The subjects were 935 colon cancer patients who had undergone resection for colorectal cancer at the First Department of Surgery, University of Fukui, Japan, between 1994 and 2010 (Table I). Surgical specimens of the peritoneal dissemination were obtained from 37 patients with peritoneal metastases as the only distant metastasis of primary colon cancer. HIPEC was performed in 22 patients with peritoneal dissemination as the only synchronous distant metastasis in cases of primary colon cancer at our hospital from 1994 to 2010. The outcome was then compared with that of patients (15 cases) who did not receive HIPEC. Cancers were reviewed and graded by two pathologists using criteria recommended by the general rules of clinical and pathological studies on cancer of colon, rectum and anus for histological type, lymphatic invasion and venous invasion (17). The research was performed in accordance with the humane and ethical rules for human experimentation that are stated in the Declaration of Helsinki.

This procedure allowed the abdominal cavity to be extended widely enough to allow perfusate to spread throughout the peritoneal cavity (18). Two liters of saline contained 150 mg of cisplatin, 20 mg mitomycin C, and 200 mg of etoposide. An additional 2 litres of the same infusate were heated in a waterbath and pumped into circulation between the abdomen and a reservoir at ~500 ml per minute. The temperature at several points of the peritoneal surface was maintained at appromimately 43°C by controlling the temperature of the water bath and the speed of the pump. Abdominal temperatures were measured at the serosal surface in the subphrenic space and the cavity of Douglas, and the temperature of the infusate was also measured in the inflow tube, outflow tube, and water bath. The thermal dose (TD) obtained during the treatment was calculated simultaneously during HIPEC and expressed in terms of equivalent time at 43°C (19). HIPEC was performed until the TD reached 40 min.

The patients with peritoneal dissemination as the only synchronous distant metastasis in cases of primary colon cancer were analyzed for MUC1, 2, 3, 4, and 5AC protein expression. Surgical specimens of the peritoneal dissemination prepared from formalin-fixed, paraffin-embedded tissues were analyzed for protein expression by the streptavidin-biotin peroxidase method (20). The expression was interpreted as positive when the protein was expressed in >30% of the cancer cells.

The following antibodies were used: anti-human MUC1, 2, and MUC5 (Novocastra, UK), MUC3 (Santa Cruz Biotechnology, CA, USA), MUC4 (Invitrogen, CA, USA).

The human colon cancer cell lines (SW620, colo205, and LoVo) were cultured at 37°C in 5% CO₂ in RPMI-1640 or DMEM medium containing 10% fetal bovine serum.

Total RNA was extracted from cells using Isogen (Wako, Japan) (21). Single strand cDNA prepared from 3 μg of total RNA using Moloney murine leukemia virus reverse transcriptase (Gibco-BRL, MD, USA) with an oligo (dT) primer-14 was used as the template for the polymerase chain reaction (PCR). The primers for PCR to amplify MUC2 gene-coding regions were: The 5′ primer, MUC2-AX: TGCCTGGCCCTGTCTTTG and the 3′ primer, MUC2-BX: CAGCTCCAGCATGAGTGC. Thirty cycles of denaturation (94°C, 1 min), annealing (50°C, 0.75 min), and extension (72°C, 2 min) were carried out in a thermal cycler (PTC-100, programmable thermal controller, NJ Research Inc., MA, USA). GAPDH amplification was used as internal PCR control with 5′-GGGGAGCCAAAAGGGTCATCATCT-3′ as the sense primer and 5′-GACGCCTGCTTCACCACCTTCTTG-3′ as the antisense primer. Thirty cycles of denaturation (94°C, 1 min), annealing (50°C, 1.5 min), and extension (72°C, 2 min) were carried out in a thermal cycler. PCR product (10 μl) was resolved by electrophoresis in 12% acrylamide gel. The sequencing was performed on PCR products that showed the bands in RT-PCR analysis. Sequence analysis showed the presence of the MUC2 gene.

The cells were cultured with RPMI-1640, 10% FBS and 1X penicillin/streptomycin at 37°C and 5% CO₂ incubator. MUC2-siRNA (Invitrogen) and scrambled (SCR)-siRNA (Invitrogen) were purchased. Cells were transfected with 100 nM of MUC2-siRNA, or SCR-siRNA with Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer’s protocol. SCR-siRNA was used as negative control.

The cells were plated onto a 96-well plate at 1×10⁴ and incubated for 12 h. The cells were treated with cisplatin of 100 μg/ml at 43°C (5% CO₂ incubator) for 24 h.

To assess cell proliferation, Cell Titer 96 Aqueous Non-radioactive Cell Proliferation Assay (Promega, Germany) was used according to the manufacturer’s instructions. MTS solution (20 μl) was added to each well (96-well plate) and the plates were incubated at 37°C for 1.5 h. The absorbance of the product formazan, which is considered to be directly proportional to the number of living cells in the culture, was measured at 490 nm using a Microplate Reader (Molecular Devices, CA, USA).

Survival time was calculated using the Kaplan-Meier method, and log-rank test was used to compare the curves of the survival times.

Other characteristics of the two treatment arms were compared using the chi-square test. Values of P<0.05 were considered as statistically significant.

Fig. 1 shows the survival rates in the HIPEC and non-HIPEC groups among all subjects with peritoneal dissemination. In the non-HIPEC group, the median survival time was 13 months and the 3-year survival rate was 15.6%, whereas in the HIPEC group the median survival time was 24 months and the 3-year survival rate was 39.2%. The HIPEC group thus had significantly better outcomes.

Fig. 2 shows images of positive and negative immunohistochemical staining using anti-MUC2 antibody in dissemination foci in patients with peritoneal dissemination of colorectal cancer, and Fig. 3 shows their incidence. Of the 22 patients, MUC1 protein expression was seen in 19 patients (86.4%), MUC2 in 10 (45.5%), MUC3 in 10 (45.5%), MUC4 in 15 (68.2%), and MUC5AC was seen in 15 (68.2%).

Expression of MUC2 protein and outcome was investigated in the patients with peritoneal dissemination of colon cancer in the HIPEC group. In patients positive for MUC2 expression, the MST was 14 months and the 3-year survival rate was 0.0%, whereas in patients negative for MUC2 expression, the 3-year survival rate was 61.1% (Fig. 3). Patients negative for MUC2 expression thus had a significantly better outcome. In the non-HIPEC group, no relationship was seen between outcome and expression of MUC2 protein. There was no significant difference between survival time and presence or absence of MUC1, 3, 4 and 5AC expression (Fig. 4a–d).

The results of an investigation of MUC2 mRNA expression in three different colon cancer cell lines are shown in Fig. 5. The highest expression of MUC2 was seen in the LoVo cell line. No expression was observed in SW620 and colo205.

When SiRNA-MUC2 was introduced into the LoVo cell line, which showed high MUC2 expression, the expression of MUC2 mRNA decreased as shown in Fig. 6. The percentage of living cells was investigated after culturing these cells for 1 day at 43°C in the presence of an anticancer agent. When the percentage of living cells among the cells with scrambled SiRNA (cultured at 37°C) was taken to be 100% (0.84O.D.), the percentage in the cells with scrambled SiRNA was 63% (0.53O.D.), and that in the cells with MUC2-SiRNA was 20% (0.17O.D.).