Higher CO2-insufflation pressure inhibits the expression of adhesion molecules and the invasion potential of colon cancer cells

Abstract

Aim: To investigate the influence of CO(2)-insufflation pressure on adhesion, invasion and metastatic potential of colon cancer cells based on adhesion molecules expression.

Methods: With an in vitro artificial pneumoperitoneum model, SW1116 human colon carcinoma cells were exposed to CO(2)-insufflation in 5 different pressure groups: 6 mmHg, 9 mmHg, 12 mmHg, 15 mmHg and control group, respectively for 1 h. Expression of E-cadherin, ICAM-1, CD44 and E-selectin was measured at 0, 12, 24, 48 and 72 h after CO(2)-insufflation using flow cytometry. The adhesion and invasion capacity of SW1116 cells before and after exposure to CO(2)-insufflation was detected by cell adhesion/invasion assay in vitro. Each group of cells was injected intraperitoneally into 16 BALB/C mice. The number of visible abdominal cavity tumor nodules, visceral metastases and survival of the mice were recorded in each group.

Results: The expression of E-cadherin, ICAM-1, CD44 and E-selectin in SW1116 cells were changed significantly following exposure to CO(2) insufflation at different pressures (P < 0.05). The expression of E-cadherin, CD44 and ICAM-1 decreased with increasing CO(2)-insufflation pressure. The adhesive/invasive cells also decreased gradually with increasing pressure as determined by the adhesion/invasion assay. In animal experiments, the number of abdominal cavity tumor nodules in the 15 mmHg group was also significantly lower than that in the 6 mmHg group (29.7 +/- 9.91 vs 41.7 +/- 14.90, P = 0.046). However, the survival in each group was not statistically different.

Conclusion: CO(2)-insufflation induced a temporary change in the adhesion and invasion capacity of cancer cells in vitro. Higher CO(2)-insufflation pressure inhibited adhesion, invasion and metastatic potential in vitro and in vivo, which was associated with reduced expression of adhesion molecules.

Figure 1

Five litres volume airtight Perspex box with two ventilating ports for pneumoperitoneum establishment in vitro.

Figure 2

Flow cytometry analysis of ICAM-1 and E-cadherin expression in SW1116. A: ICAM-1 (Room air control); B: ICAM-1 (0 h after the 6 mmHg insufflation); C: ICAM-1 (72 h after the 6 mmHg insufflation); D: E-cadherin (Room air control); E: E-cadherin (24 h after the 9 mmHg insufflation); F: E-cadherin (72 h after the 9 mmHg insufflation). Immediately after the 6 mmHg CO₂ insufflation (B), ICAM-1 expression increased significantly compared to room air controls (A) (P = 0.009), while it was significantly less at 72 h after the 6 mmHg insufflation (C) than control group. Twenty four hours after 9 mmHg CO₂ insufflation (E), E-cadherin expression increased significantly compared to controls (D) (P = 0.035), but returned to the control value at 72 h after the 9 mmHg insufflation (F).

Figure 3

The effect of CO₂ insufflation pressure on expression of adhesion molecules. A: Expression of CD44; B: Expression of E-cadherin; C: Expression of ICAM-1. With increasing CO₂-insufflation pressure, the expression of CD44 (A), E-cadherin (B), and ICAM-1 (C) decreased at some time points, especially at the start (0 h) after CO₂-insufflation.

Figure 4

Results of adhesion assay. A: 0 h after CO₂ insufflation; B: 72 h after CO₂ insufflation. At 0 h after CO₂ insufflation (A), with increasing CO₂-insufflation pressure, the adhesion of the SW1116 cell line decreased gradually (12 mmHg vs control, P < 0.001; 15 mmHg vs 12 mmHg, P < 0.001). At 72 h after CO₂ insufflation (B), cell adhesion was similar among the different groups (P > 0.05).

Figure 5

Results of invasion assay. A: SW1116, 0 h after exposure; B: SW1116, 72 h after exposure; C: Lovo, 0 h after exposure; D: Lovo, 72 h after exposure. A: ¹At 0 h after exposure to 15 mmHg CO₂ insufflation, invasive cells decreased significantly compared to the cells prior to exposure (P = 0.019); ²At 0 h after exposure, invasion of the 6 mmHg or the 15 mmHg group was significantly different from the other pressure groups (P < 0.05). B: The invasion fluorescence intensities of SW1116 before and 72 h after exposure to CO₂ insufflation were not significantly different (P > 0.05). Seventy two hours after the CO₂ insufflation, there were no significant differences between the various insufflation pressure groups (P > 0.05); C: ¹At 0 h after exposure to 15 mmHg, 12 mmHg or 6 mmHg CO₂ insufflation, invasive cells of Lovo decreased significantly compared to the cells prior to exposure (P = 0.008, P = 0.0045, P = 0.043); ²At 0 h after exposure, cell invasion at 6 mmHg was significantly different from the other pressure groups (P < 0.01); ³At 0 h after exposure, cell invasion in the 12 mmHg group was significantly less than the 9 mmHg group (P < 0.05); ⁴At 0 h after exposure, cell invasion in the 15 mmHg group was significantly less than the 6 and 9 mmHg groups (P < 0.01); D: The invasion fluorescence intensities of Lovo before and 72 h after exposure to CO₂ insufflation were not significantly different (P > 0.05). Seventy two hours after CO₂ insufflation, there were no significant differences between the various insufflation pressure groups (P > 0.05).

Figure 6

Results of intra-abdominal tumor nodules. A: Mesentery tumor nodules in the 15 mmHg group; B: Mesentery tumor nodules in the 6 mmHg group; C: Numbers of intra-abdominal tumor nodules in the various groups. The number of intra-abdominal tumor nodules in the 15 mmHg group (A) was significantly lower than those in 6 mmHg group (B) (^aP = 0.046). The mean numbers of abdominal cavity nodules formed by the cells in each group are described in (C).

Figure 7

Survival time of the mice injected with SW1116 cells in the different groups (P = 0.426).