Nafamostat mesylate prevents metastasis and dissemination of neuroblastoma through vascular endothelial growth factor inhibition

Abstract

Neuroblastoma is a highly malignant disease with a poor prognosis and few treatment options. Despite conventional chemotherapy for neuroblastoma, resistance, invasiveness, and metastatic mobility limit the treatment efficacy. Therefore, it is necessary to develop new strategies for treating neuroblastoma. The present study aimed to evaluate the anticancer effects of nafamostat mesylate, a previously known serine protease inhibitor, on neuroblastoma cells. Effects of nafamostat mesylate on neuroblastoma cell migration and proliferation were analyzed by wound healing assay and WST-8 assay, respectively. To elucidate the mechanisms underlying the effects of nafamostat mesylate on neuroblastoma, the expression levels of NF-κB were measured via western blotting, and the production of the cytokine vascular endothelial growth factor (VEGF) in the cell culture supernatants was determined via ELISA. In addition, a mouse model of hematogenous metastasis was used to investigate the effects of nafamostat mesylate on neuroblastoma. It was determined that nafamostat mesylate significantly inhibited migration and invasion of Neuro-2a cells, but it had no effect on cell proliferation at 24 h after treatment. Exposure of Neuro-2a cells to nafamostat mesylate resulted in decreased vascular endothelial growth factor production, which could be a pivotal mechanism underlying the inhibitory effects of neuroblastoma metastasis. The results of the present study suggest that nafamostat mesylate may be an effective treatment against neuroblastoma invasion and metastasis.

Keywords: metastasis; murine model; nafamostat mesylate; neuroblastoma; vascular endothelial growth factor.

Figure 1

Nafamostat mesylate inhibits cell migration and invasion. (A) Wound healing assays were performed. Neuro-2a cells were cultured in 6-well plates until they formed a 90% confluent monolayer. The monolayer was scratched with a pipette tip, and the cells were treated with nafamostat mesylate (50 µM, nafamostat-treated group) or with the vehicle only (distilled water, control group). Images of cell migration into the wound were captured at 0 and 24 h after wound initiation. Magnification, x40. (B) Cell viability was measured using a WST-8 assay 24 h after treatment. Unpaired Student's t-tests were used to analyze significant differences between the two groups. No significant difference was observed between the nafamostat-treated group and the control group.

Figure 2

Nafamostat mesylate inhibits the dissemination of Neuro-2a cells in A/J mice. (A) Growth of intravenously implanted tumors. Liver and mesentery metastases were extracted from a mouse who received an intravenous injection of Neuro-2a cells. (B) The weight of the mice in the nafamostat-treated group and control group was evaluated at each time-point. Unpaired Student's t-tests were used to analyze significant differences between the two groups. (C) The survival rate of the mice after intravenous Neuro-2a injection. The length of survival in the nafamostat-treated group was significantly longer than that in the control group (P<0.05, log-rank test).

Figure 3

Effects of nafamostat mesylate on Neuro-2a cells. (A) Neuro-2a cells were incubated with nafamostat mesylate (0 or 50 µM) for 24 h. The VEGF concentrations in cell culture supernatants were measured using ELISA. Unpaired Student's t-tests were used to analyze significant differences between the two groups. (B) Neuro-2a cells were incubated with nafamostat mesylate (0, 10, or 100 µM) for 24 h. The expression levels of NF-κB (top) and actin (bottom) were detected using western blotting. VEGF, vascular endothelial growth factor; NF-κB, nuclear factor-κB.