Expression of CIAPIN1 in human colorectal cancer and its correlation with prognosis

A total of 273 patients with primary CRC, who underwent surgery at the Department of Anorectal Surgery at Tianjin Union Medicine Centre, were recruited for this study from 2000 to 2003. The mean age was 58 years (range: 23~82 years) with 136 women and 137 men. Cancer tissues, along with normal tissues that were at least 5 cm away from the cancer, were obtained from the patients. Western blot analysis was performed on fresh samples from 40 CRC patients. All 273 patients' survival information of 60 month postoperative follow-up was received by telephone and mail. The median follow-up period was 39.7 months (range: 10~65 months). Patients' characteristics, such as gender, age, location of the tumour, UICC stage, local recurrence and tumour stage factors, were obtained from the medical records. Patient characteristics are summarised in Table 1. All resection samples were confirmed to be CRC by clinical pathology. All the patients were staged based on the UICC staging system. Of the 273 patients, 24 (8.8%) had T1-stage, 33 (12.1%) had T2-stage, 182 (66.7%) had T3-stage, and 34 (12.4%) had T4-stage CRC. Tissues were fixed in 10% formaldehyde, embedded in paraffin, cut into 4- μm sections, and mounted on slides. All patients gave informed consent to use excess pathological specimens for research purposes. The protocols used in the study were approved by the hospital's Protection of Human Subjects Committee. The use of human tissues in this study was approved by the institutional review board of the Fourth Military Medical University and was done in accordance with international guidelines. Of the 273 patients, 23 (8.4%, some CRC patients with stage IV of UICC) received neoadjuvant chemotherapy, 242 (88.6%, CRC patients with stage IIB, IIC, III and IV of UICC) underwent surgery alone and received subsequent chemotherapy, and 31 (11.4%, CRC patients with stage I and IIA of UICC) only received surgical treatment.

The cancer and normal tissues from 273 patients were embedded in paraffin and cut into sections for immunohistochemical analysis. Immunohistochemistry was performed using the Histostain-Plus SP kit, which offers superior sensitivity. Briefly, the sections were deparaffinised with xylene and rehydrated through gradient ethanol immersion. Endogenous peroxidase activity was quenched by 0.3% (v/v) hydrogen peroxide in methanol for 20 min, followed by three 5-min washes with PBS. The sections were then blocked with 10% (v/v) normal goat serum in PBS for 1 hr, followed by overnight incubation at 4°C with the anti-CIAPIN1 antibody diluted (1:200, initial concentration 1.8 mg/ml; Mouse anti-CIAPIN1 MAb was developed by our laboratory) [12, 13] in PBS containing 3% (wt/vol) BSA. A negative control was performed by replacing the primary antibody with pre-immune mouse serum. After three 5 min washes with PBS containing 0.02% (v/v) Tween-20 (PBST), the sections were treated with biotinylated goat anti-mouse antibody for 20 min at room temperature, followed by three additional 5 min washes with PBST. Then, the specimens were incubated with streptavidin-horseradish peroxidase for 20 min at room temperature followed by repeated washes, as described above. Reaction product was visualised with DAB at room temperature for 5 min. Sections were counterstained with haematoxylin for 30 sec and rinsed with tap water, immediately dehydrated by sequential immersion in gradient ethanol and xylene and were then mounted with Permount onto coverslips. Images were obtained under a light microscope (Olympus BX51; Olympus, Japan) equipped with a DP70 digital camera.

Sections without the primary antibody were used as negative controls. Clear cell renal cell carcinoma samples that previously showed immunoreactivity to the CIAPIN1 antibody were used as positive controls to confirm CIAPIN1expression. The slides were evaluated independently by two pathologists who were blinded to the study [14‐16]. Any disagreement was resolved by consensus after joint review. Expression of CIAPIN1 was evaluated as the percentage of positive cells and staining intensity as previously described. The percentage of positive cells was evaluated quantitatively and scored as 0 for staining of ≤1% of total cells counted, 1 for staining of 2~25%, 2 for staining of 26~50%, 3 for staining of 51~75%, and 4 for staining of >75% of the cells examined. Intensity was graded as follows: 0, no signal; 1, weak; 2, moderate; and 3, strong staining. A total 'staining score' of 0~12 was calculated and graded as negative (-, score 0~1), weak (+, score 2~4), moderate (++, score 5~8), or strong (+++, score 9~12) [16, 17].

The relationship between CIAPIN1 expression levels and clinicopathological factors was analysed using the Wilcoxon-Mann-Whitney test. The overall survival time of CRC patients was defined as the time from the surgery to death due to cancer. The Kaplan-Meier method was used to determine the cumulative probability of survival, and data were analysed with the log-rank test. Univariate and multivariate statistical analyses were done using the Cox regression model to investigate the effects of patients' characteristics (CIAPIN1 status, gender, age, location, extent of primary tumour, nodal status, metastasis and histological grade) on overall survival. A score was assigned to each variable for the Cox regression analysis [17, 18]. A value of P < 0.05 was considered statistically significant.

Additionally, receiver operator characteristic (ROC) curves were constructed for expression of CIAPIN1 as a predictor for 'patient death' and 'cancer local recurrence'. The ROC curve determines how effective a predicted risk value is at discriminating a bivariable outcome (in this case 'death' or 'no death', or 'local recurrence' or 'non- local recurrence') by constructing a graph with sensitivity on the Y-axis and one specificity on the X-axis. It depicts the inverse relationship between sensitivity and specificity.

Equal amounts of total proteins were loaded on 12% SDS PAGE and electroblotted onto a nitrocellulose membrane. Non-specific binding was blocked with 5% non-fat milk in PBS for 1 hr at room temperature. Then, the membrane was incubated with anti-CIAPIN1 MAb (1:400) overnight at 4°C, rinsed with TBST three times and then incubated with HRP-labelled goat anti-mouse IgG for 1 hr. After three washes with TBST, the bands were developed with the enhanced chemiluminescence reagent for 5 min [14].

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Two micrograms of RNA were used for reverse transcription. The polymerase chain reaction (PCR) primers used were as follows: for CIAPIN1, 5'-CGG AAT TCATGG CAG ATT TTG GGA TCT C-3' (forward), 5'-GGT CGA CCT AGG CAT CAA GAT TGC TAT C-3' (reverse) and for β-actin, 5'-ATG ATATCG CCG CGC TCG TC-3' (forward), 5'-CGC TCG GTG AGG ATC TTC A-3'(reverse). PCR products were separated on a 1% agarose gel, visualised and photographed under ultraviolet light [14].

The human CRC cell lines LoVo, CoLo205, HCT116, HT-29, SW620, and SW480 were obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained as recommended. Cells were cultured in DMEM with 2 mM l-glutamine and Earle's balanced salt solution (BSS) adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids, and 1.0 mM sodium pyruvate. All culture fluid was supplemented with 10% FCS. All cells were cultured with 5% CO₂ at 37°C in a humidified chamber [17, 18].

The human colon carcinoma cell lines HT29 and SW480 were cultured in L-15 medium (Sigma-Aldrich). The media contained 10% foetal bovine serum (Atlanta Biologicals), and cells were cultured at 37°C in 5% CO₂. Differentiation was induced by treatment with 2 mM sodium butyrate (Sigma Chemical) for 6 d, and cells were harvested each day from the beginning of treatment to the sixth day. The differentiation stage was assessed by transmission electron microscopy (TEM; for changes in cell structure and architecture) and degree of alkaline phosphatase (AP) activity. After removal of the culture medium, the attached cells were scraped into ice-cold PBS, centrifuged at 4000 × g, resuspended in PBS, and sonicated with an ultrasonic cell disrupter. Cellular debris was pelleted by centrifugation, and supernatants were transferred to new tubes and stored at -70°C until measurement. AP activity was measured according to the manufacturer's instructions using p-nitro-phenylphosphate as the substrate (Merck, Darmstadt, Germany) and calculated in units per milligram protein (U/mg prot). Protein content was determined using a BCA™ Protein Assay Kit.

The expression of CIAPIN1 by immunoreactivity with the specific MAb was generally localised in both the cytoplasm and the nucleus of colonic epithelial tissues Fig. 1[A(a-e)]. CIAPIN1-positive expression in CRC was 47.25% (129/273), significantly lower than 79.85% (218/273) in the unaffected tissue adjacent to the tumour (P < 0.01). CIAPIN1 protein expression in CRC was significantly decreased compared with that in normal colonic epithelial tissues. In addition, its expression level decreased from well-differentiated to poorly differentiated tumours Fig. 1[A(c-e)]. However, there was no significant difference in the expression level of CIAPIN1 protein between normal and gland hyperplasia of colonic mucous membrane tissues Fig. 1[A(a-b)]. As shown in Fig. 1A and Table 1, 273 CRC patients were subdivided into the following three subgroups based on the expression levels of CIAPIN1, 165 with none to weak expression (60.4%; -/+), 72 with moderate to locally strong expression (26.4%; ++), and 36 with strong expression (13.2%; +++).

The correlation between expression level of CIAPIN1 and patients' characteristics, such as gender, age, location of cancer, UICC stage, local recurrence and tumour stage factors was investigated. CIAPIN1 protein expression correlated with having a primary tumour (pT), tumour stage (UICC), and histological grade (Table 1; P = 0.0393, 0.0297, and 0.0397, respectively). No correlation was observed between CIAPIN1 expression and age, gender, location, local recurrence, L stage, V stage, pN and pM. The overall survival analysis using the Kaplan-Meyer method revealed that the prognosis of CRC patients with high or moderate CIAPIN1 expression was significantly better than those with no or weak CIAPIN1 expression, and high expression was better than moderate expression (Fig. 1(B); P = 0.0002).

The ROC curve for non-local recurrence within 5 years (Fig. 1(C)) showed that the score 7 of CIAPIN1 expression level provided maximal sensitivity (0.63) and specificity (0.74). Similarly, the curve for survival after 5 years (Fig. 1(C)) showed that the score 6 of CIAPIN1 expression level provided a maximal sensitivity (0.61) and specificity (0.73). The area under the curve for non-local recurrence was 0.74 and for survival was 0.71. This shows that the score of CIAPIN1 expression level did predict non-local recurrence and survival.

Both univariate and multivariate analyses showed that primary tumour (pT₃/pT₄), regional lymph node metastasis (pN₁), distant tumour metastasis (pM₁), local recurrence (yes) and low-expression CIAPIN1 (-/+) were independent, poor prognostic factors of CRC (Table 2 and Table 3); However, age (> 75 years), gender (male), location (rectum or sigmoid), adjuvant therapy (yes) and tumour grade (> G2) were not related to the prognosis of CRC (Table 2 and Table 3).

To accurately quantify CIAPIN1 expression level changes in colorectal carcinomas, we performed Western blot analysis in 40 CRC tissues and matched normal tissues. Among the 40 clinical specimens, as expected, 28 (70.0%) samples of CRC tissues showed lower expression than that in adjacent normal tissues; 7 (18.5%) samples of CRC tissues showed higher expression than that in adjacent normal tissues; and 5 (11.5%) samples of CRC tissues showed no expression. Based on these results, we confirmed that CIAPIN1 protein expression was lower in carcinomas tissues compared with adjacent normal tissues (Fig. 2(A)). We subsequently investigated whether the expression of CIAPIN1 correlated with the differentiation status and UICC stage as shown by immunohistochemistry. CIAPIN1expression was found to be increased in well-differentiated tumours (Fig. 2(B)). Furthermore, CIAPIN1 expression decreased gradually with increasing stage (Fig. 2(C)). This was consistent with the results of the immunohistochemistry experiments.

CIAPIN1 gene and protein expression levels were tested in the human CRC cell lines LoVo, CoLo205, HCT116, HT-29, SW620, and SW480. As shown in Fig. 3A, CIAPIN1 expression was detected in all CRC cell lines. To investigate the expression of CIAPIN1 during CRC cell differentiation, in vitro studies with HT-29 and SW480 CRC cell lines were performed. Cells were treated with 2 mM sodium butyrate to induce differentiation. Following incubation, the differentiation status was confirmed by TEM examination of the presence of regular brush borders and tight junctions, which are structural markers of differentiation (Fig. 3B). In addition, butyrate-induced differentiation was measured by alkaline phosphatase (AP) activity. AP activity increased significantly in HT-29 and SW480 cells after 48 h of incubation (Fig. 3C). Both protein and mRNA expression levels of CIAPIN1 were measured in HT29 and SW480 cell lines at the indicated time points after butyrate treatment. Prolonged incubation with butyrate resulted in a time-dependent induction of CIAPIN1expression (Fig. 3D).