Chitosan oligosaccharide enhances the anti-cancer effects of 5-fluorouracil on SNU-C5 colorectal cancer cells by activating ERK

Abstract

Background: Chitosan oligosaccharide (COS) is the major degradation product of chitosan by enzymatic processes. COS, with complete water solubility, exerts significant biological effects, including anti-cancer activity. We investigated the anti-tumor effects of COS on colorectal cancer as effective therapeutic methods with low side effects are lacking.

Methods: COS was obtained from low molecular weight chitosan by an enzymatic method and the anti-cancer effects were measured by cell viability assay, flow cytometry analysis, Western blotting, and xenograft.

Results: COS suppressed the proliferation of SNU-C5 cells compared to other colorectal cancer cells, but higher concentrations were required in the xenograft model. Co-treatment with 5-fluorouracil (5-FU) and COS enhanced the anti-cancer effects of 5-FU in SNU-C5 cells in vitro and in vivo. Flow cytometry revealed that COS induced cell cycle arrest at the G0/G1 phase without 5-FU or at the S and G2/M phases with 5-FU but did not affect cell death pathways. COS increased extracellular signal-regulated protein kinase (ERK) activation with or without 5-FU, whereas 5-FU treatment increased p53 activation. A low-dose of an ERK inhibitor suppressed COS-induced ERK activation and resulted in higher proliferation compared with COS.

Conclusions: These results suggest that COS might enhance the anti-cancer effects of 5-FU in SNU-C5 colorectal cancer cells by activating ERK.

Keywords: 5-Fluorouracil (5-FU); Chitosan oligosaccharide (COS); Colorectal cancer (CRC); ERK (Extracellular signal-regulated protein kinase).

Figure 1. Scheme of xenograft model.

Figure 2. Anti-cancer effects of chitosan oligosaccharide (COS) in colorectal cancer cells. Among the colorectal cancer cell lines tested, SNU-C5 cells were the most sensitive to COS treatment in a dose-dependent manner (A). The anti-proliferative effects were significant at 10 μg/mL of COS (B). *p < 0.05, ***p < 0.001 vs. vehicle.

Figure 3. Anti-cancer effects of chitosan oligosaccharide (COS) in SNU-C5 cells in vivo. Body weights of SNU-C5 xenograft mice remained stable after COS treatment. COS treatment (500 mg/kg) significantly inhibited tumor growth (volume and weight) 40 days after treatment. *p < 0.05 vs. vehicle.

Figure 4. Anti-cancer effects of 5-fluorouracil (5-FU) in colorectal cancer cells. Among the colorectal cancer cell lines tested, SNU-C5/5-FUR cells were the most resistant to 5-FU treatment (A). Co-treatment with 5-FU and chitosan oligosaccharide (COS; 1, 10, and 100 μg/mL) further decreased SNU-C5 cell viability (B). The anti-proliferative effects of co-treatment were significant at 10 μg/mL of COS. *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. 5-FU.

Figure 5. Anti-cancer effects of chitosan oligosaccharide (COS) as an adjuvant to 5-fluorouracil (5-FU) in SNU-C5 cells in vivo. Co-treatment with 5-FU and chitosan oligosaccharide (COS) further decreased SNU-C5 cell viability. The anti-proliferative effects of co-treatment were significant at 100 μg/mL of COS. *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle; ^#p < 0.05 vs. COS; ^§p < 0.05, ^§§p < 0.01 vs. 5-FU.

Figure 6. Anti-cancer effects of chitosan oligosaccharide (COS) on cell cycle arrest in SNU-C5 cells. Representative images of the vehicle, COS (100 μg/mL), 5-FU (1 μM), and 5-FU + COS-treated SNU-C5 cells and flow cytometry (A). COS did not affect cell death pathways, whereas 5-FU induced apoptosis regardless of COS co-treatment. COS induced cell cycle arrest at the G0/G1 phase without 5-FU and the S and G2/M phases with 5-FU (B). *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle; ^##p < 0.01, ^###p < 0.001 vs. COS; ^§p < 0.05 vs. 5-FU.

Figure 7. Mitogen-activated protein kinase (MAPK) signaling pathways would be feasible candidates for the mechanism of the anti-cancer effects of chitosan oligosaccharide (COS, 100 μg/mL) in SNU-C5 cells. COS treatment activated ERK with or without 5-fluorouracil (5-FU, 1 μM) among MAPK, p53, and Akt signaling pathways, whereas 5-FU treatment increased p53 phosphorylation. *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. COS; ^§p < 0.05, ^§§p < 0.01 vs. 5-FU.

Figure 8. Chitosan oligosaccharide (COS)-induced ERK activation was not observed in other colorectal cancer cells. Although ERK activation was consistent, ERK expression was higher in SNU-C5 cells (A). COS (100 μg/mL) treatment decreased proliferation in HCT116 cells but no significant changes in HT29 and SNU-C5/5-FUR cells (B). COS (100 μg/mL) treatment did not change the ERK activation in HCT116 and HT29 cells but significantly decreased in SNU-C5/5-FUR cells (C). *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle.

Figure 9. Chitosan oligosaccharide (COS)-induced ERK activation was specific in SNU-C5 cells. ERK inhibitor (ERK-I; PD98059) diminished cell viability in a dose-dependent manner but increased with low-dose in SNU-C5 cells (A). PD98059 significantly increased the relative proliferation at 5 μM. COS (100 μg/mL)-induced anti-proliferative effects were abolished by co-treatment with PD98059 at 5 and 10 μM (B). COS-dependent activation of ERK and p38 was decreased by co-treatment with PD98059 (C). *p < 0.05, ***p < 0.001 vs. vehicle; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. COS.