In the present study, 60 colon cancer specimens obtained from the Department of Pathology, The First Affiliated Hospital of Henan University of Science and Technology (Luoyang, China) between January 2006 and January 2007 were examined. The patients included 35 males and 25 females with a mean age of 56.2 years (range, 42–71 years). Pathological data were studied prospectively following surgery. The survival time was calculated from the date of surgery to the follow-up deadline (August 2016) or date of mortality. Two experienced pathologists from the Department of Pathology, the First Affiliated Hospital of Henan University of Science and Technology, were blinded to the results of the present study. Clinicopathological parameters of these patients are displayed in Table I. Tumor differentiation and Dukes' stage were classified in accordance with the report by Fukushima et al (13). Written informed consent was obtained from each patient upon collection of the samples. The study protocol was approved by the Human Ethics Review Committees of Henan University of Science and Technology (approval no. 2013-PJ152).

Staining for IL-32 was performed using 10% formalin-fixed, paraffin-embedded serial sections at 37°C. Sections (4-µm thick) were cut from the selected paraffin blocks and deparaffinized in dimethylbenzene. The slides were microwaved in sodium citrate-hydrochloric acid buffer solution for 4 min for antigen retrieval at 95°C and rehydration with phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4). IL-32 was detected with a mouse polyclonal antibody (cat. no: sc-517408, 1:150 dilution, Santa Cruz Biotechnology, Dallas, TX, USA). Ki-67 (cat. no: GT209401) were purchased from Gene Biotechnology (http://www.genetech.com.cn, Shanghai, China). The antibody reaction was conducted at 37°C for 3 h. Labeling was detected by adding biotinylated secondary antibodies (http://www.maxim.com.cn/sitecn/myzhjcxthsjh/981.html, KIT-9710, Rabbit, 37°C for 0.5 h), avidin-biotin complex and diaminobenzidine (all from Maxim-Bio, Fuzhou, China, http://www.maxim.com.cn/sitecn/myzhjcxthsjh/981.html). Sections were then counterstained with 10% hematoxylin at 37°C for 0.5 h. Expression of IL-32 was scored according to the positive percentage and staining intensity of the stained tumor cells. The percent positivity was scored as ‘0’ (0-25%), ‘1’ (26-50%), ‘2’ (51-75%) and ‘3’ (>75%). The staining intensity was scored as ‘0’ (no staining), ‘1’ (weakly stained), ‘2’ (moderately stained) and ‘3’ (strongly stained). If the product of multiplication between staining intensity and the percentage of positive cells is ≥2, it was considered as immunoreaction positive (+). Ki-67 was scored according the positive number of 100 cancer cells at high magnification by a light microscope (Olympus Corporation, Tokyo, Japan; magnification, ×400).

A total of 5 paired colon cancer and non-cancerous colonic epithelium tissues were lysed (RIPA Buffer Beyotime Institute of Biotechnology) and lysates were obtained by centrifugation at 4°C (35,000 × g, 30 min). BCA Protein assay kit was used to measure protein concentration. Following applying 30 µg total protein to 10% SDS-PAGE, the total protein was transferred to a polyvinylidene fluoride membrane. Following blocking (5% skimmed milk, 37°C for 3 h), the membranes were incubated with IL-32 antibody at 4°C for 12 h (1:200 dilution; cat. no. sc-50001; Santa Cruz Biotechnology), followed by horseradish peroxidase-conjugated secondary antibody (Rabbit anti-goat IgG, ZB 2306, 1:5,000 dilution, incubation at 37°C for 2 h). Subsequently, IL-32 expression was detected by enhanced chemiluminescent substrate (cat. no. 34580; Thermo Scientific, Inc.). GAPDH was served as the internal control (1:150 dilution; cat. no. sc-0411; Santa Cruz Biotechnology).

Statistical analyses were performed using SPSS 13.0 for Windows (SPSS, Inc., Chicago, IL, US). The association between IL-32 and clinicopathological parameters was assessed using Fisher's exact test. Results are presented as mean ± SD. Survival curves were calculated using the Kaplan-Meier method and compared by log-rank test. The Cox proportional hazards regression model was performed for multivariate survival analysis. P<0.05 was considered to indicate a statistically significant difference.

To investigate the expression of IL-32 in colon cancer, IL-32 staining in 60 colon cancer specimens was assessed using immunohistochemistry. Immunohistochemical results indicated weak immunoreactivity of IL-32 in non-cancerous colonic epithelium (Fig. 1A and B); however, IL-32 staining was distributed in the cytoplasm of cancer cells (Fig. 1C and D). Of the 60 cases, 37 (61.67%) samples exhibited positive staining of IL-32 in the cytoplasm.

To further verify the results obtained from immunohistochemistry, western blot analysis was performed to detect the IL-32 expression in 5 paired colon cancer and non-cancerous colonic epithelium tissues. The data demonstrated that there was notably increased IL-32 expression in colon cancer tissues, compared with complementary non-cancerous tissues (Fig. 2), which was consistent with the immunohistochemical results.

To investigate the role of IL-32 on proliferation of colon cancer, the association between IL-32 and PI was evaluated. As depicted in Fig. 3, there was significantly increased PI in the group positive for IL-32 expression (64.5±9.2), compared with the IL-32 negative group (33.5±5.8). These data demonstrated that IL-32 may be associated with the proliferation of colon cancer cells.

As depicted in Table I, there were 26 IL-32 positive samples with Dukes' stage C+D, compared with only 6 for IL-32 negative samples. Furthermore, positive IL-32 expression was significantly associated with tumor size ≤5 cm, compared with negative IL-32 expression (P=0.015) and this result confirmed that IL-32 may be associated with the proliferation of colon cancer. No significant association was observed between IL-32 expression and other variables, including sex, age, tumor differentiation and lymph node metastasis (P>0.05).

In the present study, the survival of 60 patients with colon cancer, divided into IL-32-positive and -negative groups, was investigated. Patients positive for IL-32 expression had less favorable prognoses, compared with patients negative for IL-32 expression, which indicates that IL-32 may be an independent prognostic factor for colon cancer (IL-32-positive group vs. IL-32-negative group: P<0.05; Fig. 4).

Multivariate analyses were performed to confirm that IL-32 is an independent prognostic predictor for patients with colon cancer. Cox proportional regression models together with other pathological features, including sex, age, tumor size, Dukes' stage, tumor differentiation and lymph node metastasis, were used. As presented in Table II, multivariate analysis indicated that IL-32 expression, Dukes' stage, tumor differentiation and lymph node metastasis could be independent prognostic factors in patients with colon cancer. Notably, lymph node metastasis had the most significant parameter (P=0.014), followed by IL-32 expression (P=0.026), Dukes' stage (P=0.035) and tumor differentiation (P=0.042).