 5′-TCG GCC ACA TTG TGA ACT TT-3′. The relative mRNA expression of RKIP was normalized against the internal control and analyzed by the 2^−ΔΔct method. The experiment was performed in triplicate and repeated three times.

Western blot analyses were performed using a standard protocol (21). Next, 24 µg of protein extracts was subjected to 12% SDS-PAGE gel electrophoresis. A rabbit anti-RKIP antibody (1:50; Santa Cruz Biotechnology) and a rabbit anti-GAPDH antibody (1:5,000; Cell Signaling Technology, Danvers, MA, USA) were used for analysis according to the manufacturer's instructions. RKIP protein levels were normalized to the total GAPDH levels on the same membrane, which were visualized using an enhanced chemiluminescence ECL detection system (KeyGen Biotech, Nanjing, China).

Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HCT116/vector and HCT116/RKIP cells were plated at a density of 2,000 cells/well, and SW480/vector and SW480/sh-RKIP cells were plated at 4,000 cells/well. The absorbance value of each sample was read at 570 nm using a microplate reader. All experiments were performed in octuplicate to calculate the average result.

To determine the effect of RKIP on cell mobility, a scratch test was performed. Monolayer cells were scrape-wounded using standard micropipette tips. The vertical distance of the inner face of the denuded zone was measured using a fluorescence inverted microscope at 0, 12, 24 and 36 h. All experiments were performed in triplicate.

To better evaluate the invasive and migratory potentials of the cells, 24-well Transwell chambers (8 µm pores) from BD Biosciences were used. For the migration assay, the tumor cells were resuspended at a density of 6×10⁵ cells in 200 µl serum-free medium and transferred to the top chamber of each insert without matrix gel. Then, 500 µl of serum was added to the lower compartment as the chemotactic factor. After incubation for 16 h, the non-migrating cells on the upper side were removed, and the cells that migrated to the undersurface were fixed and dyed with 0.1% crystal violet. In parallel, the invasion Transwell assay was performed as mentioned above with the inclusion of Matrigel mix pre-coated on the inserts and cultured for 24 h. The number of migrating or invading cells was microscopically quantified by counting five independent fields. The final results were compared using the mean of triplicate assays (22).

Female BABL/c athymic nude mice aged 5 weeks were purchased from the Animal Center of Guangdong Province (Guangzhou, China) and maintained under specific pathogen-free conditions. For the in vivo proliferation assays, a total of 10⁶ cells of HCT116/RKIP or SW480/sh-RKIP were injected subcutaneously into the left dorsal flanks of nude mice and the corresponding negative control cells were injected into the right (n=6). An algorithm, volume = length × width × length × 0.5236 (23), was used to calculate the tumor volume every 4 days. Four weeks after injection, the animals were sacrificed and the tumors were weighed. To investigate the effect of RKIP on metastasis, a concentration of 2×10⁷ cells/ml of the HCT116/RKIP, HCT116/vector, SW480/sh-RKIP and SW480/vector cells was injected into the tail veins of mice. Six weeks after injection, the mice were sacrificed, and the lungs and livers were dissected out and embedded in paraffin. The animal tissue blocks were then cut into 4 µm sections consecutively for further staining with H&E. The micrometastases in the lungs and livers were examined and counted by pathologists who had no prior knowledge of the mouse groups (24). All of the in vivo experiments were conducted in strict accordance with the National Institutes of Health guidelines. The protocol was approved by the Ethics Committee of Animal Experiments of the Sun Yat-sen University Cancer Center.

The associations between RKIP expression levels and clinical characteristics were evaluated using the Chi-square analysis. Survival curves were drawn using the Kaplan-Meier method and assessed by the log-rank test. Disease-free survival (DFS) was defined as the interval between the operation date to the date of metastasis or recurrence and OS was computed from the date of surgery to the date of death, or the last follow-up. Univariate and multivariate analyses were performed using the Cox proportional hazards regression model. A Student's t-test was used to analyze the single comparison between two means. All tests were two-sided and considered to be significant with p-values of <0.05. Data are expressed as the mean ± standard error mean (SEM) unless otherwise stated. Statistical analyses were performed using the Statistical Package for the Social Sciences software version 13.

In order to investigate the expression pattern of RKIP in human CRC, IHC was performed to detect RKIP in 129 cases of primary CRC tumor samples as well as 127 cases of adjacent non-cancerous tissues. RKIP staining had a predominantly cytoplasmic and membrane-associated distribution and was rarely nuclear (Fig. 1A and B). The average IHC score in 129 primary CRC tumor samples was 3.95, which was significantly lower than that in the 127 adjacent non-cancerous tissues (average IHC score was 5.04, p<0.001) (Fig. 1C). Moreover, 74 of the 129 primary CRC tumor samples (57.4%) had low RKIP protein expression. In contrast, in the adjacent non-cancerous tissues, only 35 of 127 cases (27.7%) had low RKIP expression staining (p<0.001; Table I). These results indicate that RKIP expression was significantly downregulated in CRC in comparison to the adjacent non-cancerous tissues.

Among the 129 cases of primary stage II CRC, 74 cases of primary CRC tumor samples had low RKIP protein expression, and reduced RKIP expression was found to have a significant correlation with tubular adenocarcinoma (p=0.015; Table II). No statistically significant association was found between RKIP expression and other clinicopathological variables including gender, age, location of primary mass, tumor size, depth of invasion and tumor differentiation (Table II).

To further investigate the prognostic significance of RKIP expression, DFS and OS analyses were performed in these 129 CRC cases using Kaplan-Meier analysis with a log-rank test (Fig. 2). At the end of the follow-up time, 17.8% of the cases (23/129) were observed to have metastasis and/or disease recurrence. Patients with low RKIP expression had a shorter DFS time than those with high RKIP expression (median DFS 125 vs. 143 months; p=0.032; Fig. 2A). Additionally, the T3 stage population (106 cases) was separately analyzed to evaluate the prognostic effect of RKIP by excluding the 23 T4 stage cases. A good correlation between the low RKIP expression and relapse independent of T stage (median DFS 106 vs. 126 months; p=0.049; Fig. 2B). Furthermore, univariate and multivariate analyses indicated that RKIP expression was an independent prognostic factor for CRC relapse (Table III). However the relationship between RKIP expression and overall survival was not statistically significant both in the entire studied population (median OS 125 vs. 107 months; p=0.058; Fig. 2C) and in the T3 stage population (median OS 126 vs. 108 months; p=0.065; Fig. 2D).

We detected both RKIP mRNA and protein levels by qPCR and western blotting in four CRC cell lines (HT29, Lovo, SW480 and HCT116) and in one normal colonic tissue. RKIP was found to be reduced in all these CRC cell lines compared to the normal tissue (Fig. 3A and B). To further investigate the influence of RKIP on malignant phenotypes in CRC in vitro, the CRC cell line HCT116 which had the relatively lowest RKIP expression level, was chosen for reconstituting RKIP expression. The CRC SW480 cells which had the relatively highest RKIP expression level were chosen for RKIP shRNA knockdown experiment. The effects of exogenous RKIP overexpression in HCT116 cells and RKIP knockdown in SW480 cells were confirmed by western blotting (Fig. 3C).

Cell motility was determined using migration and invasion assays. As shown in Fig. 4A and B, the restoration of RKIP expression inhibited HCT116/RKIP cells from penetrating the polycarbonate membrane and a collagen matrix by an average of 48.5 and 45.6%, respectively, compared to the HCT116/vector cells. Similar results were observed in the SW480 cell line in which RKIP knockdown significantly increased cell migration and invasion (Fig. 4C and D). Moreover, RKIP suppression of cell motility was confirmed by a wound healing assay. At 32 h, wound closure in the HCT116/RKIP cells was modest compared to the HCT116/vector cells (26.7±6.1 vs. 65.1±4.0%; p<0.001). Conversely, the distance closure of the SW480/vector cells was much shorter than that of the SW480/sh-RKIP cells (47.7±4.1 vs. 70.2±2.3%; p<0.001; Fig. 4E and F). Next, the effect of RKIP on proliferation of cancer cells was evaluated using the MTT assay in the transfected cells. The cell proliferation curve indicated that neither exogenous overexpression nor reduction of RKIP had generated statistically significant differences in cell proliferative rates (p>0.05).

To further verify the inhibitory effect of RKIP on metastasis in vivo, the two paired transfected cell lines (HCT116/RKIP and HCT116/vector, SW480/sh-RKIP and SW480/vector) were injected into the tail veins of nude mice. After 6 weeks, the nude mice were sacrificed to measure lung and liver metastases. Only mice injected with the HCT116/vector cells had observed macrometastases when compared to the HCT116/RKIP group, including 66.7% (4/6 mice) pulmonary, 16.7% (1/6 mice) hepatic, 50% (3/6 mice) subcutaneous and 16.7% (1/6 mice) muscle macrometastases, indicating that the overexpression of RKIP markedly decreased the incidence of metastasis. Furthermore, the lung and liver micrometastases obtained from the four groups were examined and counted under a microscope. The average number of lung micrometastases in mice injected with the HCT116/RKIP cells was 64.3% less than that in mice injected with the HCT116/vector cells (p=0.010; Fig. 5A and B). The average number of pulmonary micrometastases in mice injected with the SW480/vector cells was 79.6% less than that in those injected with the SW480/siRKIP cells (p=0.039; Fig. 5C and D), which confirmed the role of RKIP in metastasis suppression in vivo in CRC. However, there were no statistically significant differences in the comparison of the average number of liver micrometastases between the two groups (p>0.05). To explore the effect of RKIP on tumorigenesis in vivo, the reconstructed cells and their control cells were injected into the left and right dorsal flanks of nude mice, respectively, as described in Materials and methods. The tumor growth curve was generated by measuring tumor volume over time, and the data showed no statistically significant difference between the reconstructed cells and their control cell groups. RKIP neither suppressed nor promoted tumor growth (Fig. 6).