Prevention of liver metastasis by intraperitoneal 5-FU chemotherapy in nude mice inoculated with human colon cancer cells

Abstract

AIM: To prevent hepatic metastasis by regional adjuvant chemotherapy after radical surgery for colon cancer.

METHODS: A nude mouse model of human colon cancer (HCC) was used to evaluate the prevention of metastasis of HCC cells following the application of early postoperative intraperitoneal (IP) high-dose 5-fluorouracil chemotherapy.

RESULTS: The incidence of liver metastasis was decreased by 40%, and the mean number of metastatic liver nodules was reduced by 50.89%. Compared with controls, 5-FU 40 mg in NS 40 mL/kg IP for 2 consecutive days prolonged mean survival by 48.21%.

CONCLUSION: IP is a promising and effective novel regional adjuvant chemotherapy for the prevention of liver metastasis of HCC cells after radical surgery for colon cancer.

Key Words: Colonic neoplasms/surgery; Liver neoplasms/drug therapy; Fluorouracil/therapeutic use; Liver neoplasms/secondary

INTRODUCTION

The incidence of liver metastasis after radical surgery for colon cancer is very high and and significantly shortens the survival of patients. Intravenous (IV) 5-FU chemotherapy is often not effective for the prevention of liver metastasis. Therefore, early postoperative intraperitoneal (IP) chemotherapy with high-dose 5-FU was evaluated for prevention of liver metastasis by human colonic cancer (HCC) cells in a nude mouse model. HCC cells were inoculated via the spleen.

MATERIALS AND METHODS

Animals

Thirty athymic BALB/c nude mice 5-6 wk of age were provided by the Shanghai Cancer Institute. During the conduct of the study procedures, mice were maintained in a laminar flow cabinet under specific pathogen free conditions.

Cell lines

Colon cancer cell lines derived from human colonic adenocarcinoma were provided by Shanghai Immunology Institute[1]. The cells were cultured and maintained with more than 95% viability in RPMI 1640 medium with 10% fetal calf serum, penicillin (100 μg/mL), and streptomycin (100 μg/mL) at 37 °C in a 5% CO₂ atmosphere. Aliquots of 1 × 10⁶ cells were suspended in 0.03 mL in normal saline (NS) prior to use.

Splenic injection

Mice were anesthetized by intraperitoneal (IP) injection of phenobarbital and the spleen was exposed under sterile conditions by an incision in the left side of the animal. A 0.03 mL volume of NS containing 1 × 10⁶ cells was slowly injected into the splenic pulp over 1 min, then the spleen was replaced in position and the incision was closed[2]. The mice were randomly assigned to three groups of 10 each. Group A was given 5-Fu 40 mg in NS 40 mL^-1·kg^-1, for 2 consecutive days, group B was given 5-Fu 20 mg in NS 40 mL^-1·kg^-1, for 5 consecutive days, and group C (controls) was given NS 40 mL/kg, Animals were treated 24 h after tumor inoculation and followed-up to determine survival time. The animals were killed 60 days after tumor inoculation, the liver was removed and the tumor nodules were counted.

Statistics

Results were expressed as means ± standard deviation (x^-± s). Data were analyzed by analysis of variance (ANOVA) and the chi-square test . P≤ 0.05 was considered statistically significant.

RESULTS

The incidence of metastasis in groups A, B and C was 60% (6/10), 70% (7/10), and 100% (10/10), respectively. The 40% difference between group A and 30% difference between group B and controls were not statistically significant (P > 0.05).The mean number of metastatic liver nodules was 24.80 ± 8.64 in group A, 31.50 ± 8.68 in group B, and 50.50 ± 7.32 in group C. The differences of 50.89% between group A and controls and 37.62% between group B and controls were both significant (P < 0.05, Table 1).

Table 1 Metastatic nodules in the livers of experimental and control groups and controls ( x^-± s_x-, nodule).

Group	Liver metastatic nodules (n)					liver nodules (x-± s)
	0	1-20	21-40	41-60	61-80
5-FU 40 mga	4	2	1	1	2	24.80 ± 8.64
5-FU 20 mg	3	1	2	1	3	31.50 ± 8.68
Controls	0	2	1	3	4	50.50 ± 7.32

^aP < 0.05, vs controls

The mean survival times were 45.50 ± 5.12 days in group A, 43.90 ± 4.77 days in group B, and 30.70 ± 2.59 days in the control mice. Survival was prolonged by 48.21% and 43% in groups A and B, respectively (P < 0.05, Table 2). surgery

Table 2 Survival of mice in experimental and control groups.

Group	Survival time (d)				Mean survival (d)
	21-30	31-40	41-50	51-60
5-FU 40 mg	3	1	0	6	45.50 ± 5.12a
5-FU 20 mg	3	1	2	4	43.90 ± 4.77a
Controls	6	2	2	0	30.70 ± 2.59

^aP < 0.05, vs controls

DISCUSSION

Previous studies reported high concentrations of antineoplastic agents in the abdominal cavity, portal vein, and liver after IP administration, but low concentrations in the systemic circulation, indicating a pharmacokinetic advantage of IP over IV chemotherapy[3-6]. We reported in our previous studies that HCC cells survived, grew, and disseminated following IP injection, and that chemically induced peritonitis, peritoneal adhesion, and nephrotoxicity were not induced by early postoperative IP chemotherapy with 5-Fu 40 mg in NS, 40 mL^-1·kg^-1 for 2 consecutive days[7]. We used a nude mouse model of HCC cell metastasis to evaluate the effects of early postoperative IP chemotherapy with high-dose, large-volume 5-FU chemotherapy. We found a 40% reduction in the incidence of liver metastasis and a decrease in 50.89% in the mean number metastatic liver nodules. The mean survival time was prolonged by 48.21% compared with control mice. The results obtained in this experimental animal model suggest that IP chemotherapy has potential as an effective adjuvant approach in prevention of hepatic metastases in colon cancer.